Microbial proteases for cell detachment

Abstract

The present invention relates to a microbial protease for cell detachment. The present invention also relates to a composition suitable for cell detachment containing said microbial protease, the use of said microbial protease in a cell detachment process, and a method for cell detachment using said microbial protease.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to a microbial protease for cell detachment. The present invention also relates to a composition suitable for cell detachment containing said microbial protease, the use of said microbial protease in a cell detachment process, and a method for cell detachment using said microbial protease. [Background technology]

[0003] Cell detachment is an important step during the passaging of cells when grown as adherent cells as well as cell clusters. The detachment step preferably involves the use of proteolytic enzymes, as they are gentle yet effective in not only releasing cells from the surface to which they adhere, but also lysing cell clusters formed in suspension cultures.

[0004] Accutase® and Accumax® (both available, for example, from Innovative Cell Technologies, Inc.) are commercially available products for cell detachment that contain a mixture of enzymes with proteolytic and collagenolytic activity isolated from invertebrate sources. A problem associated with these products is that regulatory agencies generally do not allow animal-derived products to be used in drug development and production processes, preventing their application in cell therapy. Another problem with these products is that, as a result of the animal-derived nature of these mixtures, there is an inherent risk of batch-to-batch variability in terms of composition and activity, which results in products that are not well defined.

[0005] TrypLE™ (available, for example, from ThermoFisher Scientific) is a commercially available trypsin product that can be used for cell detachment. TrypLE™ is recombinantly produced and is therefore not of animal origin. However, a problem associated with TrypLE™ is that not all cell types are adequately detached when treated with trypsin alone, which limits the broad applicability of this product.

[0006] It is an object of the present invention to provide an enzyme solution that addresses the shortcomings associated with current products for cell detachment. In particular, it is an object of the present invention to provide an enzyme solution that meets regulatory requirements, is useful for detaching many different types of cells, and can be produced in a uniform manner without batch-to-batch variation. Summary of the Invention [Means for solving the problem]

[0007] The present invention relates to microbial proteases and their use in cell detachment and cell cluster dissociation processes. The inventors have recognized that microbial proteases with increased P1 preference for the amino acid residues Leu, Tyr, Phe, and Lys are particularly suitable for use in cell detachment. Without being bound by theory, it is speculated that the P1 preference profile exhibited by the microbial proteases of the present invention results in effective yet gentle cleavage of cell surface proteins involved in surface attachment and cell-cell adhesion. Furthermore, the microbial proteases of the present invention can be recombinantly produced, which ensures regulatory compliance as well as a highly uniform production process when used in the development and production of cells for pharmaceutical applications such as cell therapy.

[0008] In a first aspect, the present invention relates to a composition suitable for cell detachment, comprising a microbial protease.

[0009] In a second aspect, the present invention relates to the use of microbial proteases in cell detachment processes.

[0010] In a third aspect, the present invention relates to a method for cell detachment comprising contacting a cell with the composition of the first aspect, wherein the cell is attached to a surface or another cell. [Brief explanation of the drawings]

[0011] [Figure 1] 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. Lane 3: 0.1 μg / mL desalted Accutase with type IV collagen substrate. Lane 4: Desalted Accutase without substrate. Lane 5: Type IV collagen substrate only. [Figure 2] A schematic overview of the hPSC setup is shown, indicating the time points (circled) at which hPSC monolayer detachment and assessment of cluster formation were performed. [Figure 3] A schematic overview of the hPSC setup is shown, indicating the time points (circled) at which hPSC cluster dissociation and repopulation assessment were performed. DETAILED DESCRIPTION OF THE INVENTION

[0012] Array Overview SEQ ID NO: 1 is the S1 protease from Sarocladium strictum.

[0013] SEQ ID NO: 2 is the S1 protease from Nocardiopsis prasina.

[0014] SEQ ID NO: 3 is the DNA sequence encoding the S1 protease from Sarocladium strictum.

[0015] SEQ ID NO: 4 is the DNA sequence encoding the S1 protease from Nocardiopsis prasina.

[0016] SEQ ID NO: 5 is the secretion signal from Bacillus clausii.

[0017] definition cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature 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 that is processed through a series of steps, including splicing, before appearing as a spliced ​​mature mRNA.

[0018] Cell detachment: The term "cell detachment" refers to the process of detaching or releasing smaller groups of cells, or even single cells, from cell culture, particularly 2D and 3D cell culture. 2D cell culture includes adherent cell culture, in which cells are grown as a monolayer attached to the surface of a cell culture vessel (e.g., a culture flask or a Petri dish), and the cells adhere to each other and / or the surface of the cell culture vessel. 3D cell culture includes suspension culture, in which cells are grown as cell clusters suspended in agitated growth medium, and the cells adhere to each other. 3D cell culture also includes enriched medium culture (e.g., agarose culture or Matrigel culture), and scaffold culture, in which cells are grown on a structural scaffold. The terms "cell detachment" and "cell dissociation" are used interchangeably herein.

[0019] Coding sequence: The term "coding sequence" refers to 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 generally 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 may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0020] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence may be native (i.e., derived from the same gene) or heterologous (i.e., derived from different genes) to the polynucleotide encoding a polypeptide, and may be native or heterologous to each other. Such control sequences include, but are not limited to, leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include a promoter, and transcription and translation stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of a polynucleotide encoding a polypeptide.

[0021] Expression: The term "expression" refers to any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0022] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, the coding sequence operably linked to a suitable control sequence 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 a suitable ribosome binding site on the mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0023] Fragment: The term "fragment" refers to a polypeptide having one or more amino acids not present at the amino and / or carboxyl terminus of the 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.

[0024] Heterologous: The term "heterologous" with respect to a host cell means that the polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means that the regulatory sequences of the polypeptide or nucleic acid, e.g., the promoter, are not naturally associated with the polypeptide or nucleic acid, i.e., the regulatory sequences are derived from a gene other than the gene encoding the mature polypeptide.

[0025] 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 containing a polynucleotide encoding a polypeptide of the invention has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from cells.

[0026] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including, but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell, or other material is therefore in a form that is not found in nature. Isolated polypeptides include, but are not limited to, culture broths containing the polypeptide expressed and secreted in a host cell.

[0027] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0028] Nucleic Acid: The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single-stranded or double-stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode specific amino acid sequences. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0029] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that is otherwise modified to contain a segment of nucleic acid in a non-naturally occurring manner, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0030] 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 so that expression of the coding sequence is under the control of the regulatory sequence.

[0031] Passaging: The term "passaging" refers to the process of removing some or all of the cells from a culture and transferring them to fresh growth medium. Passaging of cells may also be called subculturing. In some embodiments, passaging results in a single cell suspension.

[0032] 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 contains several subgroups, including EC 3.4.21 (serine endopeptidases), which further contain several subgroups, including EC 3.4.21.62 (subtilisins). The terms "protease" and "polypeptide having protease activity" are used interchangeably herein.

[0033] For purposes of the present invention, protease activity (EC3.4) may be determined according to the protease activity assay described in the Examples herein.

[0034] For purposes of the present invention, trypsin activity (EC 3.4.21.4) may be determined according to the trypsin activity assay described in the Examples herein.

[0035] For 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.

[0036] For purposes of the present invention, type I collagenase activity may be determined according to the type I collagenase activity assay described in the Examples herein.

[0037] For purposes of the present invention, type IV collagenase activity may be determined according to the type IV collagenase activity assay described in the Examples herein.

[0038] Purified: The term "purified" refers to 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 may form a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, and typically is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more pure (e.g., on a weight percent or molar basis). In a related sense, a composition is enriched for a molecule if the concentration of the molecule is substantially increased after applying a purification or concentration procedure. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0039] In one aspect, the term "purified," as used herein, refers to a polypeptide (e.g., a microbial protease) or cell that is essentially free of components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a polypeptide that is essentially free of insoluble components, particularly insoluble components, from the natural organism from which the polypeptide is obtained. In one aspect, the polypeptide has been separated from a portion of the soluble components of the organism and culture medium from which the polypeptide is recovered. The polypeptide can be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0040] Thus, a polypeptide (e.g., a microbial protease) can be purified so that only trace amounts of other proteins (e.g., other polypeptides) are present. The term "purified," as used herein, can refer to the removal of other components, particularly other proteins, most particularly other enzymes, present in the cell from which the polypeptide originated. A polypeptide can also be "substantially pure," i.e., free from other components from the organism that produces the polypeptide (e.g., the host organism in the case of a recombinantly produced polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in a preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in a preparation (e.g., a composition suitable for cell detachment). As used herein, a "substantially pure polypeptide" can mean a polypeptide preparation that contains up to 10%, preferably up to 9%, preferably up to 8%, preferably up to 7%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, more preferably up to 2%, more preferably up to 1%, more preferably up to 0.5, more preferably up to 0.1%, more preferably up to 0.05%, more preferably up to 0.01%, even more preferably up to 0.005%, and most preferably up to 0.001%, by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0041] Thus, a substantially pure polypeptide (e.g., a microbial protease) is preferably 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, by weight of total polypeptide material present in a preparation (e.g., a composition suitable for cell detachment). Polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be achieved, for example, by preparing the polypeptide using well-known recombinant or classical purification methods.

[0042] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form a group different from that found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been altered from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes at levels or under conditions different from those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0043] Recover: The terms "recover" and "recovery" refer to removing a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, for example, by collecting polypeptide crystals, by chromatography, by filtration, such as depth filtration (using filter aids or packed filter media, chamber filter fabric filtration, rotary drum filtration, drum filtration, rotary vacuum drum filters, candle filters, horizontal leaf filters, or similar, with sheet or pad filtration in a framework or modular setup) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross-flow, dynamic cross-flow, or dead-end operation), or by centrifugation (using decanter centrifuges, disc centrifuges, hydrocyclones, or similar), or by using particle size fractionation to precipitate proteins and collect the polypeptide from the broth medium, and using related solid-liquid separation methods. Recovery encompasses isolation and / or purification of the polypeptide.

[0044] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0045] For purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The -nobrief option must be specified on the command line to cause the Needle program to report the longest identity. The Needle output, labeled "longest identity," is calculated as follows: (Identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0046] For purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), as implemented in the Needle program in the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. The nobrief option must be specified on the command line to cause the Needle program to report the longest identity. The Needle output, labeled "longest identity," is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0047] Detailed Description of the Invention The present invention relates to microbial proteases and their use in cell detachment and cell cluster dissociation processes. The inventors have recognized that microbial proteases with increased P1 preference for the amino acid residues Leu, Tyr, Phe, and Lys are particularly suitable for use in cell detachment. Without being bound by theory, it is speculated that the P1 preference profile exhibited by the microbial proteases of the present invention results in effective yet gentle cleavage of cell surface proteins involved in surface attachment and cell-cell adhesion. Furthermore, the microbial proteases of the present invention can be recombinantly produced, which ensures not only a highly uniform production process but also regulatory compliance when used in the development and production of cells for pharmaceutical applications such as cell therapy.

[0048] Compositions suitable for cell detachment The present invention relates to a composition suitable for cell detachment, 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.

[0049] The microbial protease can be a fungal protease or a bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0050] In one 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.

[0051] In one 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.

[0052] 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.

[0053] In one embodiment, the microbial protease has chymotrypsin activity. Chymotrypsin activity (EC 3.4.21.1) can be determined according to the chymotrypsin activity assay described in the Examples herein.

[0054] 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.

[0055] In one embodiment, the microbial protease has type IV collagenase activity, which may be determined according to the type I collagenase activity assay described in the Examples herein.

[0056] In a preferred embodiment, the microbial protease has chymotrypsin activity and is substantially free of trypsin activity, wherein chymotrypsin activity (EC 3.4.21.1) is determined according to the chymotrypsin activity assay described in the Examples herein, and trypsin activity (EC 3.4.21.4) is determined according to the trypsin activity assay described in the Examples herein.

[0057] In one embodiment, the composition comprises a microbial protease having a purity of at least 90%, 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 by weight of total polypeptide material present in the composition.

[0058] In preferred embodiments, the microbial protease has a purity of at least 99%, 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.

[0059] In more preferred embodiments, the microbial protease has a purity of at least 99.9%, e.g., at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of total polypeptide material present in the composition.

[0060] In a most preferred embodiment, the microbial protease has a purity of at least 99.99%, such as at least 99.995%, at least 99.999%, or greater, by weight of total polypeptide material present in the composition.

[0061] In one aspect, the composition suitable for cell detachment is a liquid composition. Preferably, the composition is an aqueous composition to ensure compatibility with media commonly used in cell culture. In some embodiments, the liquid composition is lyophilized. In another aspect, the composition is a solid composition, preferably a lyophilized composition.

[0062] To ensure that the liquid composition has a pH value compatible with cell culture conditions, the composition may contain an aqueous buffer solution. The composition may contain 1 to 99% by weight of the aqueous buffer solution, for example, 5 to 95%, 10 to 90%, 15 to 85%, 20 to 80%, or 25 to 75%. Alternatively, the composition may contain at least 5% by weight of the aqueous buffer solution, 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%, or at least 99.5% or more.

[0063] In some embodiments, the liquid composition has a pH value of 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 composition has a pH value of 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 composition has a pH value of 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 composition has a pH value of about 7.4.

[0064] 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 (e.g., PBS) buffer.

[0065] In some embodiments, the liquid composition comprises a microbial protease of the present 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.

[0066] In some embodiments, the liquid composition comprises a concentration of about 0.1 μg / ml to about 20 μg / ml, e.g., 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, The microbial protease of the present invention may be present in an amount of 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.

[0067] In some embodiments, the liquid composition comprises the microbial protease of the present 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.

[0068] In some embodiments, the liquid composition comprises a microbial protease of the present 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.

[0069] In some embodiments, the liquid composition comprises a microbial protease of the present invention in an amount of about 1 μg / ml to about 20 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, 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.

[0070] In a preferred embodiment, the liquid composition comprises the microbial protease of the present 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, and most preferably 1 μg / ml to 5 μg / ml.

[0071] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount 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.

[0072] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 0.1 mg / ml to about 20 mg / ml, e.g., 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.

[0073] 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.

[0074] 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.

[0075] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of 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.

[0076] In a preferred embodiment, the liquid composition comprises the polypeptide of the invention in an amount 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, and most preferably 1 mg / ml to 5 mg / ml.

[0077] In some embodiments, the liquid composition includes ethylenediaminetetraacetic acid (EDTA). Preferably, the liquid composition includes EDTA in an amount of about 0.01 mM to about 100 mM, e.g., 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 includes 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.

[0078] In some embodiments, the liquid composition comprises magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ In some embodiments, the liquid composition is substantially free of 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+ ) is not included.

[0079] In some embodiments, the liquid composition comprises a phosphate buffer (e.g., PBS), EDTA, and magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free of

[0080] In a preferred embodiment, the liquid composition comprises a phosphate buffer solution (e.g., PBS) having a pH value of about 7 to about 8, preferably about 7 to about 7.5, and most preferably about pH 7.4; 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 the liquid composition contains magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free of

[0081] In a preferred embodiment, the liquid composition comprises a phosphate buffer solution (e.g., PBS) having a pH value of about 7 to about 7.5, most preferably about pH 7.4; the liquid composition further comprises EDTA in an amount of about 0.5 mM to about 5 mM, most preferably about 1 mM; and the liquid composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) is not included.

[0082] In a preferred embodiment, the liquid composition comprises a phosphate buffer solution (e.g., PBS) having a pH value of about 7 to about 7.5, most preferably about pH 7.4; the liquid composition further comprises EDTA in an amount of about 0.5 mM to about 5 mM, most preferably about 1 mM; and the liquid composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) does not contain: The composition contains SEQ ID NO: 1 or SEQ ID NO: 2 in an amount of 0.1 μg / ml to 20 μg / ml.

[0083] In a preferred embodiment, the liquid composition comprises a phosphate buffer (e.g., PBS) having a pH value of about pH 7.4; the composition further comprises EDTA in an amount of about 1 mM; and the composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) is not included.

[0084] In a preferred embodiment, the liquid composition comprises a phosphate buffer (e.g., PBS) having a pH value of about pH 7.4; the composition further comprises EDTA in an amount of about 1 mM; and the composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) does not contain: The composition contains SEQ ID NO: 1 or SEQ ID NO: 2 in an amount of 1 μg / ml to 20 μg / ml.

[0085] The liquid composition may further comprise an enzyme stabilizer (examples include a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative, such as an aromatic borate ester, or a phenylboronic acid derivative, such as 4-formylphenylboronic acid).

[0086] In some embodiments, a filler or carrier material is included to increase the volume of the liquid composition. Suitable fillers and carrier materials include, but are not limited to, various salts of sulfates, carbonates, and silicates, as well as talc, clay, and the like. 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 some embodiments, the composition contains from about 5% to about 90% of such materials.

[0087] 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.

[0088] In another embodiment, the present invention provides (a) 0.001 to 25% w / w of a microbial protease of the present invention (e.g., SEQ ID NO: 1 or SEQ ID NO: 2); (b) 20-80% w / w polyol; (c) optionally 0.001 to 2% w / w of a preservative; and (d) water The present invention relates to a liquid composition comprising:

[0089] In another embodiment, the present invention provides (a) 0.001 to 25% w / w of a microbial protease of the present invention (e.g., SEQ ID NO: 1 or SEQ ID NO: 2); (b) 0.001-2% w / w of a preservative; (c) optionally 20 to 80% w / w of a polyol; and (d) water The present invention relates to a liquid composition comprising:

[0090] In another embodiment, the liquid composition comprises one or more additives 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, acetates, and phosphates, preferably 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 glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0091] In one embodiment, the liquid composition comprises glucose in an amount of about 0.1 g / L to about 10 g / L, e.g., 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.

[0092] In another embodiment, the liquid composition comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol. In one embodiment, the liquid formulation comprises 20% to 80% polyol, e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, where 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% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0093] 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 of the preservative, e.g., 0.05-1% w / w of the preservative, or 0.1-0.5% w / w of the preservative. In one embodiment, the liquid formulation comprises 0.001-2% w / w of the preservative (i.e., total amount of preservative), e.g., 0.02-1.5% w / w of the preservative, 0.05-1% w / w of the preservative, or 0.1-0.5% w / w of the preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0094] In one aspect, the composition further comprises one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. Preferably, the one or more additional enzymes are selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, DNase, 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, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0095] In a preferred embodiment, the composition further comprises DNase.

[0096] microbial proteases The present invention also relates to microbial proteases having increased P1 preference for Leu, Tyr, Phe, and Lys. In preferred embodiments, Leu, Tyr, Phe, and Lys are among the five most preferred amino acid residues at the P1 position. P1 preference can be determined according to Example 3 herein.

[0097] The microbial protease can be a fungal protease or a bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] In one embodiment, the microbial protease has chymotrypsin activity. Chymotrypsin activity (EC 3.4.21.1) can be determined according to the chymotrypsin activity assay described in the Examples herein.

[0102] 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.

[0103] In one embodiment, the microbial protease has type IV collagenase activity, which may be determined according to the type I collagenase activity assay described in the Examples herein.

[0104] In another aspect, the microbial protease is derived from SEQ ID NO: 1 or SEQ ID NO: 2 by substituting, deleting, or adding one or several 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, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- 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 net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding module.

[0105] Critical amino acids in polypeptides, e.g., microbial proteases, can be identified using art-known procedures 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 a molecule to identify amino acid residues critical to the molecule's activity and / or specificity, and the resulting molecules are tested for protease activity and / or P1 specificity (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, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., 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. Additionally, the identification of important amino acids can be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or within a polypeptide or protein family, including polypeptides / proteins typically derived from a common ancestor with 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 important 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.

[0106] 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 associated screening procedures such as those disclosed in 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; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0107] To detect the activity of cloned, mutagenized polypeptides expressed by host cells, mutagenesis / shuffling methods can be combined with high-throughput automated screening methods (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and easily sequenced using standard methods in the art. These methods allow for the rapid determination of individual important amino acid residues within a polypeptide.

[0108] In one aspect, the microbial protease is isolated.

[0109] In another embodiment, the microbial protease is purified.

[0110] Sources of microbial proteases The microbial proteases of the present invention can be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from," when used herein in reference to a given source, is intended to mean that the polypeptide encoded by the polynucleotide is produced by the source or by a strain into which a polynucleotide of the present invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0111] In another aspect, the microbial protease is obtained from a Sarocladium species, such as Sarocladium strictum.

[0112] In one aspect, the microbial protease is obtained from a Nocardiopsis species, such as Nocardiopsis prasina.

[0113] For the species described above, it will be understood that the present invention encompasses both perfect and imperfect forms, as well as other taxonomic equivalents, e.g., anamorphs, regardless of the name by which the species is known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0114] Microbial proteases may be identified and obtained using the probes described above from microorganisms isolated from nature (e.g., soil, compost, water, etc.) or from other sources, including DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. Polynucleotides encoding the microbial proteases can then be obtained by similarly screening genomic DNA or cDNA libraries of other microorganisms or mixed DNA samples. Once a polynucleotide encoding a microbial protease is detected by the probe, the polynucleotide can be isolated and cloned using techniques known to those skilled in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0115] Polynucleotides The present invention also relates to polynucleotides encoding the microbial proteases of the present invention.

[0116] The polynucleotide may be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof. The polynucleotide may be obtained from a strain of Sarocladium, e.g., Sarocladium strictum, or a related microorganism, or from a strain of Nocardiopsis, e.g., Nocardiopsis prasina, or a related microorganism.

[0117] In one embodiment, a polynucleotide encoding a microbial protease of the invention is isolated from cells of the genus Sarocladium, such as Sarocladium strictum.

[0118] In one embodiment, a polynucleotide encoding a microbial protease of the invention is isolated from cells of the genus Nocardiopsis, such as Nocardiopsis prasina.

[0119] The polynucleotide may also be mutated by introducing nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but that correspond to the codon usage of the host organism in which the enzyme is intended to be produced, or that may result in a different amino acid sequence. For a general description of nucleotide substitutions, see, e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0120] In one aspect, the polynucleotide is isolated.

[0121] In another embodiment, the polynucleotide is purified.

[0122] Nucleic Acid Constructs The present invention also relates to nucleic acid constructs comprising a polynucleotide of the 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.

[0123] Polynucleotides may be manipulated in a variety of ways to bring about expression of a microbial protease. Manipulation of the polynucleotide prior to insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0124] promoter The control sequence may be a promoter, which is a polynucleotide recognized by a host cell to direct expression of a polynucleotide encoding a microbial protease of the present invention. The promoter contains transcriptional control sequences that mediate expression of the microbial protease. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0125] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY; Davis et al., 2012, supra; and Song et al., 2016, PLOS One 11(7):e0158447.

[0126] Examples of promoters suitable for directing transcription of the polynucleotides of the invention in filamentous fungal host cells 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 Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0127] Examples of promoters useful for expression in yeast hosts 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 Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0128] Terminator The control sequence may also be a transcription terminator recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the microbial protease. Any terminator functional in the host cell may be used in the present invention.

[0129] Preferred terminators for bacterial host cells may be obtained from the genes for alkaline protease of Bacillus clausii (aprH), alpha-amylase of Bacillus licheniformis (amyL), and ribosomal RNA of Escherichia coli (rrnB).

[0130] Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as the terminators described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology, e.g., Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I.

[0131] Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0132] mRNA stabilizers A regulatory sequence may also be an mRNA stabilizing region downstream of the promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0133] Examples of suitable mRNA stabilization regions are obtained from the cryIIIA gene of Bacillus thuringiensis (WO 94 / 25612) and the SP82 gene of Bacillus subtilis (Hue et al., 1995, J. Bacteriol. 177;3465-3471).

[0134] Examples of mRNA stabilization regions in 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.

[0135] Leader sequence The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' terminus of the polynucleotide encoding the microbial protease. Any leader that is functional in the host cell may be used.

[0136] Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12):3051-3059, and Kaberdin and Blaesi, 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0137] Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0138] Suitable leaders for yeast host cells may 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).

[0139] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3' end of a polynucleotide and, upon transcription, is recognized as a signal by a host cell and adds polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0140] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0141] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0142] signal peptide The control sequence may also be a signal peptide coding region linked to the N-terminus of a polypeptide, encoding a signal peptide that directs the polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the microbial protease. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the heterologous signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the microbial protease. Any signal peptide coding sequence that directs the expressed microbial protease into the secretory pathway of the host cell may be used.

[0143] Effective signal peptide coding sequences for bacterial host cells include those obtained from the genes for maltogenic amylase from Bacillus NCIB 11837, subtilisin from Bacillus licheniformis, β-lactamase from Bacillus licheniformis, α-amylase from Bacillus stearothermophilus, neutral protease from Bacillus stearothermophilus (nprT, nprS, nprM), and prsA from Bacillus subtilis. Additional signal peptides have been described by Freudl, 2018, Microbial Cell Factories 17:52.

[0144] Effective signal peptide coding sequences for filamentous fungal host cells include those obtained from 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 those described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0145] 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.

[0146] Propeptide The regulatory sequence may 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 known as a proenzyme or propolypeptide (or, in some cases, a zymogen). Propolypeptides are generally inactive and can be converted to active polypeptides by catalytic or autocatalytic cleavage of the propeptide. Propeptide coding sequences may also be obtained from genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, laccase of Myceliophthora thermophila (WO 95 / 33836), aspartic proteinase of Rhizomucor miehei, and α-factor of Saccharomyces cerevisiae.

[0147] When both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence. Additionally, or alternatively, when both a signal peptide sequence and a propeptide sequence are present, the polypeptide may include only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide may include a mixture of mature polypeptides and polypeptides that include either partial or full-length propeptide and / or signal peptide sequences.

[0148] Regulatory sequences It may also be desirable to add regulatory sequences that regulate expression of the microbial protease relative to the growth of the host cell. Examples of regulatory sequences include those that activate or inactivate gene expression in response to chemical or physical stimuli, including the presence of a regulatory compound. Regulatory sequences for prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA α-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences include those that allow for gene amplification. In fungal systems, these regulatory sequences include dihydrofolate reductase, which is amplified in the presence of methotrexate, and metallothionein genes, which are amplified with heavy metals.

[0149] transcription factors A regulatory sequence may also be a transcription factor, 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 together 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, which often binds to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a protein of interest directly, i.e., by activating the transcription of a gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor that regulates the transcription of the gene encoding the protein of interest, for example, by binding to the promoter of the further 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 also in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0150] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. Various nucleotide and control sequences may be ligated together to create a recombinant expression vector that may contain one or more convenient restriction sites, allowing for the insertion or substitution of a polynucleotide encoding a microbial protease at such sites. Alternatively, a polynucleotide may be expressed by inserting a nucleic acid construct containing the polynucleotide into an appropriate vector for expression. In creating an expression vector, a coding sequence is placed in the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0151] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can bring about expression of a 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 may be a linear or closed circular plasmid.

[0152] The vector may be a self-replicating vector, i.e., a vector whose replication exists as an extrachromosomal element independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may include any means for ensuring self-replication. Alternatively, the vector may be such that, upon introduction into a host cell, it is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA or transposon to be introduced into the genome of the host cell, may be used.

[0153] Vectors preferably contain one or more selectable markers which permit easy selection of transformed, transfected, transduced cells, etc. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0154] The vector preferably contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously within the cell independently of the genome.

[0155] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the 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).

[0156] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to autonomously replicate in the intended host cell. The origin of replication may be any plasmid replicator that mediates autonomous replication and functions within the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0157] Two or more copies of a polynucleotide of the present invention may be inserted into a host cell to increase production of the polypeptide. For example, two, three, four, five, or more copies are inserted into the host cell. Increasing the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide; cells containing an amplified copy of the selectable marker gene, and thus cells containing additional copies of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selection agent.

[0158] Recombinant host cells The present invention also relates to recombinant host cells containing the polynucleotides of the present invention operably linked to one or more control sequences that direct the production of the microbial proteases of the present invention.

[0159] 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 that comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0160] 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 that comprises, consists essentially of, or consists of SEQ ID NO:2.

[0161] The construct or vector containing the polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The choice of host cell will largely depend on the gene encoding the microbial protease and its source. The microbial protease may be native or heterologous to the recombinant host cell. Also, at least one of the one or more regulatory sequences may be heterologous to the polynucleotide encoding the microbial protease. The recombinant host cell may contain a single copy of the polynucleotide of the invention, or at least two copies, e.g., three, four, five or more copies.

[0162] The host cell can be any microbial cell useful for the recombinant production of the microbial proteases of the present invention, for example, a prokaryotic or fungal cell.

[0163] Prokaryotic host cells can be any gram-positive or gram-negative bacterium, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0164] Bacterial host cells include Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, and Bacillus stearothermophilus. The Bacillus cell may be any Bacillus cell, including, but not limited to, a Bacillus stearothermophilus, a Bacillus subtilis, and a Bacillus thuringiensis cell. In one embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, a Bacillus licheniformis, or a Bacillus subtilis cell.

[0165] For purposes of the present invention, the class / genus / species of Bacillus shall be defined as set forth in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0166] Bacterial host cells may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0167] A bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0168] 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, and transduction, where the DNA is introduced as a linear or circular polynucleotide. Those skilled in the art will be able to easily identify a suitable method for introducing DNA into a given prokaryotic cell, for example, depending on the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology 18:56; Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294; Choi et al., 2006, J. Microbiol. Methods 64:391-397; and Donald et al., 2013, J. Bacteriol. 195(11):2612-2620.

[0169] The host cell may be a fungal cell. As used herein, "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0170] Fungal cells may be transformed by processes including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic methods, and shock wave-mediated transformation, as reviewed in Li et al., 2017, Microbial Cell Factories 16:168, and 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 linear or circular polynucleotide.

[0171] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). For purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0172] Yeast host cells include Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, and the like. The yeast host cell may be a Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0173] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivisions 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 is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0174] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neomyces, and others. The filamentous fungal host cell may be a Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell. In a more preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0175] For example, filamentous fungal host cells may be selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis girvescens. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenstrandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestrisThe cell may be a Trichoderma terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0176] In one embodiment, the host cell is isolated.

[0177] In another embodiment, the host cells are purified.

[0178] Production method The present invention also relates to methods for producing the microbial proteases of the present invention, comprising: (a) culturing a host cell that produces the microbial protease of the present invention in wild-type form under conditions conducive to production of the microbial protease; and, optionally, (b) recovering the microbial protease.

[0179] 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. P1 preference can be determined according to Example 3 herein.

[0180] 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 a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0181] 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 a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0182] The present invention also relates to methods for producing the microbial proteases of the invention, comprising: (a) culturing a recombinant host cell of the invention under conditions conducive to production of the microbial protease of the invention; and, optionally, (b) recovering the microbial protease.

[0183] 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. P1 preference can be determined according to Example 3 herein.

[0184] 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 a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0185] 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 a preferred embodiment, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0186] 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 B. subtilis cell or a B. licheniformis cell. In a preferred embodiment, the recombinant host cell is an Aspergillus cell, most preferably an A. niger cell or an A. oryzae cell. In a preferred embodiment, the recombinant host cell is a Pichia cell, most preferably a P. pastoris cell.

[0187] The host cells are cultured in a nutrient medium suitable for the production of the microbial protease using methods known in the art. For example, the cells can be cultured in a suitable medium and under conditions that allow for the expression and / or isolation of the microbial protease in shake flask cultures, or by small- or large-scale fermentation in laboratory or industrial fermentors (including continuous, batch, fed-batch, or solid-state and / or microcarrier-based fermentation). Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., compositions published in catalogs of the American Type Culture Collection). If the microbial protease is secreted into the nutrient medium, the microbial protease can be recovered directly from the medium. If the microbial protease is not secreted, it can be recovered from cell lysates.

[0188] 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, the formation of an enzyme product, the disappearance of an enzyme substrate, or assays that determine the relative or specific activity of the microbial protease.

[0189] The microbial protease may be recovered from the culture medium using methods known in the art, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, freeze drying, evaporation, or precipitation. In one aspect, the whole fermentation broth containing the microbial protease is recovered. In another aspect, the cell-free fermentation broth containing the microbial protease is recovered.

[0190] Microbial proteases may 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).

[0191] In an alternative embodiment, the microbial protease is not recovered.

[0192] Protease Granules The present invention also relates to enzyme granules / particles comprising the microbial proteases of the present invention. In one embodiment, the granules comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0193] The core may have a diameter, measured as an equivalent spherical diameter (average particle size by volume), of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm. The core diameter, measured as an equivalent spherical diameter, can be determined using laser diffraction, for example, using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0194] In one embodiment, the core comprises a microbial protease of the present invention.

[0195] The core may contain additional materials such as fillers, fibrous materials (cellulose or synthetic), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants and fragrances.

[0196] The core may include a binder such as a synthetic polymer, a wax, a fat, or a carbohydrate.

[0197] The core may include salts of multivalent cations, reducing agents, antioxidants, peroxide decomposition catalysts and / or acidic buffer components, typically as a homogeneous blend.

[0198] The core may comprise inert particles into which the polypeptide has been absorbed or to which the polypeptide has been applied, for example, by fluidized bed coating.

[0199] The core may have a diameter of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm or 250 to 1200 μm.

[0200] 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 include salt coatings or other suitable coating materials, such as polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA).

[0201] The coating may be applied in an amount of at least 0.1%, such as at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15% by weight of the core, which may be up to 100%, 70%, 50%, 40%, or 30%.

[0202] The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm, or at least 5 μm thick. In some embodiments, the coating thickness is less than 100 μm, for example less than 60 μm, or less than 40 μm.

[0203] The coating must encapsulate the core unit by forming a substantially continuous layer. By substantially continuous layer, it is to be understood that the coating is substantially free of holes, so that there are few uncoated areas of the core unit. The layer or coating must, in particular, be uniform in thickness.

[0204] The coating may further contain other materials known in the art, such as fillers, anti-adherents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0205] The salt coating can comprise at least 60% by weight salt, e.g., 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%.

[0206] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, e.g., 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, hi certain embodiments, the salt coating is less than 100 μm thick, e.g., less than 60 μm or less than 40 μm.

[0207] The salt may be added from a salt solution in which the salt is completely dissolved, or from a salt suspension in which the particles are less than 50 μm, for example less than 10 μm or less than 5 μm.

[0208] 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 of at least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 g per 100 g of water, for example at least 1 g per 100 g of water, for example at least 5 g per 100 g of water.

[0209] The salts may be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (having less than 10 carbon atoms, e.g., 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or earth alkali metal salts of sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates, or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0210] The salt in the coating may have a constant humidity of more than 60%, in particular more than 70%, 80% or more than 85% at 20° C., or may be another hydrated form of such a salt (e.g., anhydrous). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0211] Specific examples of suitable salts are NaCl (CH20°C = 76%), Na2CO3 (CH20°C = 92%), NaNO3 (CH20°C = 73%), Na2HPO4 (CH20°C = 95%), Na3PO4 (CH20°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%), KHPO4 (CH20°C = 92%), KHPO4 (CH20°C = 96.5%), KNO3 (CH20°C = 93.5%), NaSO4 (CH20°C = 93%), KSO4 (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.

[0212] The salts may be in anhydrous form or may be hydrated salts, i.e., crystalline salt hydrates containing bound water of crystallization, as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (NaSO), anhydrous magnesium sulfate (MgSO), magnesium sulfate heptahydrate (MgSO·7H2O), zinc sulfate heptahydrate (ZnSO·7H2O), sodium phosphate dibasic heptahydrate (NaHPO·7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0213] Preferably, the salt is added as a solution of the salt, for example using a fluidized bed.

[0214] The coating material can be a wax coating material or a film-forming coating material. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1,483,591.

[0215] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). Examples of multi-coated enzyme granules are described in WO 93 / 07263 and WO 97 / 23606.

[0216] The cores can be prepared by granulating a blend of ingredients by methods including granulation techniques such as, for example, crystallization, precipitation, pan coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation.

[0217] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CECapes; Vol. 1; 1980; Elsevier. Preparation methods include following known feed and granule formulation techniques. (a) A spray-dried product in which a liquid microbial protease-containing solution is atomized in a spray-drying tower to form droplets that are dried during passage through the drying tower to form a microbial protease-containing particulate material. Microparticles can be produced in this manner (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (b) Layered products in which a microbial protease is coated as a layer around preformed inert core particles, typically by atomizing the microbial protease-containing solution in a fluidized bed apparatus where the preformed core particles are fluidized, allowing the microbial protease-containing solution to adhere to the core particles and dry completely, leaving a layer of microbial protease on the surface of the core particles. If useful core particles of the desired particle size can be found, particles of the desired particle size can be obtained in this manner. Products of this type are described, for example, in WO 97 / 23606. (c) Adsorbed core particles in which the microbial protease is adsorbed onto and / or into the core rather than coated as a layer around the core, such a process is described in WO 97 / 39116. (d) Extruded or pelletized products, in which a microbial protease-containing paste is pressed into pellets or extruded under pressure through small orifices and cut into particles, which are then dried. Such particles are usually quite large because the material from which the extrusion orifices are made (usually a perforated plate) limits the allowable pressure drop across the extrusion orifices. Also, when small orifices are used, very high extrusion pressures increase the generation of heat within the microbial protease paste, which is detrimental to the microbial protease (Michael S. Showell (editor); Powdered Detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (e) Prilled products, in which a microbial protease-containing powder is suspended in molten wax and the suspension is sprayed into a cooling chamber, e.g., by a rotating disk atomizer, causing the droplets to rapidly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product has the microbial protease uniformly distributed throughout the inert material, rather than concentrated on its surface. U.S. Pat. Nos. 4,016,040 and 4,713,245 describe this technology. (f) Mixer-granulated products, in which a microbial protease-containing liquid is added to a dry powder composition of conventional granulation components. When the liquid and powder are mixed in the appropriate ratio and the moisture from the liquid is absorbed by the dry powder, the dry powder components adhere and begin to aggregate, building particles to form granules containing the microbial protease. Such processes are described in U.S. Pat. No. 4,106,991, European Patent Nos. 170360, 304332, and 304331, and International Publication Nos. 90 / 09440 and 90 / 09428. In certain embodiments of this process, various high-shear mixers can be used as granulators. Granules consisting of microbial protease, fillers, binders, etc., are mixed with cellulose fibers to reinforce the particles, producing so-called T-granules. The reinforced particles are more robust and less likely to release enzyme dust. (g) Size reduction, in which cores are 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 ground or crushed product. The large and small particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons. (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. Particles hit by the spray droplets become wet and sticky. The sticky particles collide with other particles, causing them to adhere to each other, forming granules. (i) The cores may be subjected to drying, such as in a fluidized bed dryer. Other methods known in the feed or enzyme industry for drying granules may be used by those skilled in the art. Drying is preferably carried out at a product temperature of 25 to 90°C. For some microbial proteases, it is important that the cores containing the microbial proteases contain a small amount of water before coating with salt. If water-sensitive microbial proteases are coated with salt before removing excess water, the excess water may become trapped within the cores, which may adversely affect the activity of the microbial proteases. After drying, the cores preferably contain 0.1 to 10% w / w water.

[0218] Non-dusting granules may be produced, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art.

[0219] The granules may further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. Preferably, the one or more additional enzymes are 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, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof. The more granules in which each enzyme is present, the more uniform the enzyme distribution will be, and the less physical separation of different enzymes due to different particle sizes will be. A method for purifying multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0220] Another example of the formulation of polypeptides through the use of co-granules is disclosed in WO 2013 / 188331.

[0221] The present invention also relates to protected polypeptides prepared according to the methods disclosed in EP 238216.

[0222] Fermentation Broth Composition or Cell Composition The present invention also relates to fermentation broth formulations or cell compositions comprising the microbial proteases of the present invention. The fermentation broth formulations or cell compositions further comprise additional components used in the fermentation process, such as, for example, cells (including host cells containing genes encoding the microbial proteases of the present invention used to produce the microbial proteases of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a killed whole broth containing organic acids, killed cells and / or cell debris, and culture medium.

[0223] In one embodiment, the fermentation broth formulation or cell composition comprises a microbial protease with 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.

[0224] The microbial protease can be a fungal protease or a bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0225] In one 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.

[0226] In one 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.

[0227] The term "fermentation broth," as used herein, refers to a preparation produced by cell fermentation with no or minimal recovery and / or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation and incubated under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cells) and secretion into the cell culture medium. A fermentation broth can contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, a fermentation broth is unfractionated and includes spent culture medium and cellular debris present after microbial cells (e.g., filamentous fungal cells) have been removed, for example, by centrifugation. In some embodiments, a fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0228] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component comprising an organic acid of at least 1-5 carbons and / or a salt thereof, and a second organic acid component comprising an organic acid of at least 1-6 or more carbons 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 of the foregoing, 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 of the foregoing.

[0229] In one aspect, the composition contains an organic acid and optionally further contains dead cells and / or cell debris, hi some embodiments, the dead cells and / or cell debris are removed from the cell-killed whole broth to obtain a composition free of these components.

[0230] The fermentation broth formulation or cell composition may further comprise preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0231] The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killed whole broth or cell composition contains spent culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions that allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0232] Whole broth or cell compositions as described herein are typically liquid but may contain insoluble components such as dead cells, cell debris, culture medium components, and / or insoluble enzymes, etc. In some embodiments, the insoluble components may be removed to obtain a clarified liquid composition.

[0233] The whole broth formulations and cell compositions of the present invention may be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0234] Methods and Uses The present invention also relates to methods for detaching cells, comprising contacting the cells with a microbial protease of the invention or a composition of the invention, wherein the cells are attached to a surface and / or another cell. In one embodiment, the detached cells are attached to a surface. In one embodiment, the detached cells are part of a cell monolayer. In one embodiment, the detached cells are part of a cell cluster.

[0235] 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.

[0236] The microbial protease can be a fungal protease or a bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0237] 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 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.

[0238] 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 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.

[0239] The methods of the present invention may be used to exfoliate any type of cell, including, but not limited to, A375 metastatic melanoma cells, beta 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 chick embryonic neurons, Sf9 insect cells, U251 glioma cells, vascular endothelial cells, vascular smooth muscle cells, and Vero cells.

[0240] In a preferred embodiment, the exfoliated cells are mammalian cells, preferably canine or human cells, most preferably human cells.

[0241] The method of the present invention can be used to explant any type of stem cell or stem cell derivative. Thus, the stem cells can be totipotent stem cells (e.g., fertilized egg cells), pluripotent stem cells (e.g., embryonic stem cells), multipotent 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.

[0242] In some embodiments, the exfoliated cells are pluripotent stem cells, mesenchymal stem cells, beta cells, neurons, adipocytes, epithelial cells, or kidney cells.

[0243] In some embodiments, the detached cells are attached to a surface, such as a plastic or glass surface. In some embodiments, the detached cells are attached to another cell. In some embodiments, the attached cell is part of a cell cluster.

[0244] In some embodiments, the detached cells are attached to biomaterials, extracellular matrix (ECM)-coated surfaces, and / or other scaffolds made from 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.).

[0245] In some embodiments, the exfoliated cells are stem cells or stem cell derivatives. Preferably, the cells are pluripotent stem cells, induced pluripotent stem cells, (stem cell-derived) dopaminergic progenitor cells, or (stem cell-derived) beta cells.

[0246] In one embodiment, the detached cells are pluripotent stem cells, preferably human pluripotent stem cells. Preferably, the pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0247] In one embodiment, the detached cells are induced pluripotent stem cells, preferably human induced pluripotent stem cells. Preferably, the induced pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0248] In one embodiment, the detached cells are beta cells, preferably human beta cells. Preferably, the beta cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0249] In one embodiment, the detached cells are stem cell-derived beta cells, preferably human stem cell-derived beta cells. Preferably, the stem cell-derived beta cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0250] In one embodiment, the detached cells are dopaminergic progenitor cells, preferably human dopaminergic progenitor cells. Preferably, the dopaminergic progenitor cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0251] In one embodiment, the detached cells are stem cell-derived dopaminergic progenitor cells, preferably human stem cell-derived dopaminergic progenitor cells. Preferably, the stem cell-derived dopaminergic progenitor cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0252] In one embodiment, the detached cells are bone marrow-derived mesenchymal stem cells (BM-MSCs). Preferably, the bone marrow-derived mesenchymal stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0253] In one embodiment, the detached cells are canine cells, preferably Madin-Darby canine kidney (MDCK) cells. Preferably, the canine cells, more preferably Madin-Darby canine kidney cells, are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0254] In one embodiment, the detached cells are human embryonic kidney 293 (HEK293) cells. Preferably, the human embryonic kidney 293 cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0255] In one embodiment, the detached cells are adipose stromal cells. Preferably, the adipose stromal cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0256] The present invention also relates to the use of the microbial protease of the present invention in a cell detachment process. 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.

[0257] The microbial protease can be a fungal protease or a bacterial protease. In one embodiment, the microbial protease is a fungal protease. In one embodiment, the microbial protease is a bacterial protease.

[0258] 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 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.

[0259] 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 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.

[0260] The microbial proteases of the present invention may be used in any type of cell detachment process, including, but not limited to, A375 metastatic melanoma cells, beta 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 chick embryonic neurons, Sf9 insect cells, U251 glioma cells, vascular endothelial cells, vascular smooth muscle cells, and Vero cells.

[0261] In a preferred embodiment, the exfoliated cells are mammalian cells, preferably canine or human cells, most preferably human cells.

[0262] The microbial proteases of the present invention can be used in any type of stem cell detachment process. Thus, the cells to be detached can be totipotent stem cells (e.g., fertilized egg cells), pluripotent stem cells (e.g., embryonic stem cells), multipotent 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.

[0263] In one embodiment, the cells to be exfoliated are stem cell derivatives, preferably pluripotent stem cell derivatives, most preferably human pluripotent stem cell derivatives.

[0264] In some embodiments, the exfoliated cells are pluripotent stem cells, mesenchymal stem cells, beta cells, neurons, adipocytes, epithelial cells, or kidney cells.

[0265] In some embodiments, the detached cells are attached to a surface, such as a plastic or glass surface. In some embodiments, the detached cells are attached to another cell. In some embodiments, the attached cell is part of a cell cluster.

[0266] In some embodiments, the detached cells are attached to biomaterials, extracellular matrix (ECM)-coated surfaces, and / or other scaffolds made from 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.).

[0267] In some embodiments, the exfoliated cells are stem cells or stem cell derivatives. Preferably, the cells are pluripotent stem cells, induced pluripotent stem cells, (stem cell-derived) dopaminergic progenitor cells, or (stem cell-derived) beta cells.

[0268] In one embodiment, the detached cells are pluripotent stem cells, preferably human pluripotent stem cells. Preferably, the pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0269] In one embodiment, the detached cells are induced pluripotent stem cells, preferably human induced pluripotent stem cells. Preferably, the induced pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0270] In one embodiment, the detached cells are beta cells, preferably human beta cells. Preferably, the beta cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0271] In one embodiment, the detached cells are stem cell-derived beta cells, preferably human stem cell-derived beta cells. Preferably, the stem cell-derived beta cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0272] In one embodiment, the detached cells are dopaminergic progenitor cells, preferably human dopaminergic progenitor cells. Preferably, the dopaminergic progenitor cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0273] In one embodiment, the detached cells are stem cell-derived dopaminergic progenitor cells, preferably human stem cell-derived dopaminergic progenitor cells. Preferably, the stem cell-derived dopaminergic progenitor cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0274] In one embodiment, the detached cells are bone marrow-derived mesenchymal stem cells (BM-MSCs). Preferably, the bone marrow-derived mesenchymal stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0275] In one embodiment, the detached cells are canine cells, preferably Madin-Darby canine kidney cells. Preferably, the canine cells, more preferably Madin-Darby canine kidney cells, are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0276] In one embodiment, the detached cells are human embryonic kidney 293 (HEK293) cells. Preferably, the human embryonic kidney 293 cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0277] In one embodiment, the detached cells are adipose stromal cells. Preferably, the adipose stromal cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0278] Preferred Embodiments 1) The use of microbial proteases in the cell detachment process.

[0279] 2) The use according to embodiment 1, wherein 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.

[0280] 3) The use according to embodiment 2, wherein the P1 preference is determined according to Example 3 herein.

[0281] 4) The use according to any of embodiments 1 to 3, wherein the microbial protease exhibits chymotrypsin activity as determined according to the chymotrypsin activity assay described herein.

[0282] 5) The use according to any one of embodiments 1 to 4, wherein the microbial protease is a fungal protease or a bacterial protease.

[0283] 6) The use according to any one of embodiments 1 to 5, wherein the microbial protease is a Sarocladium protease, preferably a Sarocladium strictum protease.

[0284] 7) The use according to any one of embodiments 1 to 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.

[0285] 8) The use according to any of the preceding embodiments, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0286] 9) The use according to any one of embodiments 1 to 8, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0287] 10) The use according to any one of embodiments 1 to 5, wherein the microbial protease is a Nocardiopsis protease, preferably a Nocardiopsis prasina protease.

[0288] 11) The use according to any one of embodiments 1 to 5 and 10, 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:2.

[0289] 12) Use according to any of embodiments 1 to 5 and 10 to 11, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0290] 13) The use according to any one of embodiments 1 to 5 and 10 to 12, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0291] 14) Use according to any of embodiments 1 to 13, in which the cells are detached from the surface or from cell clusters.

[0292] 15) The use according to embodiment 14, wherein the surface is a plastic surface or a glass surface.

[0293] 16) The use according to any one of embodiments 1 to 15, wherein the cells are human cells.

[0294] 17) The use according to any one of embodiments 1 to 16, wherein the cells are stem cells or stem cell derivatives; preferably, the cells are pluripotent stem cells, induced pluripotent stem cells, (stem cell-derived) dopaminergic progenitor cells, or (stem cell-derived) beta cells.

[0295] 18) A composition suitable for cell detachment, comprising a microbial protease.

[0296] 19) The composition of embodiment 18, wherein 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.

[0297] 20) The composition according to any of embodiments 18-19, wherein the P1 preference is determined according to Example 3 herein.

[0298] 21) The composition of any of embodiments 18 to 20, wherein the microbial protease exhibits chymotrypsin activity as determined according to the chymotrypsin activity assay described herein.

[0299] 22) The composition according to any one of embodiments 18 to 21, wherein the microbial protease is a fungal protease or a bacterial protease.

[0300] 23) The composition of any one of embodiments 18 to 22, wherein the microbial protease is a Sarocladium protease, preferably a Sarocladium strictum protease.

[0301] 24) The composition of any of embodiments 18 to 23, 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.

[0302] 25) The composition of any of embodiments 18 to 24, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:1.

[0303] 26) A composition according to any one of embodiments 18 to 25, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0304] 27) The composition of any one of embodiments 18 to 22, wherein the microbial protease is a Nocardiopsis protease, preferably a Nocardiopsis prasina protease.

[0305] 28) The composition of any of embodiments 18 to 22 or 27, 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:2.

[0306] 29) The composition of any of embodiments 18-22 or 27-28, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:2.

[0307] 30) A composition described in any one of embodiments 18 to 22 or 27 to 29, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0308] 31) The composition of any of embodiments 18 to 30, wherein the microbial protease has a purity of at least 90%, 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 by weight of the total polypeptide material present in the composition.

[0309] 32) The composition according to any one of embodiments 18 to 31, which is a liquid composition.

[0310] 33) The composition according to embodiment 32, wherein the liquid composition is an aqueous composition.

[0311] 34) The composition of any of embodiments 32-33, wherein the liquid composition comprises an aqueous buffer; preferably, the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate, or bicarbonate; most preferably, the aqueous buffer comprises phosphate.

[0312] 35) The composition of any one of embodiments 32 to 34, having a pH value of 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.

[0313] 36) Microbial protease is 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 36. The composition of any of embodiments 32-35, wherein the hydroxybenzoates are present in an amount of about 1 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.

[0314] 37) The composition of any one of embodiments 32 to 36, further comprising ethylenediaminetetraacetic acid (EDTA); preferably, further comprising EDTA in an amount of about 0.01 mM to about 100 mM.

[0315] 38) Magnesium ion (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free of magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ 38. The composition of any one of embodiments 18-37, wherein the composition does not comprise:

[0316] 39) A method for cell detachment comprising contacting cells with a composition suitable for cell detachment comprising a microbial protease, wherein the cells are attached to a surface or another cell.

[0317] 40) The method of embodiment 39, wherein 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.

[0318] 41) A method according to any one of embodiments 39 to 40, wherein P1 preference is determined according to Example 3 herein.

[0319] 42) The method of any one of embodiments 39 to 41, wherein the microbial protease exhibits chymotrypsin activity as determined according to the chymotrypsin activity assay described herein.

[0320] 43) The method of any one of embodiments 39 to 42, wherein the microbial protease is a fungal protease or a bacterial protease.

[0321] 44) The method of any one of embodiments 39 to 43, wherein the microbial protease is a Sarocladium protease, preferably a Sarocladium strictum protease.

[0322] 45) The method of any one of embodiments 39 to 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.

[0323] 46) The method of any one of embodiments 39 to 45, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:1.

[0324] 47) A method according to any one of embodiments 39 to 46, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 1.

[0325] 48) The method of any one of embodiments 39 to 43, wherein the microbial protease is a Nocardiopsis protease, preferably a Nocardiopsis prasina protease.

[0326] 49) The method of any one of embodiments 39 to 43 or 48, 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:2.

[0327] 50) The method of any of embodiments 39-43 or 48-49, wherein the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:2.

[0328] 51) A method according to any one of embodiments 39 to 43 or 48 to 50, wherein the microbial protease is a variant of a fragment of SEQ ID NO: 2.

[0329] 52) The method of any one of embodiments 39 to 51, wherein the microbial protease has a purity of at least 90%, 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 by weight of the total polypeptide material present in the composition.

[0330] 53) The method of any one of embodiments 39 to 52, wherein the composition is a liquid composition.

[0331] 54) The method of embodiment 53, wherein the liquid composition is an aqueous composition.

[0332] 55) The method of any of embodiments 53-54, wherein the liquid composition comprises an aqueous buffer; preferably, the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate, or bicarbonate; most preferably, the aqueous buffer comprises phosphate.

[0333] 56) The method of any of embodiments 53 to 55, wherein the liquid composition has a pH value of 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.

[0334] 57) The microbial protease is 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 57. The method of any of embodiments 53-56, wherein the IL-14 is present in an amount of 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.

[0335] 58) The method of any one of embodiments 53 to 57, wherein the liquid composition further comprises ethylenediaminetetraacetic acid (EDTA); preferably, the composition further comprises EDTA in an amount of about 0.01 mM to about 100 mM.

[0336] 59) The liquid composition contains magnesium ions (Mg 2+) and / or calcium ions (Ca 2+ ) is substantially free of magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ 59. The method of any one of embodiments 53 to 58, wherein the method does not include

[0337] 60) A method according to any one of embodiments 39 to 59, wherein the cells are human cells.

[0338] 61) A method according to any one of embodiments 39 to 60, wherein the cell 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. [Example]

[0339] Materials and Methods Accutase desalting Lyophilized Accutase® XL (Sigma Aldrich, total volume) was dissolved in 25 mL of MilliQ water, and 10 mL was loaded onto a HiPrep 26 / 10 desalting chromatography column (Sigma Aldrich) equilibrated with 50 mM Tricine, 10 mM CaCl2, pH 7.5 at 10°C and a flow rate of 10 mL / min using an AEKTA explorer 100. Elution continued in the same buffer. The first peak fraction was collected and analyzed by absorbance at 280 nm (A 280 ) and stored at −20° C. Desalted Accutase was used for proteolytic activity assays (trypsin, chymotrypsin, collagenase type I, and collagenase type IV).

[0340] Trypsin activity assay The assay was performed in a 96-well format using a total well volume of 200 μL. The enzyme concentration ranged from 0.5 to 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. 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, add MilliQ water to 1 L). Samples were tested over a concentration range of 5 to 100 μg / mL. 15 μL of diluted sample was added to 185 μL of BAEE substrate solution. The reaction was monitored by absorbance (A) at 253 nm at 27 °C. 253 ) over a 10 minute period.

[0341] Chymotrypsin activity assay Assays were performed in a 96-well format using a total well volume of 200 μL. Enzyme concentrations ranged from 20 to 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) containing 17 mM β-cyclodextrin, pH 7.5. The final BTEE substrate solution was 1.72 mg / mL. Samples were diluted in 80 mM Tris (assay buffer), pH 7.5. Samples were tested over a concentration range of 50 to 500 μg / mL. 20 μL of diluted sample was added to 120 μL of assay buffer and 60 μL of BTEE substrate solution. The reaction was monitored by absorbance (A) at 256 nm at 27 °C. 256 ) over a 10 minute period.

[0342] Type I collagenase activity assay 30 μL of 1 mg / mL collagen FITC (Merck) suspended in 1 mM acetic acid, Dulbecco's phosphate-buffered saline (DPBS, Ca 2+ and Mg 2+Twenty microliters of enzyme at 0.25–7.5 μg / mL in PBS buffer (free of ATP; Merck) was mixed with 100 μL of 0.5 M Tricine (Sigma), pH 7.4. Reactions were carried out at 37°C and stopped 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 duplicates of 100 μL of supernatant at 485 nm excitation and 535 nm emission (detection).

[0343] Type IV collagenase activity assay 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), and the pH was adjusted with 10 μL of 0.5 M Tricine pH 7.4. After 15 min, the reaction was stopped by adding 105 μL of 10% trichloroacetic acid precipitation, incubated at 5°C for 10 min, and then centrifuged (13,000 g; 3 min). The supernatant was removed, and the precipitate was solubilized 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 sample was subjected to SDS-PAGE analysis using a gel, Tris / glycine / SDS buffer, and a Bio-Rad Precision Plus protein ladder. Enzyme activity was scored as no activity (0), less activity than Accutase (desalted sample) (-), equal (+) or greater activity (++). For an example, see Figure 1, where degradation of a first band at less than 200 kDa, a second band at 150 kDa, and a third band at 100 kDa was observed.

[0344] Protease activity assay The proteolytic activity of a polypeptide can be determined by a method utilizing the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, a blocked peptide that can be cleaved by endoproteases. After proteolytic cleavage, yellow free pNA molecules are released and can be measured by visible spectrophotometry at a wavelength of 405 nm. The Suc-AAPF-PNA substrate is manufactured, for example, by Bachem (catalog number L1400, dissolved in DMSO).

[0345] The sample containing the polypeptide to be analyzed is diluted in the remaining 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 absorbance is measured at 405 nm (A 405 ) every 20 seconds for 5 minutes. Samples should be diluted to a level where the slope is linear. The slope of the time-dependent absorbance curve (absorbance per minute) is directly proportional to the proteolytic activity of the polypeptide under a given set of conditions.

[0346] Assessment of cell detachment and cell cluster dissociation Evaluation of human pluripotent stem cell detachment and cluster dissociation Human pluripotent stem cells (hPSCs) were cultured as monolayers on laminin-flake tissue flasks (T-flasks) and passaged to form clusters in suspension. Cells were then cultured in suspension for up to eight passages. See Figures 2 and 3 for a schematic overview of the process and the metrics at which evaluations were performed.

[0347] Assessment of hPSC surface detachment and cluster formation (2D): Human pluripotent stem cells (induced or embryonic stem cells) were cultured in hPSC growth medium such as NutriStem hPSC XF (Sartorius, Germany) or StemFit Basic03 (Ajinomoto Co., Inc., Japan) for 3–4 days according to the individual culture protocol. On the day of passaging, the spent medium was removed from the T-flask and washed with washing buffer (Ca). 2+ and Mg 2+ PBS without HCl; 0.04-0.3 mL / cm 2 ) was added. The cell monolayer was washed to remove the buffer. 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, followed by pre-warming to room temperature and adding 0.5 mM ethylenediaminetetraacetic acid (EDTA). Accutase was pre-warmed to room temperature before cell detachment.

[0348] Add each cell detachment solution to the cell culture vessel (0.02–0.08 mL / cm). 2 , typically 0.04 mL / cm 2 ), and the vessel was incubated at 37°C for 3–20 minutes (typically 5–10 minutes). Growth medium supplemented with 10 μM Y-27632 (Tocris, United Kingdom) was added to the vessel, and the cell suspension was mixed by pipetting to obtain a single cell suspension. After surface detachment, the cells were centrifuged, resuspended in an appropriate volume, and counted using a NucleoCounter NC202 (ChemoMetec, Denmark). The total cell number, cell viability, and percentage of aggregation (% aggregation) were recorded according to [1 and 2]. The cells were then inoculated into shake flasks and placed in a shaking incubator. To assess the seeded cell suspension and viability on day 0, a 200 μL sample was taken and analyzed approximately 30–60 minutes after seeding.

[0349] Twenty-four hours after seeding, the fold change on day 1 after passage was determined to determine cluster formation efficiency. Cells within clusters were determined by processing the cell suspension, which was then analyzed using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [3]. Briefly, 100 μL of solution A was added to 100 μL of cell suspension and vigorously pipetted until single cells could be observed (visual confirmation, usually after 5–15 min). 100 μL of solution B was added to the suspension, and the cell suspension was analyzed using an NC200 (ChemoMetec, Denmark) cell counter. Cells were further cultured in suspension for up to 3 days. To estimate cell proliferation, the daily fold change was determined on day 3 after seeding using a NucleoCounter NC202 (ChemoMetec, Denmark) according to [2]. Cluster size and size distribution (coefficient of variation of cluster diameter) were determined on day 3 after passage 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.

[0350] [Table 1]

[0351] Dissociation of hPSC clusters in shake flasks and assessment of cluster re-formation (3D): A cell detachment solution containing the polypeptide of SEQ ID NO: 1 was prepared 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, followed by preheating to room temperature and adding 0.5 mM EDTA. Accutase was preheated to room temperature before cell detachment.

[0352] hPSCs were cultured as clusters in suspension in shake flasks in a shaking incubator for 3 or 4 days before passage. On the day of passaging, hPSC clusters were centrifuged to remove the supernatant and Ca 2+ and Mg2+ The cells were washed with PBS-free (0.1–1 mL per mL of the original working volume, typically 0.25 mL / mL). The centrifuge tube containing the cells was placed horizontally on an orbital shaker and incubated at 37°C for 3–15 minutes. Growth medium supplemented with 10 μM Y-27632 (Tocris, United Kingdom) was added to the centrifuge tube, and the cell suspension was mixed by pipetting to obtain a single cell suspension. After cell clusters were dissociated, the cells were centrifuged, resuspended in an appropriate volume of growth medium supplemented with 10 μM Y-27632 (Tocris, United Kingdom), and counted using a NucleoCounter NC202 (ChemoMetec, Denmark). The total cell number, cell viability, and percentage of aggregation (aggregation%) were recorded according to [1 and 2]. To estimate the cluster formation efficiency, the fold change on day 1 after passaging was determined. Cells within clusters were determined by using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [3]. To estimate cell proliferation, the daily fold change was determined 3 days after seeding according to [1]. The size and size distribution of clusters (coefficient of variation of cluster diameter) were determined 3 days after passaging using a Biorep Islet Cell Counter according to [4] and [5]. The evaluated parameters were scored according to Table 2 below, and the average score was calculated.

[0353] After the second passage at the cluster stage, the pluripotent phenotype was confirmed by staining with antibodies specific for the surface markers Oct4 (BD) and Nanog (Nordic BioSite AP) using fluorescence-activated single-cell sorting (FACS) analysis.

[0354] [Table 2]

[0355] Assessment of hPSC cluster dissociation in bioreactors (3D): 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, preheating the solution to 37°C, and adding 0.5 mM EDTA.

[0356] hPSCs were cultured as clusters in suspension in a bioreactor (10 L DASGip, Eppendorf, Germany) for 5 days. On the day of passaging, hPSC clusters were allowed to settle, the supernatant was removed, and Ca 2+ and Mg 2+ The cells were washed with PBS containing no ATP. After washing, the wash buffer was removed, cell detachment solution was added, and the cell clusters were incubated at 37°C for 3–5 minutes. Growth medium supplemented with 10 μM Y-27632 (Tocris, United Kingdom) 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). The total cell number, cell viability, and percentage of aggregation (% aggregation) were recorded according to [1 and 2].

[0357] Assessment of dopaminergic progenitor cell detachment in 2D Detachment of the monolayer of stem cell-derived dopaminergic progenitor cells was assessed at day 11 of the dopaminergic precursor stage (where they were partially differentiated). Prior to enzymatic treatment, spent medium was removed and washed with wash buffer (Ca). 2+ and Mg 2+ PBS without PBS: 0.04-0.3 / cm 2 ) was added to wash the cell layer, and the washing buffer was removed.

[0358] A cell detachment solution was prepared by dissolving the polypeptide 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. Accutase was preheated to room temperature. The corresponding enzyme solution was added to the cell culture vessel (0.04-0.3 mL / cm). 2The vessel was incubated at 37°C for 5–15 minutes until the cells appeared round and began to float as assessed by microscopy. Growth medium was added to the vessel, and the cell suspension was mixed by pipetting to obtain a suspension of single cells and small clusters. The cell suspension was analyzed for total cell number, cell viability, and percent aggregation (% aggregation) using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [3].

[0359] Dopaminergic progenitor cells on day 11 were reseeded and expanded and further matured until day 16. Cultures were assessed for reseeding efficiency on days 11 and 12. Attachment stability was assessed daily until day 16.

[0360] On day 16, dopaminergic progenitor cells were harvested with cell detachment solution and analyzed as described above. FACS analysis was used to confirm the phenotype of dopaminergic progenitor cells on day 16 by staining with antibodies specific for the surface markers FOXA2 and Otx2 (Miltenyi Biotech).

[0361] The evaluated parameters were scored according to Table 3 below and the mean score was calculated.

[0362] [Table 3]

[0363] Assessment of beta-cell cluster dissociation in 3D Stem cell-derived β-cell clusters (immature β-cells at the β-cell day 3 stage, BC03) were allowed to settle by gravity in a falcon tube. Spent medium was removed, and the clusters were washed with 10 ml of DPBS. After washing, the clusters were again allowed to settle 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 cold DPBS to a concentration of 4 μg / mg, followed by the addition of 0.5 mM EDTA and preheating to room temperature. Accutase was preheated to room temperature.

[0364] 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. A single cell suspension was secured by vigorously 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 number, and percent aggregation (% aggregation) were recorded according to [2]. Cells were centrifuged, resuspended in cryoprotectant, and then cryopreserved.

[0365] The β-cells were thawed and the cryoprotectant was washed off using growth medium with repeated washing and centrifugation cycles. Single cells were seeded into suspension culture in a shaking incubator and allowed to reaggregate.

[0366] Reaggregation was assessed 48 hours after seeding by counting free cells in suspension and calculating the percentage of seeded single cells that formed clusters. Additionally, the total volume of clusters in the sample was assessed by the cluster volume per 1^6 seeded cells (pIEQ). Cluster volume (pIEQ) was measured using a Biorep Islet Cell Counter [4, 5].

[0367] Parameters important for the dissociation and re-formation of β-cell clusters were assessed and scored according to Table 4, and the mean score was calculated.

[0368] [Table 4]

[0369] Evaluation of bone marrow-derived mesenchymal stem cell detachment in 2D Bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured in tissue-culture-treated 24-well 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 removing the wash buffer, preheated (37°C) cell detachment solution (6 μg / mL SEQ ID NO: 1 or Accutase in DPBS supplemented with 0.5 mM EDTA or DPBS supplemented with 0.5 mM EDTA; 0.2 mL) was added to the wells, followed by incubation at 37°C for 5 minutes. Then, 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. The cells in the suspension (0.2 mL) were counted using a NucleoCounter NC200 (ChemoMetec, Denmark), and the total cell number, cell viability, and percentage of aggregation (% aggregation) were recorded according to [6].

[0370] Assessment of Madin-Darby Canine Kidney Cell Detachment in 2D Madin-Darby canine kidney (MDCK) cells were cultured in tissue-culture-treated 24-well cell culture plates (NEST Biotechnology, China) using Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS). Prior to cell detachment, spent medium was removed from the wells and wash buffer (DPBS) was added. After removing the wash buffer, cell detachment solution (18 μg / mL SEQ ID NO: 1 in DPBS supplemented with 0.5 mM EDTA; Accutase; or DPBS supplemented with 0.5 mM EDTA as a negative control; preheated to 37°C; 0.2 mL) was added to the wells, followed by incubation at 37°C for 5 minutes. Then, 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. The cells in the suspension (0.2 mL) were counted using a NucleoCounter NC200, and the total cell number, cell viability, and percentage of aggregation were recorded according to [6].

[0371] Assessment of human embryonic kidney 293 cell detachment in 2D Human embryonic kidney 293 (HEK293) cells were cultured in tissue-culture-treated 24-well cell culture plates (NEST Biotechnology, China) in DMEM supplemented with 10% FBS. Prior to cell dissociation, spent medium was removed from the wells and wash buffer (DPBS) was added. After removing the wash buffer, cell detachment solution (6 μg / mL SEQ ID NO: 1 in DPBS supplemented with 0.5 mM EDTA; Accutase; or DPBS supplemented with 0.5 mM EDTA as a negative control; preheated to 37°C; 0.2 mL) was added to the wells and incubated at 37°C for 1 minute. Then, 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 the suspension (0.2 mL) were counted using a NucleoCounter NC200, and the total cell number, cell viability, and percentage of aggregation (% aggregation) were recorded according to [6].

[0372] Assessment of adipose stromal cell detachment in 2D Adipose stromal cells (ASCs) were grown in T75 Nunc flasks in alpha modified Eagle's minimum essential medium (α-MEM) supplemented with 5% human platelet lysate (hPL) for 48 hours. Prior to cell dissociation, spent medium was removed from the flasks and washed with PBS. After removing the wash buffer, cell detachment solution (5 μg / ml SEQ ID NO: 1 or TrypLE Select, Gibco) in DPBS supplemented with 0.5 mM EDTA was added to the wells, followed by incubation at 37°C for 4–5 minutes. Two volumes of growth medium were then added, and cells were harvested and resuspended by centrifugation. Cells were then counted using a NucleoCounter NC202 (ChemoMetec, Denmark), and the total cell number, cell viability, percentage of aggregation (aggregation %), and cell debris index were recorded according to [1, 2].

[0373] Example 1: Expression of SEQ ID NO: 1 (S1 protease from Sarocladium strictum) The gene encoding the S1 protease from Sarocladium strictum 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, confirmed to be identical to the sequence shown in SEQ ID NO:3, and transformed into Aspergillus oryzae strain BECh2 (WO 2000 / 39322) by the methods described by Christensen et al., 1988, Biotechnology 6, 1419-1422 and WO 2004 / 032648. Transformants were selected during regeneration from protoplasts based on their ability to utilize acetamide as a nitrogen source, conferred by a selectable marker in the expression vector, and then re-isolated under selection. Recombinant protease production was assessed by culturing transformants in 10 ml of YPG medium (WO 05 / 066338) in sterile plastic 30 ml tubes at 34°C for 4 days with shaking at 275 rpm. Samples were analyzed for protease activity using the pNA assay as described in WO 2004 / 072279 at pH 8 and monitoring expression by SDS-PAGE. Three transformants were selected for high levels of recombinant protease expression, and one of these was further selected because it gave the highest level of expression of the three when grown in 100 ml of YPG medium in a baffled 500 ml shake flask at 37°C for 4 days at 275 RPM. Recombinant expression was monitored by SDS-PAGE.

[0374] The selected transformants were fermented in 100 ml of FG4P medium (WO 1994 / 26925) in a 500 ml baffled shake flask with shaking at 250 rpm for 4 days at 30° C. The fermentation broth was then purified according to conventional methods well known to those skilled in the art to obtain SEQ ID NO:1.

[0375] Example 2: Expression of SEQ ID NO: 2 (S1 protease from Norcardiopsis prasina) A linear integration vector system was used for the expression cloning of the S1 protease from Norcardiopsis prasina. This linear integration construct was a PCR fusion product created by fusing the gene encoding the S1 protease from Norcardiopsis prasina (SEQ ID NO: 4) between two homologous Bacillus subtilis chromosomal regions, along with a strong promoter and a chloramphenicol resistance marker. The fusion was achieved by solid-state extension 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. Gene expression was controlled by a triple promoter system (as described in WO 1999 / 43835) consisting of promoters derived from the Bacillus licheniformis α-amylase gene (amyL), the Bacillus amyloliquefaciens α-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIA promoter containing stabilizing sequences. The gene encoding chloramphenicol acetyltransferase was used as a marker (e.g., 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 Bacillus chromosome by homologous recombination into the pectate lyase locus.

[0376] The gene encoding the S1 protease (SEQ ID NO: 4) from Norcardiopsis prasina was amplified from the strain's chromosomal DNA using gene-specific primers containing overhangs to two flanking vector fragments. Expression of the S1 protease was achieved 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 the genomic DNA of strain MB1361 (based on strain PL3598 described in patent application WO 2003095658). The two linear vector fragments and the gene fragment were assembled into a single linear vector construct by solid-state enzyme PCR. An aliquot of the PCR product was transformed into Bacillus subtilis. Transformants were selected on LB plates supplemented with 6 μg of chloramphenicol per ml.

[0377] One transformant containing a sequence confirmed to have incorporated the expression construct was grown in liquid culture on a rotary shaker in 500 mL baffled Erlenmeyer flasks each containing 100 mL of yeast extract-based medium for 4 days at 30° C. The fermentation broth was then purified according to WO 2004 / 111222 to obtain SEQ ID NO:2.

[0378] Example 3: Determination of protease specificity The substrate specificity of a protease can be defined according to its P1 preference, where the P1 position is defined as the amino acid residue located N-terminal to the cleavage site of the protease (Biochemical and Biophysical Research Communications, volume 27, issue 2, 20 April 1967, pages 157-162).

[0379] Preference is defined as the observed frequency being higher than that expected by random cleavage when counting the number of cleavage sites from protease digestion performed on a complex protein substrate containing high sequence diversity.

[0380] Specifically, purified protease samples are incubated on a 10 kDa cutoff spin filter with yeast protein extract (Promega V7341) for 16 hours at 37°C in 100 mM HEPES, 1 mM CaCl2, pH 7. Prior to incubation, the substrate is denatured by trichloroacetic acid (TCA) precipitation, reduced by the addition of dithiothreitol (DTT), and alkylated by the addition of iodoacetamide (IAA).

[0381] Three reactions were performed with protease:substrate ratios of 1:1250, 1:6250, and 1:30000. The resulting protease digests were collected after centrifugation by collecting the flow-through. Additional washes may be required to improve peptide recovery. The protease digests were acidified with TFA and directly analyzed by LC-MS / MS, e.g., Evosep One (Evosep) / timsTOF Pro (Bruker Daltonik).

[0382] To identify peptides, the data are searched against the UniProt yeast reference proteome using the Mascot search engine (Matrix science) with the following search parameters: Enzymes: None Peptide mass tolerance: ±25 ppm Fragment mass tolerance: ±0.05 Da Maximum number of missed cuts: 0 The N- and C-termini of the identified peptides are used to deduce protease cleavage sites. The extent of proteolysis must be sufficiently low to reflect the initial preferred cleavage site. Knowledge of the amino acid sequence of the protein from which the peptide is derived identifies the amino acids present at the subsite (e.g., P1) at the time of proteolytic cleavage.

[0383] Preference for an amino acid in a subsite (e.g., P1) is calculated by comparing the sum of the intensities of identified peptides containing this amino acid in the subsite (e.g., P1) to the prevalence expected in random cleavage of the protein.

[0384] 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 have increased P1 preferences for the amino acid residues Leu, Tyr, Phe, and Lys. When the P1 preferences of SEQ ID NO: 1 and SEQ ID NO: 2 were determined with all 20 common amino acids and then ranked, Leu, Tyr, Phe, and Lys were among the five most preferred amino acid residues at the P1 position.

[0385] Example 4: Proteolytic activity of SEQ ID NO: 1, SEQ ID NO: 2, and Accutase 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 enzymatic activities of SEQ ID NO: 1 and SEQ ID NO: 2 are reported relative to Accutase (see Table 5).

[0386] SEQ ID NO: 1 showed increased chymotrypsin activity and similar type IV collagenase activity compared to Accutase, while trypsin activity and type I collagen activity were decreased compared to Accutase.

[0387] SEQ ID NO:2 had similar chymotryptic activity compared to Accutase, but reduced tryptic and collagenolytic activity.

[0388] [Table 5]

[0389] Example 5: hPSC monolayer detachment and cluster formation (2D) of SEQ ID NO: 1 SEQ ID NO:1 and Accutase were evaluated and scored for hPSC monolayer detachment and cluster formation (Table 6).

[0390] [Table 6]

[0391] The overall performance of SEQ ID NO: 1 was improved compared to Accutase, in particular the rate of aggregation was reduced while the cluster formation efficiency was improved.

[0392] Example 6: Dissociation and re-formation of hPSC clusters with SEQ ID NO: 1 in shake flasks (3D) SEQ ID NO:1 and Accutase were evaluated and scored for dissociation and reformation of hPSC clusters (Table 7).

[0393] [Table 7]

[0394] The overall performance of SEQ ID NO: 1 was improved compared to Accutase. In particular, cell viability, cluster formation efficiency, and cell proliferation were improved. In addition, after two passages as clusters, the coefficient of variation of cluster diameter was comparable or improved for hPSCs treated with SEQ ID NO: 1 compared to hPSCs treated with Accutase.

[0395] Pluripotency was assessed after two passages as clusters. No reduction 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).

[0396] Example 7: Dissociation and re-formation of hPSC clusters with SEQ ID NO:2 in shake flasks (3D) SEQ ID NO:2 and Accutase were evaluated and scored for dissociation and reformation of hPSC clusters (Table 8). Compared to Accutase, SEQ ID NO:2 showed improved overall performance, particularly improved cluster reformation and cluster size uniformity.

[0397] [Table 8]

[0398] Pluripotency was assessed after two passages as clusters. 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).

[0399] Example 8: Dissociation of hPSC clusters with SEQ ID NO: 1 in a bioreactor (3D) SEQ ID NO: 1 was evaluated for dissociation of hPSC clusters in a bioreactor containing a starting volume of 5 L, yielding 4.81 x 10 6 A cell viability of 97% and an aggregation rate of 9.9% out of the total cell count of cells / mL were obtained.

[0400] Example 9: Detachment of dopaminergic progenitor cells with SEQ ID NO: 1 (2D) 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).

[0401] [Table 9]

[0402] The overall performance of SEQ ID NO: 1 was improved compared to Accutase. In particular, cell yield (as evidenced by total cell number and percentage of FOXA2 / OTX2 double-positive cells, see Table 10) was improved, while the rate of aggregation was reduced (also see Table 10). In addition, adhesion stability was improved compared to Accutase.

[0403] [Table 10]

[0404] Example 10: Dissociation of beta cell clusters by SEQ ID NO: 1 and SEQ ID NO: 2 (3D) BC03 stage beta cell clusters were dissociated using either SEQ ID NO: 1, SEQ ID NO: 2, or Accutase. After dissociation, cell viability and cell yield scores were recorded and then cryopreserved. After cryopreservation, BC03 cells were assessed and scored for their ability to undergo cluster re-formation. The total volume of clusters (10 6 The pIEQ (pI per seeded cells) was determined and scored.

[0405] The scores for 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, especially in cell yield and cluster re-formation compared to Accutase.

[0406] [Table 11]

[0407] Example 11: Detachment of bone marrow-derived mesenchymal stem cells (2D) SEQ ID NO: 1 and Accutase were evaluated for detachment of bone marrow-derived mesenchymal stem cells (BM-MSCs) in 2D culture (Table 12). The overall performance of SEQ ID NO: 1 was improved compared to Accutase, particularly with improved yield and reduced rates of clumping.

[0408] [Table 12]

[0409] Example 12: Madin-Darby Canine Kidney Cell Detachment in 2D SEQ ID NO:1 and Accutase were evaluated for detachment of Madin-Darby Canine Kidney (MDCK) cells in 2D culture (Table 13). The overall performance of SEQ ID NO:1 was improved compared to Accutase. In particular, yield was improved and the rate of clumping was reduced.

[0410] [Table 13]

[0411] Example 13: Detachment of Human Embryonic Kidney 293 Cells (2D) SEQ ID NO:1 and Accutase were evaluated for detachment of human embryonic kidney 293 (HEK293) cells in 2D culture (Table 14). The overall performance of SEQ ID NO:1 was improved compared to Accutase, particularly with improved yield and reduced number of clumps.

[0412] [Table 14]

[0413] Example 14: Dissociation of adipose stromal cells (3D) SEQ ID NO:1 and Accutase were evaluated for dissociation of adipose stromal cell (ASC) clusters in 3D culture (Table 15). Compared to TrypLE Select, SEQ ID NO:1 showed improved overall performance, particularly improved yield and reduced amount of non-viable cells (cell debris indicator).

[0414]

Table 15

[0415] References 1) ChemoMetec, Application Note No. 2026. Rev. 1.4. Count&Viability-Via2-Cassette (trademark) 2) ChemoMetec, Application Note No. 2028. Rev. 1.3. Aggregated Cells-Via2-Cassette (trademark) 3) ChemoMetec, Application Note No. 0215, Rev. 1.2. Counting Aggregated Cells using the Via1-Cassette (trademark) with Reagent A100 and B3 4) BioRep User manual for AUTOMATIC ISLET CELL COUNTER 4, Ref. ICC-04: 5) Fully Automated Islet Cell Counter (ICC) for the Assessment of Islet Mass, Purity, and Size Distribution by Digital Image Analysis. Peter Buchwald, Andres Bernal, Felipe Echeverri, Alejandro Tamayo-Garcia, Elina Linetsky and Camillo Ricordi. Cell Transplantation, Vol. 25, pp., pp. 1747-1761, 2016. 6) ChemoMetec, Application Note No. 0201, Rev. 1.6. Mammalian Cells-Viability and Cell Counting using the Via1-Casette (trademark)

Claims

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

2. 2. The composition of claim 1, wherein 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.

3. 3. The composition of any one of claims 1 to 2, 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, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:

1.

4. 4. The composition of any one of claims 1 to 3, 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, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:

2.

5. Use of microbial proteases in cell detachment processes.

6. The use of claim 5, wherein 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.

7. 7. The use according to any one of claims 5 to 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, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:

1.

8. 7. The use according to any one of claims 5 to 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, the microbial protease comprises, consists essentially of, or consists of SEQ ID NO:

2.

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

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

11. The use according to any one of claims 5 to 10, wherein the cells are mammalian cells, preferably human or canine cells.

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

13. The use according to any one of claims 5 to 11, wherein the cells are pluripotent stem cells, mesenchymal stem cells, beta cells, neurons, adipocytes, epithelial cells, or kidney cells.

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

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

16. 16. The method according to any one of claims 14 to 15, wherein the cell 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.

17. The method according to any one of claims 14 to 15, wherein the cells are pluripotent stem cells, mesenchymal stem cells, beta cells, neurons, adipocytes, epithelial cells, or kidney cells.

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