Microbial proteases for cell isolation

Abstract

The present invention relates to microbial proteases for cell isolation. The invention also relates to a composition which contains the microbial protease and is suitable for cell separation, application of the microbial protease in a cell separation process, and a cell separation method adopting the microbial protease.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a microbial protease for cell separation, a composition suitable for cell separation comprising the microbial protease, use of the microbial protease in a cell separation process, and a cell separation method using the microbial protease. Background Art

[0004] Cell detachment is a key step in the process of cell passaging when cells are grown as adherent cells and cell clusters. The detachment step preferably involves the use of proteolytic enzymes, as these enzymes are gentle yet effective in releasing cells from surfaces to which they are attached and in dissolving cell clusters formed in suspension culture.

[0005] Accutase® and Accumax® (both available, for example, from Innovative Cell Technologies, Inc.) are commercially available products for cell isolation that include a mixture of enzymes with proteolytic and collagenolytic activity isolated from invertebrate sources. A drawback associated with these products is that regulatory agencies generally do not permit the use of animal-derived products during drug development and manufacturing, which hinders their applicability for cell therapy. Another disadvantage of these products is that, because these mixtures are animal-derived, there is an inherent risk of batch-to-batch variability in composition and activity, resulting in less well-defined products.

[0006] TrypLE™ (available from, for example, ThermoFisher Scientific) is a commercially available trypsin product that can be used for cell separation. TrypLE™ is recombinantly produced and therefore not of animal origin. However, a disadvantage associated with TrypLE™ is that not all cell types can be adequately separated when subjected to trypsin treatment alone, which limits the broad applicability of this product.

[0007] The object of the present invention is to provide an enzymatic solution that addresses the shortcomings associated with current products for cell separation. In particular, the object of the present invention is to provide an enzymatic solution that meets regulatory requirements, can be used for the separation of a variety of different cell types, and can be produced in a uniform manner without batch-to-batch variability. Summary of the Invention

[0008] The present invention relates to microbial proteases and their use in cell separation and cell cluster dissociation processes. The present inventors have recognized that microbial proteases with increased P1 preferences for amino acid residues Leu, Tyr, Phe, and Lys are particularly suitable for use in cell separation. Without being bound by theory, it is speculated that the P1 preference characteristics exhibited by the microbial proteases of the present invention provide effective and gentle cleavage of cell surface proteins involved in surface attachment and intercellular adhesion. In addition, the microbial proteases of the present invention can be recombinantly produced, which ensures a highly unified production process and regulatory compliance when used to develop and produce cells for pharmaceutical applications such as cell therapy.

[0009] In a first aspect, the present invention relates to a composition suitable for cell separation comprising a microbial protease.

[0010] In a second aspect, the present invention relates to the use of microbial proteases in a cell separation process.

[0011] In a third aspect, the invention relates to methods for cell separation, the methods comprising contacting a cell with a composition of the first aspect, wherein the cell is attached to a surface or another cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown is an SDS-PAGE analysis of type IV collagen degradation. Lane 1: Protein ladder. Lane 2: 0.44 µg / mL desalted Accutase with type IV collagen substrate added. Lane 3: 0.1 µg / mL desalted Accutase with type IV collagen substrate added. Lane 4: Desalted Accutase without substrate. Lane 5: Type IV collagen substrate alone.

[0013] Figure 2 A schematic diagram of the hPSC setup is shown, indicating when hPSC monolayer dissociation and assessment of cell cluster formation were performed (circled passaging stages).

[0014] Figure 3 A schematic diagram of the hPSC setup is shown, indicating when assessment of hPSC cluster dissociation and re-formation was performed (circled passaging stages).

[0015] Sequence review

[0016] SEQ ID NO: 1 is the S1 protease from Sarocladium strictum.

[0017] SEQ ID NO: 2 is the S1 protease from Nocardiopsis prasina.

[0018] SEQ ID NO: 3 is a DNA sequence encoding the S1 protease from Sclerotium compactum.

[0019] SEQ ID NO: 4 is a DNA sequence encoding the S1 protease from Nocardiopsis cepa.

[0020] SEQ ID NO: 5 is the secretion signal from Bacillus clausii.

[0021] definition

[0022] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA, which is processed through a series of steps, including splicing, before appearing as mature, spliced mRNA.

[0023] Cell separation: The term "cell separation" refers to the process of separating or releasing smaller cell populations or even single cells from cell cultures, particularly 2D and 3D cell cultures. 2D cell cultures include adherent cell cultures, in which cells grow as a monolayer attached to the surface of a cell culture vessel (e.g., a flask or dish) and are attached to each other and / or to the surface of the cell culture vessel. 3D cell cultures include suspension cultures, in which cells grow as clusters suspended in an agitated growth medium and are attached to each other. 3D cell cultures also include enriched medium cultures (e.g., agarose or Matrigel) and scaffold cultures, in which cells are grown on a structural support. The terms "cell separation" and "cell dissociation" are used interchangeably herein.

[0024] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0025] Control sequences: The term "control sequence" means a nucleic acid sequence that is involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, and can be native or heterologous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, prepropeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, a control sequence includes a promoter and transcription and translation termination signals. These control sequences can be provided with linkers for the purpose of introducing specific restriction sites that facilitate connection of the control sequence to the coding region of the polynucleotide encoding the polypeptide.

[0026] Expression: The term "expression" means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0027] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide operably linked to appropriate control sequences capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers, and sequences that control termination of transcription and translation.

[0028] Fragment: The term "fragment" means a polypeptide having one or more amino acids missing from the amino and / or carboxyl terminus of a mature polypeptide, wherein the fragment has protease activity. In one aspect, the fragment has chymotrypsin activity. In one aspect, the fragment has type I collagenase activity. In one aspect, the fragment has type IV collagenase activity.

[0029] Heterologous: With respect to a host cell, the term "heterologous" means that the polypeptide or nucleic acid is not naturally present in the host cell. With respect to a polypeptide or nucleic acid, the term "heterologous" means that the control sequences (e.g., promoter) of the polypeptide or nucleic acid are not naturally associated with the polypeptide or nucleic acid, i.e., the control sequences are from a gene other than the gene encoding the mature polypeptide.

[0030] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct (including a polynucleotide encoding a polypeptide of the present invention) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by the cell.

[0031] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.). Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form not found in nature. Isolated polypeptides include, but are not limited to, culture fluid containing secreted polypeptides expressed in host cells.

[0032] Native: The term "native" means a nucleic acid or polypeptide that occurs naturally in a host cell.

[0033] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the present compositions and methods encompass nucleotide sequences that encode a specific amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5' to 3' orientation.

[0034] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner not originally found in nature to contain a segment of nucleic acid or is synthetic and comprises one or more control sequences operably linked to the nucleic acid sequence.

[0035] Operably linked: The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0036] Passaging: The term "passaging" refers to the process of removing some or all cells from a culture and transferring them to fresh growth medium. Cell passaging can also be referred to as secondary culture. In some embodiments, passaging produces a single-cell suspension.

[0037] Protease: The term "protease" refers to a polypeptide having protease activity (EC 3.4; also known as peptidase activity) that catalyzes the hydrolysis of peptide bonds. The EC 3.4 group includes several subgroups, including EC 3.4.21 (serine endopeptidases), which further contains several subgroups, including EC 3.4.21.62 (subtilisin). The term "protease" and the expression "polypeptide having protease activity" are used interchangeably herein.

[0038] For the purposes of the present invention, protease activity (EC 3.4) may be determined according to the protease activity assay described in the Examples herein.

[0039] For the purposes of the present invention, trypsin activity (EC 3.4.21.4) can be determined according to the trypsin activity assay described in the Examples herein.

[0040] For the purposes of the present invention, chymotrypsin activity (EC 3.4.21.1) may be determined according to the chymotrypsin activity assay described in the Examples herein.

[0041] For the purposes of the present invention, type I collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0042] For the purposes of the present invention, type IV collagenase activity can be determined according to the type IV collagenase activity assay described in the Examples herein.

[0043] Purified: The term "purified" means a nucleic acid, polypeptide (e.g., a microbial protease), or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid forms discrete bands in an electrophoretic gel, a chromatography eluate, and / or a culture medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9% or more pure (e.g., by weight or by molar percentage). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is substantially increased following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other designated material or component in a composition at a relative or absolute concentration greater than that of the starting composition.

[0044] In one aspect, the term "purified" as used herein refers to a polypeptide (e.g., a microbial protease) or cell that is substantially free of components (especially insoluble components) from the producing organism. In other aspects, the term "purified" refers to a polypeptide that is substantially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some soluble components of the organism and culture medium from which it is recovered. The polypeptide can be purified (i.e., isolated) by one or more of the unit operations of filtration, precipitation, or chromatography.

[0045] In one aspect, the polypeptide can be purified (e.g., a microbial protease) so that only a small amount of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" can refer to removing other components, particularly other proteins, and most particularly other enzymes, present in the cell from which the polypeptide originates. The polypeptide can be "substantially pure," i.e., free of other components from the organism from which it is produced (e.g., a host organism for recombinantly producing the polypeptide). In one aspect, the polypeptide can be at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide can be at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the preparation (e.g., a composition suitable for cell separation). As used herein, "substantially pure polypeptide" can refer to a preparation of polypeptide that contains at most 10%, preferably at most 9%, preferably at most 8%, preferably at most 7%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, more preferably at most 2%, more preferably at most 1%, more preferably at most 0.5%, more preferably at most 0.1%, more preferably at most 0.05%, more preferably at most 0.01%, even more preferably at most 0.005%, and most preferably at most 0.001% by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0046] Thus, it is preferred that a substantially pure polypeptide (e.g., a microbial protease) is at least 90% pure, preferably at least 91%, more preferably at least 92% pure, more preferably at least 93% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, more preferably at least 99% pure, more preferably at least 99.5% pure, more preferably at least 99.9% pure, more preferably at least 99.95%, more preferably at least 99.99% pure, even more preferably at least 99.995% pure and most preferably at least 99.999% pure, based on the weight of the total polypeptide material present in a formulation (e.g., a composition suitable for cell separation). Polypeptides of the present invention are preferably in substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated therewith, whether native or recombinant). For example, this can be achieved by preparing the polypeptide using well-known recombinant methods or by using classical purification methods.

[0047] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation (e.g., cleavage and rejoining) of nucleic acid sequences to produce a population of sequences that differs from that found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0048] Recovery: The term "recover" or "recovery" means removing the polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials, e.g., recovering the polypeptide from whole fermentation broth or from cell-free fermentation broth by harvesting the polypeptide crystals, by chromatography, by filtration (e.g., depth filtration (by using filter aids or packed filter media, cloth filtration in box filters, rotary drum filtration, drum filtration, rotary vacuum drum filtration, candle filters, horizontal leaf filters or the like, sheet or pad filtration in frame or modular units), or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, ultrafiltration in crossflow, dynamic crossflow or dead-end operation)) or by centrifugation (using a horizontal centrifuge, a disk stack centrifuge, a hydro cyclone or the like) or by precipitating the polypeptide and using related solid-liquid separation methods to harvest the polypeptide from the broth by using size fractionation. Recovery encompasses isolation and / or purification of the polypeptide.

[0049] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0050] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferred 6.6.0 version or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needleman program to report the longest identity, the non-simplified (-nobrief) option must be specified in the command line. The output of the "longest identity" labeled Needleman is calculated as follows:

[0051] (identical residues × 100) / (alignment length - total number of gaps in the alignment)

[0052] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same), as implemented in the Needleman program of the EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, the same) (preferred 6.6.0 version or later). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needleman program to report the longest identity, non-simplified options must be specified in the command line. The output of the "longest identity" labeled Needleman is calculated as follows:

[0053] (number of identical deoxyribonucleotides × 100) / (length of alignment – total number of gaps in the alignment) DETAILED DESCRIPTION

[0054] The present invention relates to microbial proteases and their use in cell separation and cell cluster dissociation processes. The present inventors have recognized that microbial proteases with increased P1 preferences for amino acid residues Leu, Tyr, Phe, and Lys are particularly suitable for use in cell separation. Without being bound by theory, it is speculated that the P1 preference characteristics exhibited by the microbial proteases of the present invention provide effective and gentle cleavage of cell surface proteins involved in surface attachment and intercellular adhesion. In addition, the microbial proteases of the present invention can be recombinantly produced, which ensures a highly unified production process and regulatory compliance when used to develop and produce cells for pharmaceutical applications such as cell therapy.

[0055] Compositions suitable for cell separation

[0056] The present invention relates to compositions suitable for cell separation comprising a microbial protease. In one embodiment, the microbial protease has an increased P1 preference for Leu, Tyr, Phe, and Lys. Preferably, Leu, Tyr, Phe, and Lys are among the five most preferred amino acid residues at the P1 position. Preferably, the P1 preference is determined according to Example 3 herein.

[0057] The microbial protease can be a fungal or bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0058] In an embodiment, the composition comprises a microbial protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1. In a preferred embodiment, the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0059] In an embodiment, the composition comprises a microbial protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2. In a preferred embodiment, the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0060] In one embodiment, the microbial protease has trypsin activity.Trypsin activity (EC 3.4.21.4) can be determined according to the trypsin activity assay described in the Examples herein.

[0061] In one embodiment, the microbial protease has chymotrypsin activity.Chyotrypsin activity (EC 3.4.21.1) can be determined according to the chymotrypsin activity assay described in the Examples herein.

[0062] In one embodiment, the microbial protease has type I collagenase activity. Type I collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0063] In one embodiment, the microbial protease has type IV collagenase activity. Type IV collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0064] In preferred embodiments, the microbial protease has chymotrypsin activity and substantially no trypsin activity, wherein the chymotrypsin activity (EC 3.4.21.1) is determined according to the chymotrypsin activity assay described in the Examples herein, and wherein the trypsin activity (EC 3.4.21.4) is determined according to the trypsin activity assay described in the Examples herein.

[0065] In one embodiment, the composition comprises a microbial protease that is at least 90% pure, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more, based on weight of the total polypeptide material present in the composition.

[0066] In preferred embodiments, the microbial protease is at least 99% pure, e.g., at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more, by weight of the total polypeptide material present in the composition.

[0067] In more preferred embodiments, the microbial protease is at least 99.9% pure, e.g., at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more, by weight of the total polypeptide material present in the composition.

[0068] In a most preferred embodiment, the microbial protease is at least 99.99% pure, e.g., at least 99.995%, at least 99.999% or more, by weight of the total polypeptide material present in the composition.

[0069] In one aspect, the composition suitable for cell separation is a liquid composition. Preferably, the composition is an aqueous composition to ensure compatibility with the culture medium commonly used for cell culture. In certain embodiments, the liquid composition is freeze-dried. On the other hand, the composition is a solid composition, preferably a freeze-dried composition.

[0070] In order to ensure that the liquid composition has a pH value compatible with cell culture conditions, said composition can include an aqueous buffer.Composition can include the aqueous buffer of the amount of 1%-99% by weight, for example, the aqueous buffer of 5%-95%, 10%-90%, 15%-85%, 20%-80% or 25%-75% by weight. Alternatively, composition can include at least 5% by weight, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or higher aqueous buffer.

[0071] In some embodiments, the pH of the liquid composition is from about 5 to about 9, e.g., pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, pH 8, pH 8.5, or pH 9. More preferably, the pH of the composition is from about 7 to about 8, e.g., pH 7, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, or pH 8. Even more preferably, the pH of the composition is from about 7 to about 7.5, e.g., pH 7.1, pH 7.2, pH 7.3, pH 7.4, or pH 7.5. Most preferably, the pH of the composition is about 7.4.

[0072] In some embodiments, the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate, or bicarbonate. Preferably, the aqueous buffer is a HEPES buffer, a TRIS buffer, or a phosphate buffer (e.g., PBS).

[0073] In some embodiments, the liquid composition comprises a microbial protease of the invention in an amount of about 0.1 μg / ml to about 100 μg / ml, e.g., about 0.5 μg / ml to about 50 μg / ml, about 1 μg / ml to about 20 μg / ml, or about 1 μg / ml to about 10 μg / ml.

[0074] In some embodiments, the liquid composition comprises a microbial protease of the invention in an amount of about 0.1 μg / ml to about 20 μg / ml, for example, about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 µg / ml, about 19 µg / ml, or about 20 µg / ml.

[0075] In some embodiments, the liquid composition comprises a microbial protease of the invention in an amount of about 0.5 μg / ml to about 5 μg / ml, e.g., about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, or about 4 μg / ml, or about 5 μg / ml.

[0076] In some embodiments, the liquid composition comprises a microbial protease of the invention in an amount of about 1 μg / ml to about 10 μg / ml, e.g., about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, or about 10 μg / ml.

[0077] In some embodiments, the liquid composition comprises a microbial protease of the invention in an amount of about 1 µg / ml to about 20 µg / ml, for example, about 1 µg / ml, about 2 µg / ml, about 3 µg / ml, about 4 µg / ml, about 5 µg / ml, about 6 µg / ml, about 7 µg / ml, about 8 µg / ml, about 9 µg / ml, about 10 µg / ml, about 11 µg / ml, about 12 µg / ml, about 13 µg / ml, about 14 µg / ml, about 15 µg / ml, about 16 µg / ml, about 17 µg / ml, about 18 µg / ml, about 19 µg / ml, or about 20 µg / ml.

[0078] In a preferred embodiment, the liquid composition comprises the microbial protease of the invention in an amount of 1 µg / ml to 20 µg / ml, for example 1 µg / ml, 2 µg / ml, 3 µg / ml, 4 µg / ml, 5 µg / ml, 6 µg / ml, 7 µg / ml, 8 µg / ml, 9 µg / ml, 10 µg / ml, 11 µg / ml, 12 µg / ml, 13 µg / ml, 14 µg / ml, 15 µg / ml, 16 µg / ml, 17 µg / ml, 18 µg / ml, 19 µg / ml or 20 µg / ml, more preferably 1 µg / ml to 10 µg / ml, most preferably 1 µg / ml to 5 µg / ml.

[0079] In some embodiments, the liquid composition comprises an amount of a polypeptide of the invention of about 0.1 mg / ml to about 100 mg / ml, e.g., about 0.5 mg / ml to about 50 mg / ml, about 1 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 10 mg / ml.

[0080] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount from about 0.1 mg / ml to about 20 mg / ml, for example, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, or about 20 mg / ml.

[0081] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 0.5 mg / ml to about 5 mg / ml, e.g., about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, or about 4 mg / ml, or about 5 mg / ml.

[0082] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 1 mg / ml to about 10 mg / ml, e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml.

[0083] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount from about 1 mg / ml to about 20 mg / ml, e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, or about 20 mg / ml.

[0084] In a preferred embodiment, the liquid composition comprises an amount of the polypeptide of the invention of 1 mg / ml to 20 mg / ml, for example 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 μg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml or 20 mg / ml, more preferably 1 mg / ml to 10 mg / ml, most preferably 1 mg / ml to 5 mg / ml.

[0085] In some embodiments, the liquid composition comprises ethylenediaminetetraacetic acid (EDTA). Preferably, the liquid composition comprises EDTA in an amount of about 0.01 mM to about 100 mM, such as about 0.05 mM to about 50 mM, about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM. Preferably, the liquid composition comprises EDTA in an amount of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 0.95 mM, about 1 mM, about 1.5 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM. More preferably, the liquid composition comprises EDTA in an amount of about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 0.95 mM, about 1 mM, about 1.5 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. Most preferably, the liquid composition comprises EDTA in an amount of about 1 mM.

[0086] In some embodiments, the liquid composition contains substantially no magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ In some embodiments, the liquid composition does not contain magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ In some embodiments, the liquid composition does not contain magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ).

[0087] In some embodiments, the liquid composition comprises phosphate buffered saline (e.g., PBS), EDTA, and is substantially free of magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ).

[0088] In preferred embodiments, the liquid composition comprises a phosphate buffer (e.g., PBS) having a pH of about 7 to about 8, preferably about 7 to about 7.5, and most preferably about pH 7.4; wherein the liquid composition further comprises EDTA in an amount of about 0.1 mM to about 10 mM, preferably about 0.5 mM to about 5 mM, and most preferably about 1 mM; and wherein the liquid composition is substantially free of magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ).

[0089] In preferred embodiments, the liquid composition comprises a phosphate buffer (e.g., PBS) having a pH of about 7 to about 7.5, most preferably about pH 7.4; wherein the liquid composition further comprises EDTA in an amount of about 0.5 mM to about 5 mM, most preferably about 1 mM; and wherein the liquid composition does not comprise magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ).

[0090] In a preferred embodiment, the liquid composition comprises a phosphate buffer (e.g., PBS) having a pH of about 7 to about 7.5, most preferably about pH 7.4; wherein the liquid composition further comprises EDTA in an amount of about 0.5 mM to about 5 mM, most preferably about 1 mM; wherein the liquid composition does not contain magnesium ions (Mg 2+ ) or calcium ions (Ca 2 + ); and wherein the composition comprises SEQ ID NO: 1 or SEQ ID NO: 2 in an amount of 0.1 μg / ml to 20 μg / ml.

[0091] In a preferred embodiment, the liquid composition comprises a phosphate buffered saline (e.g., PBS) having a pH of about pH 7.4; wherein the composition further comprises EDTA in an amount of about 1 mM; and wherein the composition does not comprise magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ).

[0092] In a preferred embodiment, the liquid composition comprises a phosphate buffered saline (e.g., PBS) having a pH of about pH 7.4; wherein the composition further comprises EDTA in an amount of about 1 mM; wherein the composition does not comprise magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ); and wherein the composition comprises SEQ ID NO: 1 or SEQ ID NO: 2 in an amount of 1 μg / ml to 20 μg / ml.

[0093] The liquid composition may further comprise an enzyme stabilizer (examples of the enzyme stabilizer include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives such as aromatic borate esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid).

[0094] In certain embodiments, one or more fillers or one or more carrier materials are included to increase the volume of the liquid composition. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate, as well as talc, clay, etc. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In certain embodiments, the composition contains about 5% to about 90% of such materials.

[0095] In one aspect, the liquid composition comprises 20%-80% w / w of a polyol. In one embodiment, the liquid composition comprises 0.001%-2% w / w of a preservative.

[0096] In another embodiment, the present invention is directed to liquid compositions comprising:

[0097] (a) 0.001%-25% w / w of a microbial protease of the invention (e.g., SEQ ID NO: 1 or SEQ ID NO: 2);

[0098] (b) 20%-80% w / w polyol;

[0099] (c) optionally 0.001%-2% w / w preservative; and

[0100] (d) Water.

[0101] In another embodiment, the present invention is directed to liquid compositions comprising:

[0102] (a) 0.001%-25% w / w of a microbial protease of the invention (e.g., SEQ ID NO: 1 or SEQ ID NO: 2);

[0103] (b) 0.001%-2% w / w preservative;

[0104] (c) optionally 20% to 80% w / w of a polyol; and

[0105] (d) Water.

[0106] In another embodiment, the liquid composition comprises one or more formulating agents, such as a formulating agent selected from the group consisting of: polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600 and polypropylene glycol (PPG) having an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0107] In one embodiment, the liquid composition comprises glucose in an amount of about 0.1 g / L to about 10 g / L, such as about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, or about 10 g / L. In a preferred embodiment, the liquid composition comprises glucose in an amount of about 0.5 g / L to about 5 g / L, most preferably about 1 g / L.

[0108] In another embodiment, the liquid composition comprises 20%-80% polyol (i.e., the total amount of polyol), such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol. In one embodiment, the liquid formulation comprises 20%-80% polyol, such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0109] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid composition comprises 0.02%-1.5% w / w preservative, such as 0.05%-1% w / w preservative or 0.1%-0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001%-2% w / w preservative (i.e., the total amount of preservative), such as 0.02%-1.5% w / w preservative, 0.05%-1% w / w preservative, or 0.1%-0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0110] In one aspect, the composition further comprises one or more additional enzymes, e.g., hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, DNA enzyme, feruloyl esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0111] In a preferred embodiment, the composition further comprises DNase.

[0112] Microbial proteases

[0113] The present invention also relates to microbial proteases that have an increased P1 preference for Leu, Tyr, Phe, and Lys. In a preferred embodiment, Leu, Tyr, Phe, and Lys are among the five most preferred amino acid residues at the P1 position. The P1 preference can be determined according to Example 3 herein.

[0114] The microbial protease can be a fungal or bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0115] In preferred embodiments, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0116] In preferred embodiments, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0117] In one embodiment, the microbial protease has trypsin activity.Trypsin activity (EC 3.4.21.4) can be determined according to the trypsin activity assay described in the Examples herein.

[0118] In one embodiment, the microbial protease has chymotrypsin activity.Chyotrypsin activity (EC 3.4.21.1) can be determined according to the chymotrypsin activity assay described in the Examples herein.

[0119] In one embodiment, the microbial protease has type I collagenase activity. Type I collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0120] In one embodiment, the microbial protease has type IV collagenase activity. Type IV collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0121] In another aspect, the microbial protease is derived from SEQ ID NO: 1 or SEQ ID NO: 2 by substitution, deletion or addition of one or more amino acids. In some embodiments, the microbial protease is a variant of SEQ ID NO: 1 or SEQ ID NO: 2 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 is up to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine stretch, antigenic epitope or binding module).

[0122] Essential amino acids in polypeptides (e.g., microbial proteases) can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting molecules are tested for protease activity and / or P1 specificity to identify amino acid residues that are critical to the activity and / or specificity of the molecule (see also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708). The active site of a microbial protease can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutating putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. Essential amino acid identity can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins within a family of polypeptides or proteins derived from a common ancestor (typically having similar three-dimensional structure, function, and significant sequence similarity). Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold,” Nature 596: 583-589.

[0123] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0124] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0125] In one aspect, the microbial protease is isolated.

[0126] In another aspect, the microbial protease is purified.

[0127] Sources of microbial proteases

[0128] The microbial proteases of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in conjunction with a given source should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain into which the polynucleotide of the present invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0129] In another aspect, the microbial protease is obtained from a species of the genus Scopulariopsis, such as Scopulariopsis compactus.

[0130] In one aspect, the microbial protease is obtained from a species of the genus Nocardiopsis, such as Nocardiopsis violaceus.

[0131] It will be understood that for the aforementioned species, the present invention encompasses the perfect and imperfect stages and other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0132] Can use above-mentioned probe from other sources, comprise from nature (for example, soil, compost, water etc.) isolated microorganism or directly from the DNA sample identification and acquisition microbial protease of natural material (for example, soil, compost, water etc.).The technology that is used for directly isolating microorganism and DNA from natural habitat is well known in the art.Then can obtain the polynucleotide of coding microbial protease by similarly screening the genomic DNA or cDNA library of another microorganism or the DNA sample of mixing.In case the polynucleotide of coding microbial protease has been detected with probe, just can separate or clone this polynucleotide (referring to for example, people such as Davis, 2012, Basic Methods in Molecular Biology [the basic method of molecular biology], Elsevier [Elsevier Publishing Company]).

[0133] polynucleotides

[0134] The present invention also relates to polynucleotides encoding the microbial proteases of the present invention.

[0135] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof. The polynucleotide can be cloned from a strain of the genus Scopulariopsis (e.g., Scopulariopsis compactus) or a related organism, or cloned from a strain of the genus Nocardia (e.g., Nocardia viridis) or a related organism.

[0136] In one embodiment, a polynucleotide encoding a microbial protease of the invention is isolated from a Sclerotium (eg, Sclerotium compactum) cell.

[0137] In one embodiment, a polynucleotide encoding a microbial protease of the invention is isolated from a Nocardiopsis sp. (eg, Nocardiopsis violaceus) cell.

[0138] Polynucleotides can also be mutated by introducing nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but correspond to the codon usage of the host organism intended for production of the enzyme, or by introducing nucleotide substitutions that may result in a different amino acid sequence. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

[0139] In one aspect, the polynucleotide is isolated.

[0140] In another aspect, the polynucleotide is purified.

[0141] Nucleic acid constructs

[0142] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0143] Polynucleotides can be manipulated in a variety of ways to provide expression of a microbial protease. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for utilizing recombinant DNA methods to modify polynucleotides are well known in the art.

[0144] promoter

[0145] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of a polynucleotide encoding the microbial protease of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the microbial protease. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0146] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York; Davis et al., 2012, supra; and Song et al., 2016, PLOS One 11(7): e0158447.

[0147] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0148] For expression in yeast hosts, examples of useful promoters are described by Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae) and Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0149] terminator

[0150] The control sequence can also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3'-end of the polynucleotide encoding the microbial protease. Any terminator that is functional in the host cell can be used in the present invention.

[0151] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0152] Preferred terminators for filamentous fungal host cells are obtainable from Aspergillus or Trichoderma species, such as from the genes for Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0153] Preferred terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

[0154] mRNA stabilizer

[0155] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0156] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177: 3465-3471).

[0157] Examples of mRNA stabilizer regions of fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11): 1838-1846.

[0158] leader sequence

[0159] The control sequence can also be a leader sequence, i.e., an untranslated region of an mRNA that is important for host cell translation. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the microbial protease. Any leader sequence that is functional in the host cell can be used.

[0160] Suitable leader sequences for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059 and by Kaberdin and Bläsi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.

[0161] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0162] Suitable leaders for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0163] polyadenylation sequence

[0164] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide that, when transcribed, is recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0165] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0166] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0167] signal peptide

[0168] Control sequence can also be the signal peptide that coding is connected with the N-terminal of polypeptide and instructs the signal peptide coding region of the secretory pathway of polypeptide to enter cell.The 5 '-end of the coding sequence of polynucleotide can contain signal peptide coding sequence inherently, and this signal peptide coding sequence links together naturally in translation reading frame with the section of the coding sequence of coding microbial protease.Alternately, the 5 '-end of coding sequence can contain the signal peptide coding sequence that is heterologous for coding sequence.Under the situation that coding sequence does not contain signal peptide coding sequence naturally, may need heterologous signal peptide coding sequence.Alternately, heterologous signal peptide coding sequence can simply substitute natural signal peptide coding sequence so that strengthen the secretion of microbial protease.Can use the microbial protease of instructing expression to enter any signal peptide coding sequence of the secretory pathway of host cell.

[0169] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes for Bacillus sp. NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories 17: 52.

[0170] An effective signal peptide coding sequence for a filamentous fungal host cell is the signal peptide coding sequence obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955.

[0171] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0172] propeptide

[0173] The control sequence can also be a propeptide coding sequence encoding a propeptide located at the N-terminus of the microbial protease of the present invention. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases, a zymogen). A propolypeptide is generally inactive and can be converted into an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease, and Saccharomyces cerevisiae α-factor.

[0174] In the case where both the signal peptide sequence and the propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence. Additionally or alternatively, when both the signal peptide sequence and the propeptide sequence are present, the polypeptide may comprise only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of a mature polypeptide and a polypeptide comprising a partial or full-length propeptide sequence and / or a signal peptide sequence.

[0175] Regulatory sequence

[0176] It may also be desirable to add regulatory sequences that regulate the expression of microbial proteases associated with host cell growth. Examples of regulatory sequences are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA α-amylase promoter, and the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals.

[0177] transcription factors

[0178] The control sequence can also be a transcription factor, i.e., a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can function alone and / or in combination with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain that is typically attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of the target protein directly (i.e., by binding to its promoter to activate transcription of the gene encoding the target protein) or indirectly (i.e., by binding to the promoter of another transcription factor (which regulates the transcription of the gene encoding the target protein) to activate the transcription of the other transcription factor). Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52: 7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.

[0179] expression vector

[0180] The invention still further relates to the recombinant expression vector that comprises polynucleotide of the present invention, promotor and transcription and translation termination signal.Various Nucleotide and control sequence can link together to produce the recombinant expression vector, and this recombinant expression vector can comprise one or more suitable restriction sites to allow to insert or replace the polynucleotide of coding microbial protease at such site.Alternately, can be by polynucleotide or the nucleic acid construct that comprises these polynucleotide be inserted in the suitable vector that is used to express and express these polynucleotide.When producing expression vector, encoding sequence so is positioned in the carrier, makes encoding sequence be operably connected with the suitable control sequence that is used to express.

[0181] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0182] The carrier can be an autonomous replicating vector, i.e. a carrier existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can contain any means for ensuring self-replication. Alternatively, the carrier can be a carrier that is integrated into the genome and replicates with the chromosome into which it has been integrated when it is introduced into the host cell. Moreover, a single carrier or plasmid or two or more carriers or plasmids can be used that contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0183] The vector preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0184] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0185] For integration into the host cell genome, the vector may rely on a polynucleotide sequence encoding a microbial protease or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0186] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator that functions in the cell and mediates autonomous replication. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0187] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to improve the production of the polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into the host cell. The increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene and thus an additional copy of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0188] Recombinant host cells

[0189] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a microbial protease of the present invention.

[0190] In one embodiment, the recombinant host cell comprises a polynucleotide encoding a microbial protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the recombinant host cell comprises a polynucleotide encoding a microbial protease comprising, consisting essentially of, or consisting of SEQ ID NO: 1.

[0191] In one embodiment, the recombinant host cell comprises a polynucleotide encoding a microbial protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the recombinant host cell comprises a polynucleotide encoding a microbial protease comprising, consisting essentially of, or consisting of SEQ ID NO: 2.

[0192] Construct or the vector that will comprise polynucleotide are introduced in the host cell so that this construct or vector are maintained as chromosomal integrant or as a self-replicating extrachromosomal vector, as described earlier.The selection of host cell will depend on gene and source thereof of encoding microbial protease to a great extent.Microbial protease can be natural or heterologous for recombinant host cell.In addition, at least one in one or more control sequences can be heterologous for the polynucleotide of encoding microbial protease.Recombinant host cell can comprise single copy or at least two copies of polynucleotide of the present invention, for example three, four, five or more copies.

[0193] The host cell can be any microbial cell, such as a prokaryotic cell or a fungal cell, that can be used for the recombinant production of the microbial protease of the present invention.

[0194] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0195] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In embodiments, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus subtilis cell.

[0196] For the purposes of the present invention, the Bacillus genus / genus / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.

[0197] The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus cells.

[0198] The bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0199] Methods for introducing DNA into prokaryotic host cells are well known in the art, and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, wherein the DNA is introduced as a linearized or circular polynucleotide. Those skilled in the art will be able to easily determine the appropriate method for introducing DNA into a given prokaryotic cell according to, for example, the genus. Methods for introducing DNA into prokaryotic host cells are, for example, described in Heinze et al., 2018, BMC Microbiology [BMC Microbiology] 18:56; Burke et al., 2001, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 98: 6289-6294; Choi et al., 2006, J. Microbiol. Methods [Microbial Methods Magazine] 64: 391-397 and Donald et al., 2013, J. Bacteriol. [Bacteriology Magazine] 195(11): 2612-2620.

[0200] The host cell can be a fungal cell. "Fungi," as used herein, include Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitospore fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0201] Fungal cells can be transformed by processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (reviewed in Li et al., 2017, Microbial Cell Factories 16: 168), as well as procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474; Christensen et al., 1988, Bio / Technology 6: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into fungal host cells may be used, and the DNA may be introduced as a linearized or circular polynucleotide.

[0202] The fungal host cell can be a yeast cell. As used herein, "yeast" includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to Fungi Imperfecti (Blastomycetes). For the purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0203] The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or a Yarrowia lipolytica cell. In preferred embodiments, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0204] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeasts (e.g., Saccharomyces cerevisiae) occurs by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0205] The filamentous fungal host cell can be Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neomyces In some embodiments, the filamentous fungal host cell is a cell of the genus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a preferred embodiment, the filamentous fungal host cell is a cell of the genus Aspergillus, Trichoderma, or Fusarium. In another preferred embodiment, the filamentous fungal host cell is a cell of Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum.

[0206] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops), Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum), Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium rosaceae, Fusarium rubrum, Fusarium rubrum var.roseum), Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venezuelae, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris terrestris), Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0207] In one aspect, the host cell is isolated.

[0208] In another aspect, the host cell is purified.

[0209] Generation method

[0210] The present invention also relates to methods of producing the microbial proteases of the present invention, comprising (a) cultivating a host cell under conditions conducive for production of the microbial protease of the present invention, the host cell producing the microbial protease in its wild-type form; and optionally, (b) recovering the microbial protease.

[0211] In one embodiment, the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys. In a preferred embodiment, Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues. The P1 preference can be determined according to Example 3 herein.

[0212] In one embodiment, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0213] In one embodiment, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0214] The present invention also relates to methods of producing the microbial proteases of the invention, comprising (a) cultivating a recombinant host cell of the invention under conditions conducive for production of the microbial protease of the invention; and optionally, (b) recovering the microbial protease.

[0215] In one embodiment, the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys. In a preferred embodiment, Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues. The P1 preference can be determined according to Example 3 herein.

[0216] In one embodiment, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0217] In one embodiment, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0218] The recombinant host cell can be a bacterial host cell or a fungal host cell. In a preferred embodiment, the recombinant host cell is a Bacillus cell, most preferably a Bacillus subtilis cell or a Bacillus licheniformis cell. In a preferred embodiment, the recombinant host cell is an Aspergillus cell, most preferably an Aspergillus niger cell or an Aspergillus oryzae cell. In a preferred embodiment, the recombinant host cell is a Pichia cell, most preferably a Pichia pastoris cell.

[0219] Use methods known in the art to cultivate host cells in the nutrient medium that is applicable to produce the microbial protease.For example, can be by shaking flask culture, perhaps in suitable substratum and under the condition that allows to express and / or separate the microbial protease, carry out small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, fed-batch fermentation or solid-state and / or based on the fermentation of microcarrier) and culture cell.Suitable substratum can obtain from commercial supplier or can be prepared according to disclosed composition (for example, in the catalogue of American Type Culture Collection).If the microbial protease is secreted in the nutrient medium, then can directly reclaim the microbial protease from the substratum.If the microbial protease is not secreted, then can reclaim from the cell pyrolysis liquid.

[0220] Microbial proteases can be detected using methods known in the art that are specific for microbial proteases, including but not limited to the use of specific antibodies, enzyme product formation, disappearance of enzyme substrate, or assays that determine the relative or specific activity of the microbial protease.

[0221] Can use method known in the art (including but not limited to collection, centrifugation, filtration, extraction, spray drying, freeze drying, evaporation or precipitation) to reclaim microbial protease from substratum.In one aspect, reclaim the full fermentation broth that comprises microbial protease.In another aspect, reclaim the cell-free fermentation broth that comprises microbial protease.

[0222] Microbial proteases can be purified by various procedures known in the art to obtain substantially pure microbial proteases and / or microbial protease fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0223] In alternative aspects, the microbial protease is not recovered.

[0224] Protease Granules

[0225] The present invention also relates to enzyme granules / particles comprising the microbial protease of the invention.In an embodiment, the granule comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0226] The diameter of the core (measured as the equivalent spherical diameter (average particle size based on volume)) may be 20-2000 μm, in particular 50-1500 μm, 100-1500 μm or 250-1200 μm. The core diameter measured as the equivalent spherical diameter can be determined using laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0227] In embodiments, the core comprises a microbial protease of the invention.

[0228] The core may include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0229] The core may include a binder such as a synthetic polymer, wax, fat or carbohydrate.

[0230] The core may include, typically as a homogeneous blend, a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposition catalyst, and / or an acidic buffer component.

[0231] The core may comprise inert particles into which the polypeptide is adsorbed or applied (eg by fluid bed coating) to the surface of the inert particles.

[0232] The diameter of the core may be 20-2000 µm, in particular 50-1500 µm, 100-1500 µm or 250-1200 µm.

[0233] The core may be surrounded by at least one coating, for example to improve storage stability, reduce dust formation during handling or to color the granules. Optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).

[0234] The coating may be applied in an amount of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) by weight of the core. The amount may be up to 100%, 70%, 50%, 40%, or 30%.

[0235] The coating is preferably at least 0.1 μm thick, in particular at least 0.5 μm, at least 1 μm or at least 5 μm thick. In some embodiments, the thickness of the coating is less than 100 μm, such as less than 60 μm or less than 40 μm.

[0236] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer is understood to be a coating with few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0237] The coating may further contain other materials as known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0238] The salt coating may comprise at least 60% by weight salt, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

[0239] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, for example at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm or at least 8 μm. In particular embodiments, the thickness of the salt coating is less than 100 μm, such as less than 60 μm or less than 40 μm.

[0240] Salt may be added from a salt solution (wherein the salt is completely dissolved) or from a salt suspension (wherein the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm).

[0241] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water-soluble, in particular having a solubility in 100 g of water at 20°C of at least 0.1 g, preferably at least 0.5 g / 100 g of water, such as at least 1 g / 100 g of water, for example at least 5 g / 100 g of water.

[0242] The salt can be an inorganic salt, for example a salt of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or a simple organic acid (less than 10 carbon atoms, for example 6 or less carbon atoms) such as citrate, malonate or acetate. The example of the cation in these salts is a metal ion of an alkali or alkaline earth metal ion, ammonium ion or the first transition series, for example sodium, potassium, magnesium, calcium, zinc or aluminum. The example of an anion comprises chlorine, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrates, malonates or acetates can be used.

[0243] The salt in the coating may have a constant humidity of 60% or more, in particular 70% or more, 80% or more or 85% or more at 20° C., or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0244] Specific examples of suitable salts are NaCl (CH20°C = 76%), Na2CO3 (CH20°C = 92%), NaNO3 (CH20°C = 73%), Na2HPO4 (CH20°C = 95%), Na3PO4 (CH25°C = 92%), NH4Cl (CH20°C = 79.5%), (NH4)2HPO4 (CH20°C = 93.0%), NH4H2PO4 (CH20°C = 93.1%), (NH4)2SO4 (CH20°C = 81.1%), KCl (CH20°C = 85%), K2HPO4 (CH20°C = 92%), KH2PO4 (CH20°C = 96.5%), KNO3 (CH20°C = 93.5%), Na2SO4 (CH20°C = 81.1%), 93%), K2SO4 (CH20°C = 98%), KHSO4 (CH20°C = 86%), MgSO4 (CH20°C = 90%), ZnSO4 (CH20°C = 90%), and sodium citrate (CH25°C = 86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0245] The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate having one or more waters of crystallization, as described, for example, in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO4.7H2O), zinc sulfate heptahydrate (ZnSO4.7H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4.7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0246] Preferably, the salt is used as a salt solution, for example using a fluidized bed.

[0247] The coating material may be a waxy coating material and a film-forming coating material. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and wherein there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1483591.

[0248] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxy-propylcellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with various coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0249] The core may be prepared by granulating a blend of ingredients, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, particle size reduction, drum granulation and / or high shear granulation.

[0250] Methods for preparing the cores can be found in Handbook of Powder Technology; CE Capes, Particle size enlargement; Vol. 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as:

[0251] (a) Spray-dried products, where a solution containing a microbial protease is atomized in a spray drying tower to form small droplets which are dried during their descent in the drying tower to form particulate material containing the microbial protease. In this way, very small particles can be produced (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0252] (b) Layered products, where the microbial protease is coated in layers around preformed inert core particles. Typically, a solution containing the microbial protease is atomized in a fluidized bed apparatus where the preformed core particles are fluidized and the solution containing the microbial protease adheres to the core particles and is dried until a dry layer of the microbial protease remains on the surface of the core particles. If useful core particles of the desired size can be found, particles of the desired size can be obtained in this way. Products of this type are described, for example, in WO 97 / 23606.

[0253] (c) Absorbed core particles, where instead of coating the microbial protease in layers around the core, the microbial protease is absorbed on the surface and / or in the surface of the core. Such a method is described in WO 97 / 39116.

[0254] (d) Extruded or granulated products, where a paste containing the microbial protease is pressed into pellets or extruded under pressure through small openings and cut into particles, which are then dried. Such particles usually have a rather large size because the material with the extrusion openings (usually a plate with drilled holes) limits the pressure drop allowed through the extrusion openings. In addition, when using small openings, very high extrusion pressures increase the heat generation in the microbial protease paste, which is harmful to the microbial protease (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0255] (e) Spray granulation products, in which a powder containing the microbial protease is suspended in molten wax and the suspension is sprayed (e.g., by a rotating disk atomizer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the microbial protease is evenly distributed throughout the inert material rather than concentrated on its surface. U.S. Pat. No. 4,016,040 and U.S. Pat. No. 4,713,245 describe this technique.

[0256] (f) mixer granulation product, wherein the liquid containing microbial protease is added to the dry powder composition of conventional granulation components.Liquid and powder are mixed in a suitable ratio, and along with the moisture of the liquid being absorbed in the dry powder, the components of the dry powder will begin to adhere and condense, and particles will accumulate to form particles comprising microbial protease. Such method is described in US 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440 and WO90 / 09428. In the particular aspects of this process, various high shear mixers can be used as granulators. The particles composed of microbial protease, filler and adhesive etc. are mixed with cellulose fiber to strengthen particles, thereby producing so-called T-granules. The strengthened particles are more solid and release less enzyme dust.

[0257] (g) Particle size reduction, wherein the core is produced by grinding or crushing larger particles, pellets, tablets, briquettes, etc. containing the microbial protease. The desired core particle fraction is obtained by sieving the milled or crushed product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0258] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending fine particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. The particles struck by the sprayed liquid droplets become wet and sticky. The sticky particles collide with other particles and attach to them to form granules.

[0259] (i) These cores can be dried, for example in a fluidized bed dryer. Those skilled in the art can use other known methods for drying granules in the feed or enzyme industry. Drying is preferably carried out at a product temperature of 25°C to 90°C. For some microbial proteases, it is important that the core containing the microbial protease contains a small amount of water before being coated with salt. If a water-sensitive microbial protease is coated with salt before removing the excess water, the excess water will be trapped in the core and may have a negative impact on the activity of the microbial protease. After drying, the core preferably contains 0.1%-10% w / w water.

[0260] Non-dusting granules may be produced, for example, as disclosed in US 4,106,991 and US 4,661,452, and may optionally be coated by methods known in the art.

[0261] The granule can further comprise one or more other enzymes, for example hydrolases, isomerases, ligases, lyases, oxidoreductases and transferases. The one or more other enzymes are preferably selected from the group consisting of: acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, β-xylosidase or its any combination. Then, every kind of enzyme will be present in more granules, ensuring a more even distribution of the enzyme, and also reducing the physical separation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granules are disclosed in ip.com disclosure IPCOM000200739D.

[0262] Another example of formulating polypeptides by using co-particles is disclosed in WO 2013 / 188331.

[0263] The present invention also relates to protected polypeptides prepared according to the method disclosed in EP 238216.

[0264] Fermentation broth preparation or cell composition

[0265] The invention still further relates to fermentation liquid preparation or the cell composition that comprise microbial protease of the present invention.Fermentation liquid preparation or cell composition are further included in the other composition that uses in the fermentation process, for example as cell (comprising the host cell that contains the gene of encoding microbial protease of the present invention, these host cells are used to produce purpose microbial protease), cell debris, biomass, fermentation medium and / or zymoplast.In certain embodiments, composition is the cell-killing full nutrient solution that contains organic acid, the cell of killing and / or cell debris and substratum.

[0266] In one embodiment, the fermentation broth formulation or cell composition comprises a microbial protease with an increased P1 preference for Leu, Tyr, Phe, and Lys. Preferably, Leu, Tyr, Phe, and Lys are among the five most preferred amino acid residues at the P1 position. Preferably, the P1 preference is determined according to Example 3 herein.

[0267] The microbial protease can be a fungal or bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0268] In an embodiment, the fermentation broth formulation or cell composition comprises a microbial protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1. In a preferred embodiment, the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0269] In an embodiment, the fermentation broth formulation or cell composition comprises a microbial protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2. In a preferred embodiment, the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0270] As used herein, term " fermented liquid " refers to the preparation produced by cell fermentation, not experiencing or experiencing minimum recovery and / or purification.For example, when microbial culture is hatched and grown to saturation under the carbon restriction condition that allows protein synthesis (for example, by host cell expression enzyme) and protein secretion in the cell culture medium, fermented liquid is produced.Fermented liquid can contain the unfractionated or fractionated content of the fermentation material derived when fermentation ends.Typically, fermented liquid is unfractionated and comprises the cell debris that exists after spent substratum and for example by centrifugal removal microbial cells (for example, filamentous fungal cells).In certain embodiments, fermented liquid contains spent cell culture medium, extracellular enzyme and vigorous and / or non-viable microbial cells.

[0271] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component (comprising at least one organic acid of 1-5 carbon atoms and / or a salt thereof) and a second organic acid component (comprising at least one organic acid of 6 or more carbon atoms and / or a salt thereof). In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more thereof; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more thereof.

[0272] In one aspect, the composition comprises one or more organic acids and optionally further comprises killed cells and / or cell debris. In some embodiments, these killed cells and / or cell debris are removed from the whole culture medium of cell killing to provide a composition without these components.

[0273] The fermentation broth formulation or cell composition may further comprise preservatives and / or antimicrobial (eg, bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0274] The whole culture fluid or cell composition of cell killing can contain the unfractionated contents of the fermentation material derived at the end of fermentation.Typically, the whole culture fluid or cell composition of cell killing contains spent culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) are grown to saturation and incubated under carbon restriction conditions to allow protein synthesis.In certain embodiments, the whole culture fluid or cell composition of cell killing contains spent cell culture medium, extracellular enzymes and the filamentous fungal cells killed.In certain embodiments, methods known in the art can be used to permeabilize and / or lyse the microbial cells present in the whole culture fluid or composition of cell killing.

[0275] As described herein, whole culture fluid or cell composition is typically liquid, but can contain insoluble components, such as killed cells, cell debris, culture medium components and / or one or more insoluble enzymes.In some embodiments, insoluble components can be removed to provide a clarified liquid composition.

[0276] The whole culture broth formulation and cell composition of the present invention can be produced by the method described in WO 90 / 15861 or WO 2010 / 096673.

[0277] Methods and uses

[0278] The invention further relates to methods for cell separation, which methods comprise contacting a cell with a microbial protease of the present invention or a composition of the present invention, wherein the cell is attached to a surface and / or another cell. In one embodiment, the cell to be separated is attached to a surface. In one embodiment, the cell to be separated is a part of a cell monolayer. In one embodiment, the cell to be separated is a part of a cell cluster.

[0279] In one embodiment, the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys. Preferably, Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues at the P1 position. Preferably, the P1 preference is determined according to Example 3 herein.

[0280] The microbial protease can be a fungal or bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0281] In embodiments, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0282] In embodiments, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0283] The methods of the present invention can be used to isolate any type of cell, including but not limited to A375 metastatic melanoma cells, β cells, BHK cells, bone marrow stem cells, cardiomyocytes, CHO cells, COS cells, D54 glioma cells, dopaminergic progenitor cells, fibroblasts, HEK293 cells, HeLa cells, hepatocytes, hepatocyte progenitor cells, human stem cells, HT1080 fibrosarcoma cells, immortalized mouse testicular germ cells, keratinocytes, L929 cells, M24 metastatic melanoma cells, macrophages, Madin-Darby canine kidney cells, mesenchymal stem cells, MG63 cells, NIH / 3T3 cells, NT2 cells, primary chicken embryonic neuronal cells, Sf9 insect cells, U251 glioma cells, vascular endothelial cells, vascular smooth muscle cells and Vero cells.

[0284] In preferred embodiments, the cells to be isolated are mammalian cells, preferably canine or human cells, most preferably human cells.

[0285] The methods of the present invention can be used to isolate any type of stem cell or stem cell derivative. Thus, the stem cell can be a totipotent stem cell (e.g., a fertilized egg cell), a pluripotent stem cell (e.g., an embryonic stem cell), a multipotent stem cell (e.g., a mesenchymal stem cell), an oligopotent stem cell (e.g., a hematopoietic stem cell), or a unipotent stem cell (e.g., a muscle stem cell). In one embodiment, the stem cell is a human stem cell. In one embodiment, the stem cell is a human pluripotent stem cell, a human multipotent stem cell, a human oligopotent stem cell, or a human unipotent stem cell. In a preferred embodiment, the stem cell is a human pluripotent stem cell. In another preferred embodiment, the stem cell is a human induced pluripotent stem cell.

[0286] In some embodiments, the cells to be isolated are pluripotent stem cells, mesenchymal stem cells, beta cells, neurons, adipocytes, epithelial cells, or kidney cells.

[0287] In some embodiments, the cell to be separated is attached to a surface, such as a plastic surface or a glass surface. In some embodiments, the cell to be separated is attached to another cell. In some embodiments, the cell to be attached is part of a cell cluster.

[0288] In some embodiments, the cells to be isolated are attached to a surface coated with a biomaterial, an extracellular matrix (ECM), and / or other scaffolds made of natural polymers (e.g., collagen, hyaluronic acid, fibrin, alginate, gelatin, etc.) or synthetic polymers (e.g., poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL), etc.).

[0289] In some embodiments, the cell to be isolated is a stem cell or a stem cell derivative. Preferably, the cell is a pluripotent stem cell, an induced pluripotent stem cell, a (stem cell-derived) dopaminergic progenitor cell, or a (stem cell-derived) beta cell.

[0290] In one embodiment, the cells to be isolated are pluripotent stem cells, preferably human pluripotent stem cells.Preferably, the pluripotent stem cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0291] In one embodiment, the cells to be isolated are induced pluripotent stem cells, preferably human induced pluripotent stem cells.Preferably, the induced pluripotent stem cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0292] In one embodiment, the cells to be isolated are beta cells, preferably human beta cells.Preferably, the beta cells are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0293] In one embodiment, the cells to be isolated are stem cell-derived beta cells, preferably human stem cell-derived beta cells.Preferably, the stem cell-derived beta cells are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0294] In one embodiment, the cells to be isolated are dopaminergic progenitor cells, preferably human dopaminergic progenitor cells.Preferably, the dopaminergic progenitor cells are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0295] In one embodiment, the cells to be isolated are stem cell-derived dopaminergic progenitor cells, preferably human stem cell-derived dopaminergic progenitor cells. Preferably, the stem cell-derived dopaminergic progenitor cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0296] In one embodiment, the cells to be isolated are bone marrow-derived mesenchymal stem cells (BM-MSCs).Preferably, the bone marrow-derived mesenchymal stem cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0297] In one embodiment, the cells to be isolated are canine cells, preferably Madin-Darby canine kidney (MDCK) cells.Preferably, the canine cells, more preferably Madin-Darby canine kidney cells, are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0298] In one embodiment, the cells to be isolated are human embryonic kidney 293 (HEK293) cells.Preferably, the human embryonic kidney 293 cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0299] In one embodiment, the cells to be isolated are adipose stromal cells.Preferably, the adipose stromal cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0300] The present invention further relates to the purposes of the microbial protease of the present invention in cell separation process.In one embodiment, the microbial protease has an increased P1 preference to Leu, Tyr, Phe and Lys.Preferably, Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues in the P1 position.Preferably, the P1 preference is determined according to Example 3 herein.

[0301] The microbial protease can be a fungal or bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0302] In embodiments, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0303] In embodiments, the microbial protease has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In preferred embodiments, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0304] The microbial proteases of the present invention can be used in any type of cell separation process, including but not limited to the separation of the following: A375 metastatic melanoma cells, β cells, BHK cells, bone marrow stem cells, cardiomyocytes, CHO cells, COS cells, D54 glioma cells, dopaminergic progenitor cells, fibroblasts, HEK293 cells, HeLa cells, hepatocytes, hepatocyte progenitor cells, human stem cells, HT1080 fibrosarcoma cells, immortalized mouse testicular germ cells, keratinocytes, L929 cells, M24 metastatic melanoma cells, macrophages, Madin-Darby canine kidney cells, mesenchymal stem cells, MG63 cells, NIH / 3T3 cells, NT2 cells, primary chicken embryonic neuronal cells, Sf9 insect cells, U251 glioma cells, vascular endothelial cells, vascular smooth muscle cells and Vero cells.

[0305] In preferred embodiments, the cells to be isolated are mammalian cells, preferably canine or human cells, most preferably human cells.

[0306] The microbial proteases of the present invention can be used in any type of stem cell isolation process. Thus, the stem cells to be isolated can be totipotent stem cells (e.g., fertilized egg cells), multipotent stem cells (e.g., embryonic stem cells), pluripotent stem cells (e.g., mesenchymal stem cells), oligopotent stem cells (e.g., hematopoietic stem cells), or unipotent stem cells (e.g., muscle stem cells). In one embodiment, the stem cells are human stem cells. In one embodiment, the stem cells are human pluripotent stem cells, human multipotent stem cells, human oligopotent stem cells, or human unipotent stem cells. In a preferred embodiment, the stem cells are human pluripotent stem cells. In another preferred embodiment, the stem cells are human induced pluripotent stem cells.

[0307] In one embodiment, the cells to be isolated are stem cell derivatives, preferably pluripotent stem cell derivatives, most preferably human pluripotent stem cell derivatives.

[0308] In some embodiments, the cells to be isolated are pluripotent stem cells, mesenchymal stem cells, beta cells, neurons, adipocytes, epithelial cells, or kidney cells.

[0309] In some embodiments, the cell to be separated is attached to a surface, such as a plastic surface or a glass surface. In some embodiments, the cell to be separated is attached to another cell. In some embodiments, the cell to be attached is part of a cell cluster.

[0310] In some embodiments, the cells to be isolated are attached to a surface coated with a biomaterial, an extracellular matrix (ECM), and / or other scaffolds made of natural polymers (e.g., collagen, hyaluronic acid, fibrin, alginate, gelatin, etc.) or synthetic polymers (e.g., poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL), etc.).

[0311] In some embodiments, the cell to be isolated is a stem cell or a stem cell derivative. Preferably, the cell is a pluripotent stem cell, an induced pluripotent stem cell, a (stem cell-derived) dopaminergic progenitor cell, or a (stem cell-derived) beta cell.

[0312] In one embodiment, the cells to be isolated are pluripotent stem cells, preferably human pluripotent stem cells.Preferably, the pluripotent stem cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0313] In one embodiment, the cells to be isolated are induced pluripotent stem cells, preferably human induced pluripotent stem cells.Preferably, the induced pluripotent stem cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0314] In one embodiment, the cells to be isolated are beta cells, preferably human beta cells.Preferably, the beta cells are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0315] In one embodiment, the cells to be isolated are stem cell-derived beta cells, preferably human stem cell-derived beta cells.Preferably, the stem cell-derived beta cells are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0316] In one embodiment, the cells to be isolated are dopaminergic progenitor cells, preferably human dopaminergic progenitor cells.Preferably, the dopaminergic progenitor cells are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0317] In one embodiment, the cells to be isolated are stem cell-derived dopaminergic progenitor cells, preferably human stem cell-derived dopaminergic progenitor cells. Preferably, the stem cell-derived dopaminergic progenitor cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0318] In one embodiment, the cells to be isolated are bone marrow-derived mesenchymal stem cells (BM-MSCs).Preferably, the bone marrow-derived mesenchymal stem cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0319] In one embodiment, the cells to be isolated are canine cells, preferably Madin-Darby canine kidney cells. Preferably, the canine cells, more preferably Madin-Darby canine kidney cells, are isolated from a surface, preferably a plastic surface or a glass surface, or from a cell cluster.

[0320] In one embodiment, the cells to be isolated are human embryonic kidney 293 (HEK293) cells.Preferably, the human embryonic kidney 293 cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0321] In one embodiment, the cells to be isolated are adipose stromal cells.Preferably, the adipose stromal cells are isolated from a surface (preferably a plastic surface or a glass surface) or from a cell cluster.

[0322] Preferred embodiment

[0323] 1) The use of microbial proteases in cell separation processes.

[0324] 2) The use according to embodiment 1, wherein the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys; preferably wherein Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues at the P1 position.

[0325] 3) The use according to embodiment 2, wherein the P1 preference is determined according to Example 3 herein.

[0326] 4) The use according to any one of embodiments 1-3, wherein the microbial protease exhibits chymotrypsin activity as determined according to the chymotrypsin activity assay described herein.

[0327] 5) The use according to any one of the preceding embodiments, wherein the microbial protease is a fungal protease or a bacterial protease.

[0328] 6) The use according to any one of the preceding embodiments, wherein the microbial protease is a Sauropoda protease; preferably wherein the microbial protease is a Sauropoda compacta protease.

[0329] 7) The use according to any one of the preceding embodiments, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1.

[0330] 8) The use according to any one of the preceding embodiments, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0331] 9) The use according to any one of the preceding embodiments, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0332] 10) The use according to any one of embodiments 1-5, wherein the microbial protease is a Nocardiopsis protease; preferably wherein the microbial protease is a Nocardiopsis violaceus protease.

[0333] 11) The use according to any one of embodiments 1-5 and 10, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2.

[0334] 12) The use according to any one of embodiments 1-5 and 10-11, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0335] 13) The use according to any one of embodiments 1-5 and 10-12, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0336] 14) The use according to any preceding embodiment, wherein the cells are isolated from a surface or cell cluster.

[0337] 15) The use according to embodiment 14, wherein the surface is a plastic surface or a glass surface.

[0338] 16) The use according to any one of the preceding embodiments, wherein the cell is a human cell.

[0339] 17) The use according to any one of the preceding embodiments, wherein the cell is a stem cell or a stem cell derivative; preferably wherein the cell is a pluripotent stem cell, an induced pluripotent stem cell, a (stem cell-derived) dopaminergic progenitor cell or a (stem cell-derived) β cell.

[0340] 18) A composition suitable for cell separation, comprising a microbial protease.

[0341] 19) The composition of embodiment 18, wherein the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys; preferably wherein Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues at the P1 position.

[0342] 20) The composition of any one of Examples 18-19, wherein the P1 preference is determined according to Example 3 herein.

[0343] 21) The composition of any of embodiments 18-20, wherein the microbial protease exhibits chymotrypsin activity as determined according to the chymotrypsin activity assay described herein.

[0344] 22) The composition of any one of embodiments 18-21, wherein the microbial protease is a fungal protease or a bacterial protease.

[0345] 23) The composition according to any one of embodiments 18-22, wherein the microbial protease is a Scopulariopsis protease; preferably wherein the microbial protease is a Scopulariopsis compactus protease.

[0346] 24) A composition according to any one of embodiments 18-23, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1.

[0347] 25) The composition of any one of embodiments 18-24, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0348] 26) The composition of any one of embodiments 18-25, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0349] 27) The composition according to any one of embodiments 18-22, wherein the microbial protease is a Nocardiopsis protease; preferably wherein the microbial protease is a Nocardiopsis violaceus protease.

[0350] 28) A composition according to any one of embodiments 18-22 or 27, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2.

[0351] 29) The composition of any one of embodiments 18-22 or 27-28, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0352] 30) The composition of any one of embodiments 18-22 or 27-29, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0353] 31) A composition according to any of embodiments 18-30, wherein the purity of the microbial protease is at least 90%, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more, based on the weight of the total polypeptide material present in the composition.

[0354] 32) The composition according to any one of embodiments 18-31, which is a liquid composition.

[0355] 33) The composition according to embodiment 32, wherein the liquid composition is an aqueous composition.

[0356] 34) A composition according to any one of embodiments 32-33, wherein the liquid composition comprises an aqueous buffer; preferably wherein the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate or bicarbonate; most preferably wherein the aqueous buffer comprises phosphate.

[0357] 35) The composition of any one of embodiments 32-34, wherein the composition has a pH of about 7 to about 8, such as pH 7, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9 or pH 8.

[0358] 36) The composition according to any one of embodiments 32-35, wherein the microbial protease is present in an amount of about 0.1 μg / ml to about 20 μg / ml, for example, about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 µg / ml, about 18 µg / ml, about 19 µg / ml, or about 20 µg / ml.

[0359] 37) The composition of any one of embodiments 32-36, further comprising ethylenediaminetetraacetic acid (EDTA); preferably wherein the composition further comprises EDTA in an amount of about 0.01 mM to about 100 mM.

[0360] 38) The composition according to any one of embodiments 18-37, wherein the composition is substantially free of magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ); preferably wherein the composition does not contain magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ).

[0361] 39) A method for cell separation comprising contacting a cell with a composition suitable for cell separation comprising a microbial protease, wherein the cell is attached to a surface or another cell.

[0362] 40) The method of embodiment 39, wherein the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys; preferably wherein Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues at the P1 position.

[0363] 41) A method according to any one of Examples 39-40, wherein the P1 preference is determined according to Example 3 herein.

[0364] 42) The method of any one of embodiments 39-41, wherein the microbial protease exhibits chymotrypsin activity as determined according to the chymotrypsin activity assay described herein.

[0365] 43) The method of any one of embodiments 39-42, wherein the microbial protease is a fungal protease or a bacterial protease.

[0366] 44) The method according to any one of embodiments 39-43, wherein the microbial protease is a Scopulariopsis protease; preferably wherein the microbial protease is a Scopulariopsis compactus protease.

[0367] 45) The method according to any one of embodiments 39-44, wherein the microbial protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1.

[0368] 46) The method of any one of embodiments 39-45, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0369] 47) The method according to any one of embodiments 39-46, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0370] 48) The method according to any one of embodiments 39-43, wherein the microbial protease is a Nocardiopsis protease; preferably wherein the microbial protease is a Nocardiopsis violaceus protease.

[0371] 49) A method according to any one of embodiments 39-43 or 48, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:2.

[0372] 50) The method of any one of embodiments 39-43 or 48-49, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0373] 51) The method according to any one of embodiments 39-43 or 48-50, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0374] 52) The method of any one of embodiments 39-51, wherein the purity of the microbial protease is at least 90%, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more, based on the weight of the total polypeptide material present in the composition.

[0375] 53) The method according to any one of embodiments 39-52, wherein the composition is a liquid composition.

[0376] 54) The method according to embodiment 53, wherein the liquid composition is an aqueous composition.

[0377] 55) The method according to any one of embodiments 53-54, wherein the liquid composition comprises an aqueous buffer; preferably wherein the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate or bicarbonate; most preferably wherein the aqueous buffer comprises phosphate.

[0378] 56) The method according to any one of embodiments 53-55, wherein the pH of the liquid composition is about 7 to about 8, for example, pH 7, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9 or pH 8.

[0379] 57) The method according to any one of embodiments 53-56, wherein the microbial protease is present in an amount of about 0.1 µg / ml to about 20 µg / ml, for example, about 0.1 µg / ml, about 0.2 µg / ml, about 0.3 µg / ml, about 0.4 µg / ml, about 0.5 µg / ml, about 0.6 µg / ml, about 0.7 µg / ml, about 0.8 µg / ml, about 0.9 µg / ml, about 1 µg / ml, about 2 µg / ml, about 3 µg / ml, about 4 µg / ml, about 5 µg / ml, about 6 µg / ml, about 7 µg / ml, about 8 µg / ml, about 9 µg / ml, about 10 µg / ml, about 11 µg / ml, about 12 µg / ml, about 13 µg / ml, about 14 µg / ml, about 15 µg / ml, about 16 µg / ml, about 17 µg / ml, about 18 µg / ml, about 19 µg / ml, or about 20 µg / ml.

[0380] 58) The method according to any one of embodiments 53-57, wherein the liquid composition further comprises ethylenediaminetetraacetic acid (EDTA); preferably wherein the composition further comprises EDTA in an amount of about 0.01 mM to about 100 mM.

[0381] 59) The method according to any one of embodiments 53-58, wherein the liquid composition contains substantially no magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ); preferably wherein the composition does not contain magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ).

[0382] 60) The method of any one of embodiments 39-59, wherein the cell is a human cell.

[0383] 61) The method of any one of embodiments 39-60, wherein the cell is a stem cell or a stem cell derivative; preferably wherein the cell is a pluripotent stem cell, an induced pluripotent stem cell, a (stem cell-derived) dopaminergic progenitor cell or a (stem cell-derived) β cell.

[0384] Examples

[0385] Materials and Methods

[0386] Desalting of Accutase

[0387] Lyophilized Accutase® XL (Sigma Aldrich, full volume) was dissolved in 25 mL of MilliQ water, and 10 mL of the solution was loaded onto a HiPrep 26 / 10 desalting column (Sigma Aldrich) equilibrated with 50 mM Tricine, 10 mM CaCl2 (pH 7.5) at 10°C using an ÄKTAexplorer 100 at a flow rate of 10 mL / min. Elution was continued in the same buffer. The first peak fraction was collected and quantified based on the absorbance at 280 nm (A 280 ) to determine protein concentration and store at -20°C. Desalted Accutase was used for proteolytic activity assays (trypsin, chymotrypsin, collagenase type I, and collagenase type IV).

[0388] Trypsin activity assay

[0389] The assay was performed in a 96-well format with a total well volume of 200 µL. The enzyme concentration range was 0.5–200 µg / mL. The substrate (Nα-benzoyl-L-arginine ethyl ester, BAEE, Merck) was diluted in 67 mM phosphate buffer, pH 7.5. The final BAEE substrate solution was 0.28 mg / mL. The samples were diluted in 67 mM phosphate buffer, pH 7.5 (13.544 g sodium phosphate dibasic heptahydrate, 2.274 g sodium phosphate monobasic monohydrate, and added to 1 L with MilliQ water). The samples were tested in a concentration range of 5 to 100 µg / mL. 15 µL of the diluted sample was added to 185 µL of the BAEE substrate solution. The enzyme was detected by measuring the absorbance at 253 nm (A) at 27°C. 253 ), the reaction was monitored over 10 minutes.

[0390] Chymotrypsin activity assay

[0391] The assay was performed in a 96-well format with a total well volume of 200 µL. The enzyme concentration range was 20–500 µg / mL. The substrate (Nα-benzoyl-L-tyrosine ethyl ester, BTEE, Sigma) was dissolved in 1 mL of 96% ethanol and diluted in 4 mL of 80 mM Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol) (pH 7.5) containing 17 mM β-cyclodextrin. The final BTEE substrate solution was 1.72 mg / mL. The samples were diluted in 80 mM Tris, pH 7.5 (assay buffer). The samples were tested in a concentration range of 50 to 500 µg / mL. 20 µL of the diluted sample was added to 120 µL of assay buffer and 60 µL of BTEE substrate solution. The enzyme was detected by measuring the absorbance at 256 nm (A) at 27°C. 256 ), the reaction was monitored over 10 minutes.

[0392] Type I collagenase activity assay

[0393] 30 µL of 1 mg / mL collagen FITC (Merck) suspended in 1 mM acetic acid was added to the mixture in Dulbecco's phosphate-buffered saline (DPBS, without Ca2+). 2+ and Mg 2+ 20 µL of 0.25–7.5 µg / mL enzyme in DPBS buffer (Merck; Merck) was mixed with 100 µL of 0.5 M Tricine (Sigma) (pH 7.4). Reactions were performed at 37°C and terminated after 30, 60, or 90 minutes by adding 150 µL of ice-cold DPBS, followed by centrifugation (13,000 g; 5 minutes). Reactions were quantified by measuring duplicate 100 µL of supernatant at 485 nm for excitation and 535 nm for emission (detection).

[0394] Type IV collagenase activity assay

[0395] The enzyme was diluted to 1, 0.25, and 0.1 mg / mL in 100 mM Tricine (pH 7.4) and mixed with 40 µL of 5 mg / mL type IV collagen (human placenta, Sigma-Aldrich). The pH was adjusted with 10 µL of 0.5 M Tricine (pH 7.4). After 15 minutes, the reaction was terminated by precipitation with 105 µL of 10% trichloroacetic acid and incubated at 5°C for 10 minutes, followed by centrifugation (13,000 g; 3 minutes). The supernatant was removed, and the pellet was dissolved in 50 µL of sample buffer (200 µL of 4x Laemmli sample buffer (Bio-Rad), 40 µL of reducing agent (Bio-Rad), 30 µL of 2 M Tris, and 130 µL of MilliQ water). The samples were analyzed by SDS-PAGE using gels, Tris / glycine / SDS buffer, and Precision Plus Protein Standards from Bio-Rad. Enzyme activity was scored as no (0), less than (-), equal to (+), or greater than (++) the activity of Accutase (desalted samples). For example, see Figure 1 , where a first band of >200 kDa, a second band of 150 kDa, and a third band of 100 kDa were observed to be degraded.

[0396] Protease activity assay

[0397] The proteolytic activity of a polypeptide can be determined using a method employing the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and is a blocked peptide cleaved by endoproteases. Following proteolytic cleavage, free pNA molecules with a yellow color are released and can be measured by visible light spectrophotometry at a wavelength of 405 nm. The Suc-AAPF-PNA substrate is manufactured, for example, by Bachem (Cat. No. L1400, dissolved in DMSO).

[0398] Samples containing the peptide to be analyzed are diluted in residual activity buffer (100 mM Tris, pH 8.6). The assay is performed by transferring 30 µl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 µl of substrate working solution (0.72 mg / ml in 100 mM Tris, pH 8.6). The solution is mixed at room temperature and the absorbance (A) is measured at 405 nm every 20 seconds for 5 minutes. 405The sample should be diluted to a level where the slope is linear. Under a given set of conditions, the slope of the time-dependent absorbance curve (absorbance / minute) is directly proportional to the proteolytic activity of the peptide.

[0399] Assessment of cell detachment and cell cluster dissociation

[0400] Human Pluripotent Stem Cell Isolation and Assessment of Cell Cluster Dissociation

[0401] Human pluripotent stem cells (hPSCs) are cultured as a monolayer on laminin fragment tissue flasks (T-flasks) and passaged to form suspension cell clusters. The cells are then cultured in suspension for up to 8 passages. For a schematic diagram of this process and instructions on when to perform assessments, see Figure 2 and Figure 3 .

[0402] Assessment of hPSC Surface Detachment and Cluster Formation (2D):

[0403] Human pluripotent stem cells (induced or embryonic stem cells) are cultured in hPSC expansion medium (e.g., NutriStem hPSC XF (Sartorius, Germany) or StemFit Basic03 (Ajinomoto, Japan)) for 3–4 days according to individual culture protocols. On the day of passaging, the spent medium is removed from the T-flask and wash buffer (without Ca) is added. 2+ and Mg 2+ PBS; 0.04–0.3 mL / cm 2 The cell monolayer was washed and the buffer removed. A cell detachment solution containing the polypeptide of SEQ ID NO: 1 was prepared by diluting SEQ ID NO: 1 to a concentration of 5 µg / mL in DPBS, which was then pre-warmed to room temperature and supplemented with 0.5 mM ethylenediaminetetraacetic acid (EDTA). Accutase was pre-warmed to room temperature prior to cell detachment.

[0404] Add the corresponding cell detachment solution to the cell culture vessel (0.02–0.08 mL / cm 2 , typically 0.04 mL / cm 2) and incubate the vessel at 37°C for 3–20 min (typically 5–10 min). Expansion medium supplemented with 10 µM Y-27632 (Tocris, UK) was added to the vessel, and the cell suspension was mixed by pipetting to obtain a single-cell suspension. After surface detachment, cells were centrifuged, resuspended in an appropriate volume, and counted using a NucleoCounter NC202 (ChemoMetec, Denmark). Total cell count, cell viability, and aggregate fraction (% aggregates) were recorded according to [1 and 2]. Cells were then seeded in shake flasks and placed in a shaker incubator. Approximately 30–60 min after seeding, a 200 µl sample was removed and analyzed to assess the seeded cell suspension and viability at day 0.

[0405] 24 h after seeding, the efficiency of cell cluster formation was determined as the fold change on day 1 after seeding. The cells in the clusters were determined by processing the cell suspension and then analyzing it using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [3]. Briefly, 100 µl of solution A was added to 100 µl of cell suspension and pipetted vigorously until a single cell suspension could be observed (visual inspection, usually after 5-15 min). 100 µl of solution B was added to the suspension and the cell suspension was analyzed using a NC200 (ChemoMetec, Denmark) cell counter. The cells were further cultured in suspension for up to 3 days. To estimate cell growth, the fold change was determined daily on day 3 after seeding using a NucleoCounter NC202 (ChemoMetec, Denmark) according to [2]. On day 3 after seeding, the cell cluster size and size distribution (coefficient of variation of cell cluster diameter) were determined using a Biorep islet cell counter according to [4] and [5]. The evaluated parameters were scored according to Table 1 below and the average score was calculated.

[0406]

[0407] Assessment of hPSC Cluster Dissociation and Cluster Re-formation (3D) in Shake Flasks:

[0408] Prepare a cell detachment solution containing the polypeptide of SEQ ID NO: 1 by dissolving SEQ ID NO: 1 or SEQ ID NO: 2 in DPBS to a concentration of 4–5 µg / mL or 1.5 µg / mL, respectively, then pre-warm to room temperature and add 0.5 mM EDTA. Pre-warm Accutase to room temperature prior to cell detachment.

[0409] hPSCs were cultured as suspension cell clusters in a shaker flask in a shaking incubator for three or four days before cell passage. On the day of passage, the hPSC clusters were centrifuged, the supernatant removed, and the cells were washed with Ca-free water. 2+ and Mg 2+ The cells were washed with PBS (0.1–1 mL / mL original working volume, typically 0.25 mL / mL). The centrifuge tube containing the cells was placed horizontally on an orbital shaker and the cells were incubated at 37°C for 3–15 min. Expansion medium supplemented with 10 µM Y-27632 (Tocris, UK) was added to the centrifuge tube and the cell suspension was mixed by pipetting to obtain a single-cell suspension. After cluster dissociation, the cells were centrifuged, resuspended in an appropriate volume of expansion medium supplemented with 10 µM Y-27632 (Tocris, UK) and counted using a NucleoCounter NC202 (ChemoMetec, Denmark) and the total cell count, cell viability and aggregate fraction (% aggregates) were recorded according to [1 and 2]. To estimate the efficiency of cell cluster formation, the fold change on day 1 after passaging was determined. The cells in the cell clusters were identified using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [3]. To estimate cell growth, daily fold changes were determined on day 3 after seeding according to [1]. On day 3 after passaging, cluster size and size distribution (coefficient of variation of cluster diameter) were determined using a Biorep islet cell counter according to [4] and [5]. The evaluated parameters were scored according to Table 2 below, and the mean score was calculated.

[0410] After the second passage at the cluster stage, the pluripotent phenotype was confirmed by staining with antibodies specific for the surface markers Oct4 (BD Biosciences) and Nanog (Nordic BioSite ApS) using fluorescence-activated single-cell sorting (FACS) analysis.

[0411]

[0412] Assessment of hPSC Cluster Dissociation (3D) in Bioreactors:

[0413] A cell detachment solution containing the polypeptide of SEQ ID NO: 1 was prepared by dissolving the polypeptide of SEQ ID NO: 1 in DPBS to a concentration of 5 µg / mL, followed by preheating to 37°C and adding 0.5 mM EDTA.

[0414] hPSCs were cultured as suspension cell clusters in a bioreactor (10 L DASGip, Eppendorf, Germany) for 5 days. On the day of passage, the hPSC clusters were pelleted, the supernatant was removed, and the cells were washed with Ca-free water. 2+ and Mg 2+ After washing, the wash buffer was removed, cell detachment solution was added, and the cell clusters were incubated at 37°C for 3–5 min. Expansion medium supplemented with 10 µM Y-27632 (Tocris, UK) was added to the bioreactor, and the cell suspension was mixed by agitation to obtain a single-cell suspension. The cells were then centrifuged, resuspended in an appropriate volume, and counted using a NucleoCounter NC202 (ChemoMetec, Denmark). Total cell count, cell viability, and aggregate fraction (% aggregates) were recorded according to [1, 2].

[0415] Assessment of Dopaminergic Progenitor Cell Isolation in 2D

[0416] The detachment of stem cell-derived dopaminergic progenitor monolayers was assessed at the dopaminergic progenitor stage (partially differentiated at this time) on day 11. Prior to enzyme treatment, the spent culture medium was removed and wash buffer (without Ca) was added. 2+ and Mg 2+ PBS; 0.04-0.3 mL / cm 2 ), the cell layer was washed and the washing buffer was removed.

[0417] Prepare the cell detachment solution by dissolving the peptide of SEQ ID NO: 1 in DPBS to a final concentration of 5 µg / mL, preheating to room temperature, and adding 0.5 mM EDTA. Preheat Accutase to room temperature. Add the corresponding enzyme solution to the cell culture vessel (0.04-0.3 mL / cm 2 ) and incubate the container at 37°C for 5–15 min until the cells become rounded and begin to float, as assessed by microscopy. Expansion medium is added to the container, and the cell suspension is mixed by pipetting to obtain a suspension of single cells and small cell clusters. The total cells, cell viability, and aggregate fraction (% aggregates) in the cell suspension are analyzed using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [3].

[0418] Day 11 dopaminergic progenitor cells were replated, expanded, and further matured until day 16. Cultures were assessed for replating efficiency on days 11 and 12. Attachment stability was assessed daily until day 16.

[0419] On day 16, dopaminergic progenitor cells were harvested with cell detachment solution and analyzed as described above. The phenotype of day 16 dopaminergic progenitor cells was confirmed by staining with antibodies specific for the surface markers FOXA2 and OTX2 (Miltenyi Biotech) using FACS analysis.

[0420] The evaluated parameters were scored according to Table 3 below and the average score was calculated.

[0421]

[0422] Assessment of β-cell cluster dissociation in 3D

[0423] Stem cell-derived β cell clusters (immature β cells at the β cell stage day 3, BC03) were sedimented by gravity in a falcon tube. The spent medium was removed, and the cell clusters were washed in 10 ml of DPBS. After washing, the cell clusters were again sedimented by gravity, and the DPBS was removed. A cell detachment solution containing the polypeptide of SEQ ID NO:1 was prepared by dissolving SEQ ID NO:1 in ice-cold DPBS to a concentration of 4 µg / mg, then adding 0.5 mM EDTA and prewarming to room temperature. Accutase was prewarmed to room temperature.

[0424] Cell detachment solution (3 ml) was added to the β-cell clusters, and the cells were incubated horizontally in a shaking incubator at 37°C for 5–7 minutes. Single-cell suspension was ensured by vigorous pipetting 3–10 times, followed by the addition of 7 mL of knockout serum replacement (KOSR). Cells were counted using a NucleoCounter NC202 (ChemoMetec, Denmark), and cell viability, total cell count, and aggregate fraction (% aggregates) were recorded according to [2]. The cells were centrifuged and resuspended in cryoprotectant and then cryopreserved.

[0425] β cells were thawed and the cryoprotectant was washed off using expansion medium through repeated wash and centrifugation cycles. Single cells were seeded into suspension cultures in a shaker incubator to reaggregate the cells.

[0426] Reaggregation was assessed 48 h after seeding by counting free cells in suspension and calculating the percentage of seeded single cells that formed clusters. In addition, the total volume of clusters in the sample was estimated by the cluster volume per 16 seeded cells (pIEQ). Cluster volume (pIEQ) was measured on a Biorep islet cell counter [4, 5].

[0427] Important parameters of β-cell cluster dissociation and reformation were assessed and scored according to Table 4, and the average score was calculated.

[0428]

[0429] Evaluation of bone marrow-derived mesenchymal stem cell isolation in 2D

[0430] Bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured in 24-well tissue culture-treated cell culture plates (NEST Biotechnology, China) in MSC Nutristem XF medium containing 2.5% human platelet lysate (Sartorius, Germany). Prior to cell detachment, spent medium was removed from the wells, and wash buffer (DPBS) was added. After removal of the wash buffer, prewarmed (37°C) cell detachment solution (6 µg / mL SEQ ID NO:1, or Accutase in DPBS supplemented with 0.5 mM EDTA; 0.2 mL) was added to the wells, followed by incubation at 37°C for 5 minutes. Subsequently, 0.2 mL of MSC Nutristem XF medium containing 2.5% human platelet lysate was added to the wells, and the cell suspension was mixed by pipetting to obtain a single-cell suspension. Cells in suspension (0.2 mL) were counted using a NucleoCounter NC200 (ChemoMetec, Denmark), and total cell count, cell viability, and aggregate fraction (% aggregates) were recorded according to [6].

[0431] Evaluation of Madin-Darby Canine Kidney Cell Isolation in 2D

[0432] Madin-Darby canine kidney (MDCK) cells were cultured in 24-well tissue culture-treated cell culture plates (Nice Biotech, China) using Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS). Prior to cell detachment, spent culture medium was removed from the wells, and wash buffer (DPBS) was added. After removal of the wash buffer, cell detachment solution (SEQ ID NO:1 at 18 µg / mL, Accutase, or DPBS supplemented with 0.5 mM EDTA (as a negative control); prewarmed to 37°C; 0.2 mL) was added to the wells, followed by incubation at 37°C for 5 minutes. Subsequently, 0.2 mL of DMEM supplemented with 10% FBS was added to the wells, and the cell suspension was mixed by pipetting to obtain a single-cell suspension. Cells in suspension (0.2 mL) were counted using a NucleoCounter NC200, and total cell count, cell viability, and aggregate fraction were recorded according to [6].

[0433] Evaluation of Human Embryonic Kidney 293 Cell Isolation in 2D

[0434] Human embryonic kidney 293 (HEK293) cells were cultured in tissue culture-treated 24-well cell culture plates (Nice Biotech, China) in DMEM supplemented with 10% FBS. Prior to cell dissociation, spent culture medium was removed from the wells, and wash buffer (DPBS) was added. After removal of the wash buffer, cell detachment solution (SEQ ID NO:1 at 6 µg / mL, Accutase in DPBS supplemented with 0.5 mM EDTA, or DPBS supplemented with 0.5 mM EDTA (as a negative control); prewarmed to 37°C; 0.2 mL) was added to the wells, followed by incubation at 37°C for 1 minute. Subsequently, 0.2 mL of DMEM supplemented with 10% FBS was added to the wells, and the cell suspension was mixed by pipetting to obtain a single-cell suspension. Cells in suspension (0.2 mL) were counted using a NucleoCounter NC200, and total cell count, cell viability, and aggregate fraction (% aggregate) were recorded according to [6].

[0435] Evaluation of Adipose Stromal Cell Isolation in 2D

[0436] Adipose-derived stromal cells (ASCs) were expanded for 48 h in minimal essential medium Eagle-α modified (α-MEM) supplemented with 5% human platelet lysate (hPL) in T75 Nunc flasks. Prior to cell dissociation, spent medium was removed from the wells and washed with PBS. After removal of the wash buffer, cell detachment solution (5 µg / mL of SEQ ID NO:1, Gibotec, in DPBS supplemented with 0.5 mM EDTA or TrypLE Select) was added to the wells and incubated at 37°C for 4–5 min. Subsequently, two volumes of expansion medium were added, and the cells were harvested by centrifugation and resuspended. Cells were counted using a NucleoCounter NC202 (ChemoMetec, Denmark), and total cell count, cell viability, aggregate fraction (% aggregates), and debris index were recorded according to [1, 2].

[0437] Example 1: Expression of SEQ ID NO: 1 (S1 protease from S. compacta)

[0438] The gene encoding the S1 protease from S. compacta was PCR amplified from a genomic clone using gene-specific primers and cloned into the Aspergillus expression vector pMStr57 (WO 04 / 032648) digested with BamHI and XhoI. The cloned gene was sequenced and confirmed to be identical to the gene shown in SEQ ID NO:3. It was then transformed into Aspergillus oryzae strain BECh2 (WO 2000 / 39322) by the methods described in Christensen et al., 1988, Biotechnology 6, 1419-1422 and WO 2004 / 032648. Transformants were selected during regeneration from protoplasts based on the ability conferred by the selectable marker in the expression vector to utilize acetamide as a nitrogen source, and these transformants were subsequently reisolated under selection. Production of the recombinant protease was assessed by culturing the transformants in 10 ml of YPG medium (WO 05 / 066338) in 30 ml sterile plastic tubes at 34°C with shaking at 275 rpm for four days. Samples were analyzed for protease activity at pH 8 using the pNA assay described in WO 2004 / 072279, and expression was monitored by SDS-PAGE. Three transformants were selected for high-level expression of the recombinant protease, and of these, one was selected for providing the highest expression of the three transformants when cultured in 100 ml of YPG medium in a baffled 500 ml shake flask at 37°C, 275 rpm for four days. Recombinant expression was monitored by SDS-PAGE.

[0439] The selected transformants were fermented in 100 ml of FG4P medium (WO 1994 / 26925) in a baffled 500 ml shake flask at 30° C. with shaking at 250 rpm for four days. The fermentation broth was then purified according to conventional methods well known to those skilled in the art to provide SEQ ID NO: 1.

[0440] Example 2: Expression of SEQ ID NO: 2 (S1 protease from Nocardia cepacia)

[0441] A linear integration vector system was used to clone the expression of the S1 protease from Nocardia viridans. The linear integration construct is a PCR fusion product composed of the gene encoding the S1 protease from Nocardia viridans (SEQ ID NO:4) fused between two homologous chromosomal regions of Bacillus subtilis to a strong promoter and a chloramphenicol resistance marker. The fusion was performed using SOE PCR (Horton, RM, Hunt, HD, Ho, SN, Pullen, JK, and Pease, LR (1989) Engineering hybrid genes without the use of restriction enzymes, gene splicing by overlap extension. Gene 77: 61-68). The SOE PCR method is also described in patent application WO 2003 / 095658. The gene was expressed under the control of a triple promoter system (described in WO 1999 / 43835) consisting of the Bacillus licheniformis α-amylase gene (amyL) promoter, the Bacillus amyloliquefaciens α-amylase gene (amyQ) promoter, and the Bacillus thuringiensis cryIIIA promoter, containing a stabilizing sequence. A gene encoding chloramphenicol acetyltransferase was used as a marker (described, for example, in Diderichsen, B.; Poulsen, GB; Joergensen, ST 1993, Plasmid A useful cloning vector for Bacillus subtilis 30:312). The final gene construct was integrated into the pectate lyase locus on the Bacillus chromosome by homologous recombination.

[0442] The gene encoding the S1 protease from Nocardia cereus (SEQ ID NO:4) was amplified from chromosomal DNA using gene-specific primers containing overhangs to two flanking vector fragments. The S1 protease was expressed by replacing the gene's native secretion signal with the Bacillus clausii secretion signal (MKKPLGKIVASTALLISVAFSSSIASA; SEQ ID NO:5). The upstream and downstream vector fragments were amplified from genomic DNA of strain MB1361 (based on strain PL3598 described in patent application WO2003095658). The two linear vector fragments and the gene fragment were assembled into a single linear vector construct using SOE PCR. Aliquots of the PCR product were transformed into Bacillus subtilis. Transformants were selected on LB plates supplemented with 6 µg of chloramphenicol per ml.

[0443] One transformant containing the sequence-verified, integrated expression construct was grown in liquid culture in 500 mL baffled Erlenmeyer flasks on a rotary shaker at 30° C. for 4 days, each containing 100 ml of yeast extract-based medium. The fermentation broth was subsequently purified according to WO 2004 / 111222 to provide SEQ ID NO: 2.

[0444] Example 3: Determination of protease specificity

[0445] Protease substrate specificity can be defined based on P1 preference. The P1 position is defined as the amino acid residue located N-terminal to the protease cleavage site (Biochemical and Biophysical Research Communications, Vol. 27, No. 2, April 20, 1967, pp. 157–162).

[0446] Bias is defined as the observed rate of occurrence relative to that expected from random cleavage when counting the number of cleavage sites resulting from protease digestion on a complex protein substrate comprising a high sequence diversity.

[0447] Specifically, purified protease samples were incubated with yeast protein extract (Promega V7341) in 100 mM HEPES, 1 mM CaCl2 (pH 7) on a 10 kDa cutoff spin filter at 37°C for 16 hours. Prior to incubation, the substrate was denatured by precipitation with trichloroacetic acid (TCA), reduced by the addition of dithiothreitol (DTT), and alkylated by the addition of iodoacetamide (IAA).

[0448] Three reactions were performed with protease:substrate ratios of 1:1250, 1:6250, and 1:30,000. The resulting protease digest was collected by centrifugation and flowthrough. Additional washes should be included to increase peptide recovery. The protease digest was acidified with TFA and analyzed directly by LC-MS / MS (e.g., Evosep One (Evosep) / timsTOFPro (Bruker Daltonik)).

[0449] For peptide identification, the data were searched against the UniProt yeast reference proteome using the Mascot search engine (Matrix Science) with the following search parameters:

[0450] Enzyme: No

[0451] Peptide mass tolerance: ± 25 ppm

[0452] Fragment mass tolerance: ± 0.05 Da

[0453] Maximum missed cut: 0

[0454] Protease cleavage sites are inferred from the N- and C-termini of the identified peptides. The extent of proteolysis should be low enough to reflect the initial preferred cleavage site. Based on knowledge of the amino acid sequence of the protein from which the peptide is derived, identify amino acids present in the subsite (e.g., P1) upon proteolytic cleavage.

[0455] The preference of an amino acid in a subsite (e.g., P1) is calculated by comparing the sum of the intensities of identified peptides containing that amino acid in the subsite (e.g., P1) to the expected frequency of occurrence from random cleavage of the protein.

[0456] Based on this method, the P1 preferences of SEQ ID NO: 1 and SEQ ID NO: 2 were evaluated. Both SEQ ID NO: 1 and SEQ ID NO: 2 had an increased P1 preference for the amino acid residues Leu, Tyr, Phe, and Lys. When the P1 preferences of SEQ ID NO: 1 and SEQ ID NO: 2 for all twenty typical amino acids were determined and then ranked, Leu, Tyr, Phe, and Lys were among the five most preferred amino acid residues at the P1 position.

[0457] Example 4: Proteolytic activity of SEQ ID NO: 1, SEQ ID NO: 2, and Accutase

[0458] The total protein concentration of Accutase was determined to be 20 µg / mL. The trypsin, chymotrypsin, collagenase type I, and collagenase type IV activities of Accutase were assessed and normalized to 100%, and the enzyme activities of SEQ ID NO:1 and SEQ ID NO:2 relative to Accutase were reported (see Table 5).

[0459] SEQ ID NO: 1 exhibits increased chymotrypsin activity and comparable type IV collagenase activity compared to Accutase, while reduced trypsin activity and type I collagenase activity compared to Accutase.

[0460] Compared to Accutase, SEQ ID NO: 2 has comparable chymotrypsin activity, but reduced trypsin and collagenolytic activities.

[0461]

[0462] Example 5: hPSC Monolayer Dissociation and Cell Cluster Formation (2D) of SEQ ID NO: 1

[0463] hPSC monolayer detachment and cell cluster formation were evaluated and scored for SEQ ID NO: 1 and Accutase (Table 6).

[0464]

[0465] Compared to Accutase, the overall performance of SEQ ID NO: 1 was improved. In particular, the aggregate fraction was reduced, while the efficiency of cell cluster formation was improved.

[0466] Example 6: Dissociation and Re-formation of hPSC Clusters Using SEQ ID NO: 1 in Shake Flasks (3D)

[0467] hPSC cluster dissociation and reformation were evaluated and scored for SEQ ID NO: 1 and Accutase (Table 7).

[0468]

[0469] Compared to Accutase, SEQ ID NO: 1 demonstrated improved overall performance. Specifically, cell viability, cluster formation efficiency, and cell growth were improved. Furthermore, after two passages as clusters, the coefficient of variation of cluster diameter for hPSCs treated with SEQ ID NO: 1 was comparable to or improved compared to hPSCs treated with Accutase.

[0470] After two passages as cell clusters, pluripotency was assessed. No decrease in pluripotency was observed in hPSCs treated with SEQ ID NO: 1 (5 µg / mL) compared to hPSCs treated with Accutase (94.2% vs. 93.7% Oct4 and Nanog double-positive hPSCs).

[0471] Example 7: Dissociation and Re-formation of hPSC Clusters Using SEQ ID NO: 2 in Shake Flasks (3D)

[0472] SEQ ID NO: 2 and Accutase were evaluated and scored for hPSC cluster dissociation and reformation (Table 8). SEQ ID NO: 2 demonstrated improved overall performance compared to Accutase. Specifically, cluster reformation and cluster size uniformity were improved.

[0473]

[0474] After two passages as cell clusters, pluripotency was assessed. No reduction in pluripotency was observed in hPSCs treated with SEQ ID NO: 2 compared to hPSCs treated with Accutase (95.5% vs. 93.7% Oct4 and Nanog double-positive hPSCs).

[0475] Example 8: Dissociation of hPSC Clusters Using SEQ ID NO: 1 in a Bioreactor (3D)

[0476] hPSC cluster dissociation of SEQ ID NO: 1 was evaluated in a bioreactor with a starting volume of 5 L, and the results showed that 4.81 x 10 6 The total cell count was 97% and the aggregate fraction was 9.9%.

[0477] Example 9: Isolation of dopaminergic progenitor cells (2D) using SEQ ID NO: 1

[0478] SEQ ID NO: 1 and Accutase were evaluated and scored for yield, aggregate formation, cell viability, replating efficiency, and attachment stability of dopaminergic progenitor cells grown as 3D cultures (Table 9).

[0479]

[0480] Compared to Accutase, SEQ ID NO: 1 showed improved overall performance. Specifically, cell yield (as demonstrated by total cell counts and the fraction of FOXA2 / OTX2 double-positive cells, see Table 10) was improved, while the fraction of aggregates was reduced (see also Table 10). Furthermore, attachment stability was improved compared to Accutase.

[0481]

[0482] Example 10: Dissociation of β-cell clusters using SEQ ID NO: 1 and SEQ ID NO: 2 (3D)

[0483] BC03 stage beta cell clusters were dissociated using SEQ ID NO: 1, SEQ ID NO: 2, or Accutase. Cell viability and cell yield were scored before and after dissociation. After cryopreservation, the ability of BC03 cells to re-form cell clusters was assessed and scored. The total volume of the cell clusters (per 10 6 The pIEQ of each seeded cell was determined and scored.

[0484] The scores of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in Table 11. The overall performance of SEQ ID NO: 1 and SEQ ID NO: 2 was improved compared to Accutase. In particular, the cell yield and cell cluster re-formation were improved compared to Accutase.

[0485]

[0486] Example 11: Isolation of bone marrow-derived mesenchymal stem cells (2D)

[0487] SEQ ID NO: 1 and Accutase were evaluated for the isolation of bone marrow-derived mesenchymal stem cells (BM-MSCs) in 2D culture (Table 12). SEQ ID NO: 1 demonstrated improved overall performance compared to Accutase. Specifically, yield was improved, while the aggregate fraction was reduced.

[0488]

[0489] Example 12: Isolation of Madin-Darby Canine Kidney Cells in 2D

[0490] SEQ ID NO: 1 and Accutase were evaluated for the separation of Madin-Darby canine kidney (MDCK) cells in 2D culture (Table 13). SEQ ID NO: 1 demonstrated improved overall performance compared to Accutase. Specifically, yield was improved, while the aggregate fraction was reduced.

[0491]

[0492] Example 13: Isolation of Human Embryonic Kidney 293 Cells (2D)

[0493] SEQ ID NO: 1 and Accutase were evaluated for the separation of human embryonic kidney 293 (HEK293) cells in 2D culture (Table 14). SEQ ID NO: 1 demonstrated improved overall performance compared to Accutase. Specifically, yield was improved, while the number of aggregates was reduced.

[0494]

[0495] Example 14: Dissociation of Adipose Stromal Cells (3D)

[0496] SEQ ID NO: 1 and Accutase were evaluated for dissociation of adipose-derived stromal cell (ASC) clusters in 3D culture (Table 15). SEQ ID NO: 1 demonstrated improved overall performance compared to TrypLE Select. Specifically, yield was improved, while the amount of non-viable cells (debris index) was reduced.

[0497]

[0498] References

[0499] 1)ChemoMetec, Application Note No. 2026. Rev. 1.4. Count & Viability- Via2-Cassette™

[0500] 2)ChemoMetec, Application Note No. 2028. Rev. 1.3. Aggregated Cells -Via2-Cassette™

[0501] 3)ChemoMetec, Application Note No. 0215, Rev. 1.2. CountingAggregated Cells using the Via1-Cassette™ with Reagent A100 and B3

[0502] 4)BioRep User manual for AUTOMATIC ISLET CELL COUNTER 4, Ref. ICC-04:

[0503] 5)Fully Automated Islet Cell Counter (ICC) for the Assessment ofIslet Mass, Purity, and Size Distribution by Digital Image Analysis. PeterBuchwald, Andres Bernal, Felipe Echeverri, Alejandro Tamayo-Garcia, ElinaLinetsky and Camillo Ricordi. Cell Transplantation, Vol. 25, pp. 1747–1761,2016.

[0504] 6)ChemoMetec, Application Note No. 0201, Rev. 1.6. Mammalian Cells –Viability and Cell Counting using the Via1-Casette™

Claims

1. A composition suitable for cell separation, comprising a microbial protease.

2. The composition of claim 1, wherein the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys; preferably wherein Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues at the P1 position.

3. The composition of any preceding claim, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1; preferably wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:

1.

4. The composition of any preceding claim, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2; preferably wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:

2.

5. The use of microbial proteases in cell separation processes.

6. Use according to claim 5, wherein the microbial protease has an increased P1 preference for Leu, Tyr, Phe and Lys; preferably wherein Leu, Tyr, Phe and Lys are among the five most preferred amino acid residues at the P1 position.

7. Use according to any one of claims 5-6, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1; preferably wherein the microbial protease comprises, consists essentially of or consists of SEQ ID NO:

1.

8. The use according to any one of claims 5-6, wherein the microbial protease has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2; preferably wherein the microbial protease comprises, consists essentially of or consists of SEQ ID NO:

2.

9. Use according to any one of claims 5 to 8, wherein the cells are isolated from a surface or cell cluster.

10. The use according to claim 9, wherein the surface is a plastic surface or a glass surface.

11. Use according to any one of claims 5 to 10, wherein the cell is a mammalian cell, preferably a human or canine cell.

12. The use according to any one of claims 5 to 11, wherein the cell is a stem cell or a stem cell derivative; preferably wherein the cell is a pluripotent stem cell, an induced pluripotent stem cell, a (stem cell-derived) dopaminergic progenitor cell or a (stem cell-derived) beta cell.

13. The use according to any one of claims 5 to 11, wherein the cell is a pluripotent stem cell, a mesenchymal stem cell, a β cell, a neuron, an adipocyte, an epithelial cell or a kidney cell.

14. A method for cell separation, the method comprising contacting a cell with a composition according to any one of claims 1 to 4, wherein the cell is attached to a surface or another cell.

15. The method according to claim 14, wherein the cell is a mammalian cell, preferably a human or canine cell.

16. The method according to any one of claims 14-15, wherein the cell is a stem cell or a stem cell derivative; preferably wherein the cell is a pluripotent stem cell, an induced pluripotent stem cell, a (stem cell-derived) dopaminergic progenitor cell or a (stem cell-derived) beta cell.

17. The method according to any one of claims 14-15, wherein the cell is a pluripotent stem cell, a mesenchymal stem cell, a beta cell, a neuron, an adipocyte, an epithelial cell or a kidney cell.

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