Alkaline protease with high enzyme activity and application thereof

Abstract

The application provides a basic protease with significantly improved enzyme activity. Specifically, the application provides a basic protease APRE-Y and a mature active form thereof, the enzyme activity of the basic protease APRE-Y is significantly improved compared with that of a wild-type basic protease, and the basic protease APRE-Y can still maintain high enzyme activity under neutral conditions. In addition, the thermal stability of the basic protease APRE-Y is significantly improved.

Description

Technical Field

[0001] This invention relates to the fields of protein engineering and industrial enzyme preparation technology, specifically to an alkaline protease with high enzyme activity and its applications. Background Technology

[0002] Alkaline protease (EC 3.4.21.62) is one of the most widely used industrial hydrolases globally, with applications in detergents, textile desizing, leather depilation, and food processing. Among them, serine proteases (subtilisin-like proteases) are the most important and common type of alkaline protease.

[0003] Existing wild-type alkaline proteases exhibit high activity under alkaline and high-temperature conditions, but their activity rapidly declines upon entering a neutral or weakly alkaline environment (pH 7.0-7.5), making them unsuitable for applications such as gentle washing, medical wound debridement, and food enzymatic hydrolysis. Furthermore, in industrial applications, the complex composition of reaction substrates demands high hydrolytic efficiency and resistance to inhibition from alkaline proteases. However, traditional directed evolution methods, due to their vast mutation space (up to 20 to the power of 275) and insufficient understanding of structure-function relationships, struggle to achieve synergistic optimization of activity, stability, and substrate profile. Therefore, providing a serine protease with a significantly different primary sequence from known proteases and maintaining high catalytic activity under neutral conditions, along with its supporting technical solutions, has significant industrial application value.

[0004] In recent years, deep learning sequence generation models (such as CARBonAra) can generate millions of high-quality mutant sequence libraries within seconds based on protein structure, while molecular docking technology can accurately assess the binding of enzymes to substrates. Both have opened up new avenues for the rational modification of enzyme proteins. However, there is currently no mature solution in the existing technology that deeply integrates these two technologies and systematically applies them to the synergistic enhancement of multiple properties of alkaline proteases.

[0005] In summary, there is an urgent need in this field to develop alkaline proteases with high catalytic activity, especially those that can maintain enzyme activity under neutral conditions. Summary of the Invention

[0006] The purpose of this invention is to provide an alkaline protease with high enzyme activity and its applications.

[0007] In a first aspect, the present invention provides an isolated polypeptide selected from the group consisting of: (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO:1 or 2; (2) A polypeptide formed by substituting, deleting or adding one or more amino acid residues, preferably 1-20, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue, or formed by adding a signal peptide sequence, and having alkaline protease activity. (3) A derivative polypeptide whose sequence contains the polypeptide sequence described in (1) or (2); and (4) A polypeptide whose amino acid sequence is ≥85% homology with the amino acid sequence shown in SEQ ID NO:1 or 2 (preferably ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%) and which has alkaline protease activity.

[0008] In another preferred embodiment, (3) is a fusion protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to (1) or (2).

[0009] In another preferred embodiment, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1 or 2.

[0010] In another preferred embodiment, the polypeptide is a serine protease.

[0011] In another preferred embodiment, the polypeptide is used to hydrolyze proteins.

[0012] In another preferred embodiment, under alkaline conditions, the enzyme activity of the polypeptide is ≥10000 U / mL, more preferably ≥15000 U / mL, more preferably ≥18000 U / mL, and most preferably ≥20000 U / mL.

[0013] In another preferred embodiment, the alkaline condition is a pH of 8.0-11.5.

[0014] In another preferred embodiment, under neutral conditions, the enzyme activity of the polypeptide is ≥8000 U / mL, more preferably ≥10000 U / mL, and even more preferably ≥12000 U / mL.

[0015] In another preferred embodiment, the neutral condition is a pH of 6.5-7.5.

[0016] In another preferred embodiment, the protein denaturation temperature Tm of the polypeptide is ≥60°C.

[0017] In another preferred embodiment, the polypeptide is chemically modified and / or immobilized.

[0018] In another preferred embodiment, the chemical modification is selected from the group consisting of acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, or combinations thereof.

[0019] In another preferred embodiment, the polypeptide is attached with a tag for detection and / or purification.

[0020] In another preferred embodiment, the tag is selected from the group consisting of His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, cMyc, ProfinityeXact, or combinations thereof.

[0021] In a second aspect, the present invention provides an isolated polynucleotide encoding a polypeptide as described in the first aspect of the present invention.

[0022] In another preferred embodiment, the polynucleotide is selected from the group consisting of: (a) A polynucleotide with a nucleotide sequence as shown in SEQ ID NO:9; (b) The nucleotide sequence is ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or ≥99.5% identical to the nucleotide sequence shown in SEQ ID NO:9, and encodes a polynucleotide of the polypeptide as described in the first aspect of the invention; (c) A polynucleotide that truncates or adds 1-60 (preferably 1-30, more preferably 1-10) nucleotides to the 5' and / or 3' ends of the nucleotide sequence shown in SEQ ID NO:9, and encodes a polypeptide as described in the first aspect of the invention; or (d) A polynucleotide complementary to any of the polynucleotides described in (a)-(c).

[0023] In another preferred embodiment, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO:9.

[0024] In another preferred embodiment, the polynucleotide further comprises, flanking the polypeptide, an auxiliary element selected from the group consisting of: signal peptides, secretory peptides, tag sequences (such as 6His), or combinations thereof.

[0025] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA sequences, RNA sequences, or combinations thereof.

[0026] In a third aspect, the present invention provides a carrier containing the polynucleotides as described in the second aspect of the present invention.

[0027] In another preferred embodiment, the carrier includes an expression carrier, a shuttle carrier, and an integration carrier.

[0028] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0029] In a fourth aspect, the present invention provides a genetically engineered host cell containing a vector as described in the third aspect of the present invention, or having a genome containing polynucleotides as described in the second aspect of the present invention.

[0030] In another preferred embodiment, the cell is a prokaryotic cell or a eukaryotic cell.

[0031] In another preferred embodiment, the host cell is a prokaryotic cell, such as Bacillus.

[0032] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

[0033] In another preferred embodiment, the host cell is Bacillus.

[0034] In another preferred embodiment, the host cell is Bacillus licheniformis (Bacillus licheniformis). Bacillus licheniformis ) or Bacillus subtilis ( Bacillus subtilis ).

[0035] A fifth aspect of the present invention provides a method for preparing a polypeptide as described in the first aspect of the present invention, comprising the following steps: (1) Under conditions suitable for protein expression, genetically engineered host cells as described in the fourth aspect of the present invention are cultured to obtain a culture containing the polypeptide; and (2) Optionally, isolate and / or purify the polypeptide from the culture.

[0036] In a sixth aspect, the invention provides the use of the polypeptide as described in the first aspect of the invention for hydrolyzing proteins or for preparing enzyme preparations of hydrolyzed proteins.

[0037] In a seventh aspect, the present invention provides an enzyme preparation comprising a polypeptide as described in the first aspect of the present invention.

[0038] In another preferred embodiment, the enzyme preparation is an immobilized enzyme preparation.

[0039] An eighth aspect of the present invention provides a method for hydrolyzing proteins, comprising the steps of: Proteins are hydrolyzed in the presence of a polypeptide as described in the first aspect of the invention or an enzyme preparation as described in the seventh aspect of the invention.

[0040] In another preferred embodiment, the hydrolyzed protein is carried out under alkaline conditions.

[0041] In another preferred embodiment, the alkaline condition is a pH of 8.0-11.5.

[0042] In another preferred embodiment, the protein hydrolysis is carried out under neutral conditions.

[0043] In another preferred embodiment, the neutral condition is a pH of 6.5-7.5.

[0044] In another preferred embodiment, the hydrolysis of protein is carried out at 30-65°C, more preferably at 35-60°C, and even more preferably at 40-55°C.

[0045] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0046] Figure 1 A schematic diagram of the pHY-300-ALK recombinant expression vector structure is shown.

[0047] Figure 2 The diagram shows a comparison of the enzyme activities of wild-type alkaline protease and APRE-Y under different conditions.

[0048] Figure 3 The relative enzyme activity curves of wild-type alkaline protease and APRE-Y at different temperatures are shown.

[0049] Figure 4 The image shows an amino acid identity comparison between APRE-Y and the most similar sequences in the NCBI nr database.

[0050] Figure 5 The diagram shows the domain partitioning of APRE-Y. Detailed Implementation

[0051] Through extensive and in-depth research and screening, the inventors have developed an alkaline protease with high enzyme activity and its applications. Specifically, this invention, through deep learning sequence design and molecular docking screening, obtained an alkaline protease APRE-Y with a nucleotide sequence significantly different from known alkaline proteases and its mature active form. The enzyme activity of APRE-Y is significantly improved compared to the wild-type alkaline protease, and it maintains high enzyme activity even under neutral conditions. Furthermore, the thermostability of APRE-Y is significantly improved. Based on these findings, the inventors completed this invention.

[0052] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0053] The polypeptide of the present invention As used herein, the terms "active polypeptide", "polypeptide of the present invention and its derivatives", "alkaline protease of the present invention", "enzyme of the present invention", and "alkaline protease APRE-Y" all refer to the polypeptide described in the first aspect of the present invention.

[0054] As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify the polypeptide using standard protein purification techniques. A substantially pure polypeptide will produce a single master band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using its amino acid sequence.

[0055] The active polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptides of the present invention can be dehydrated or non-dehydrated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0056] The present invention also includes fragments, derivatives, and analogs of the said polypeptide. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the said polypeptide.

[0057] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify the polypeptide or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0058] The preferred sequence of the polypeptide is the polypeptide shown in SEQ ID NO: 1 or 2. This term also includes variants and derived polypeptides having the same function as the polypeptide shown, with the sequence of SEQ ID NO: 1 or 2. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 150, preferably 130, more preferably 120, most preferably 110), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus.

[0059] For example, in the art, substitution with amino acids of similar or identical properties typically does not alter the function of a protein. Similarly, adding one or more amino acids to the C-terminus and / or N-terminus typically does not change the function of a protein. This terminology also includes the active fragments and active derivatives of the alkaline protease of the present invention. The present invention also provides analogues of the stated polypeptides.

[0060] These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include those having residues different from naturally occurring L amino acids (such as D amino acids), and those having non-naturally occurring or synthetic amino acids (such as β, γ amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0061] Modifications (typically without altering the primary structure) include: chemically derived forms of peptides, either in vivo or in vitro, such as acetylation or carboxylation. Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). They also include peptides modified to improve their resistance to proteolysis or optimize their solubility.

[0062] The amino or carboxyl terminus of the protein of this invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in this invention. For example, the tags may be FLAG, HA, HA1, cMyc, PolyHis, PolyArg, StrepTagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0063] To enable the translated protein to be expressed secretively (e.g., secreted extracellularly), a signal peptide sequence, such as pelB signal peptide, can be added to the amino terminus of the alkaline protease. The signal peptide can be cleaved during the secretion of the polypeptide from the cell.

[0064] The polynucleotides, vectors, and host cells of the present invention The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0065] The polynucleotides encoding the polypeptides described in this invention include: coding sequences that encode only the mature polypeptide; coding sequences of the mature polypeptide and various additional coding sequences; coding sequences of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0066] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0067] This invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring isoforms or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0068] The polypeptides and polynucleotides in this invention are preferably provided in isolated form and more preferably purified to homogenization. The full-length nucleotide sequence or fragments of the alkaline protease of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0069] Once the relevant sequence is obtained, recombinant proteins can be prepared using bioengineering methods. This typically involves cloning the polynucleotide sequence encoding the recombinant protein into an expression vector, then transforming the expression vector into cells for expression, harvesting and purifying the recombinant protein.

[0070] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0071] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing plasmids (or vectors) known in the art, transformed into cells, expressed, purified, and the protein obtained.

[0072] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0073] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the coding sequences of the vectors of the present invention or the polypeptides of the present invention or their derivatives, and methods for generating the polypeptides of the present invention by recombinant technology.

[0074] Using conventional recombinant DNA techniques, the polynucleotide sequences of this invention can be used to express or produce recombinant polypeptides of this invention or their derivatives. Generally, the following steps are involved: (1) Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the polypeptide of the present invention or a derivative thereof, or with a recombinant expression vector containing the polynucleotide; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0075] In this invention, the polynucleotide sequence encoding the polypeptide or a derivative thereof can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0076] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the polypeptide or a derivative thereof of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0077] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0078] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0079] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: bacterial cells of Escherichia coli, Streptomyces, and Chlamydia trachomatis; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0080] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0081] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0082] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0083] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0084] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0085] The main advantages of this invention include: (1) The present invention provides an alkaline protease APRE-Y and its mature active form that are significantly different from the primary sequence of existing alkaline proteases. The alkaline protease has only about 52.4% full-length similarity to the known AprE reference protein.

[0086] (2) The alkaline protease APRE-Y of the present invention has significantly higher enzyme activity than wild-type alkaline protease, with enzyme activity increased by 35.7% under the conditions of 40°C and pH 10.5.

[0087] (3) The alkaline protease APRE-Y of the present invention can still maintain high enzyme activity under neutral conditions, and its pH 7.4 / pH 10.5 activity ratio is about 3.15 times that of the wild type.

[0088] (4) The thermal stability of the alkaline protease APRE-Y of the present invention is significantly improved, and its Tm is 6°C higher than that of wild-type alkaline protease.

[0089] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0090] Example 1: Design of alkaline protease based on deep learning 1.1 Sequence Design and Computational Screening Using the wild-type M-protease (aprE, amino acid sequence as shown in SEQ ID NO:10) with UniProtKB accession number Q99405 as the target for modification, the crystal structure was first predicted using AlphaFold2 based on its amino acid sequence. Then, the deep learning model Carbonara was used to generate at least 1,000,000 sequences, which were scored by the model and the top 20,000 high-scoring sequences were retained. The structure of each of these sequences was predicted using AlphaFold2, and Rosetta Ligand semi-flexible docking was performed using AAPF (Suc-Ala-Ala-Pro-Phe-pNA) as the ligand. The binding free energy (ΔΔG) between each candidate sequence and the substrate was calculated. Based on a threshold of ΔΔG ≤ -1.5 kcal / mol, 1,200 candidates were screened out. The catalytic pocket was then checked for changes, and finally, 20 sequences with unchanged pockets and high binding affinity were retained for further testing.

[0091] 1.2 Recombinant Expression of Candidate Proteins (1) Synthesis of candidate genes The 20 candidate amino acid sequences obtained from screening were reverse translated into corresponding coding nucleotide sequences, and the corresponding coding genes were artificially synthesized. During synthesis, an NdeI restriction site matching the expression backbone was introduced at the 5′ end of each coding gene, and a stop codon TAA and a NotI restriction site were introduced sequentially at the 3′ end.

[0092] (2) Construction of candidate protein recombinant expression vectors The pHY-300-ALK recombinant expression vector (structure shown in Figure 1) was constructed and preserved in the laboratory. Figure 1 (As shown) serves as the expression backbone. The pHY-300-ALK expression backbone contains a promoter, a signal peptide coding sequence, and a propeptide coding sequence for driving the secretion and expression of alkaline protease in Bacillus licheniformis, all of which are located upstream of the mature peptide coding region.

[0093] When constructing recombinant expression vectors for each candidate protein, the promoter, signal peptide coding sequence, and propeptide coding sequence in the pHY-300-ALK expression backbone were retained, and the mature peptide coding region in the original expression backbone was replaced with the coding gene of the corresponding candidate protein.

[0094] The candidate protein-coding genes and the pHY-300-ALK expression backbone were double-digested using restriction endonucleases NdeI and NotI, respectively. After digestion, agarose gel electrophoresis was performed to recover the target gene fragment and the linearized vector fragment, respectively. T4 DNA ligase was used to ligate the recovered target gene fragment and the linearized vector fragment, ensuring that the candidate protein-coding sequence maintained the correct reading frame with the upstream propeptide-coding sequence, yielding the corresponding ligation products.

[0095] (3) E. coli transformation and recombinant plasmid verification The above ligation products were transformed into Escherichia coli DH5α competent cells, and after recovery, they were plated on LB solid medium containing 20 μg / mL tetracycline and cultured overnight at 37°C.

[0096] DNA was extracted from the *E. coli* strains, and the coding sequences of candidate proteins were amplified by PCR. The PCR products were subjected to agarose gel electrophoresis, and transformants exhibiting the desired target band size were selected for amplification and plasmid extraction. The resulting plasmids were sequenced, covering the complete coding region of the candidate proteins and the junctions between the coding regions and the vector. Sequencing results showed that the coding regions and junctional regions of each candidate protein were consistent with the designed sequences.

[0097] (4) Construction of recombinant Bacillus licheniformis The recombinant plasmids of candidate proteins, validated by sequencing, were electroporated into Bacillus licheniformis DW2 competent cells: 60 μL of Bacillus licheniformis DW2 competent cells were mixed with 1 μL of recombinant plasmid at a concentration of 50 ng / μL, and transferred to a pre-chilled 2 mm electroporation cuvette. The mixture was placed on ice for 1–1.5 min. Electroporation was performed at 21 kV / cm for 4.5–5 ms. Immediately after electroporation, 1 mL of recovery medium was added, and the cells were incubated at 37°C for 3 h.

[0098] After recovery culture, the culture medium was spread onto LB solid medium containing 20 μg / mL tetracycline and incubated overnight at 37°C. Transformants were picked and subjected to colony PCR verification. Transformants that obtained the target band of the expected size were recombinant Bacillus licheniformis containing the corresponding candidate protein coding gene.

[0099] 1.3 Fermentation expression and iterative screening of candidate proteins Recombinant Bacillus licheniformis containing different candidate protein-coding genes were inoculated into liquid culture medium containing 20 μg / mL tetracycline and activated by shaking culture at 37°C. The activated seed culture was then transferred to fermentation medium at a 1% inoculation rate and fermented at 37°C and 230 rpm for 48 h. The fermentation medium consisted of 45 g / L corn starch, 50 g / L soybean meal, 6 g / L calcium carbonate, and 5 g / L ammonium sulfate, with the culture medium at its natural pH. After fermentation, the supernatant was collected by centrifugation, and the protease activity in the supernatant of each recombinant strain was measured.

[0100] Gaussian process regression (GPR) surrogate models were constructed using the obtained candidate protease activity experimental data, and batch Bayesian optimization (q=20) was used to determine the candidate sequences to be experimentally validated in the next round. The coding genes of the candidate sequences in the next round were synthesized, recombinant expression vectors were constructed, Bacillus licheniformis was transformed, fermented and expressed, and enzyme activity was measured according to the above methods.

[0101] After three rounds of iteration, the optimal sequence APRE-Y was obtained. Its amino acid sequence, as shown in SEQ ID NO:1, consists of 269 amino acids, and its nucleotide sequence, as shown in SEQ ID NO:9, is 807 bp in length and does not contain a stop codon. The recombinant plasmid APRE-Y was named pHY-300-APRE-Y, and the positive transformant containing the recombinant plasmid pHY-300-APRE-Y was named recombinant Bacillus licheniformis DW2 / pHY-300-APRE-Y.

[0102] Example 2: Enzyme activity assay of APRE-Y 2.1 Recombinant Expression of APRE-Y Recombinant Bacillus licheniformis expressing wild-type alkaline protease (amino acid sequence as shown in SEQ ID NO:10) and APRE-Y (amino acid sequence as shown in SEQ ID NO:1) were inoculated into liquid culture medium containing antibiotics, activated by shaking at 37°C, and then transferred to fermentation medium at an inoculum of 1%. Fermentation was carried out at 37°C and 230 rpm for 48 h. The supernatant was collected by centrifugation to obtain fermentation broth containing wild-type alkaline protease or APRE-Y. After purification, pure wild-type alkaline protease or APRE-Y was obtained.

[0103] 2.2 Enzyme activity assay APRE-Y protease activity was determined according to GB / T 23527.1-2023, using casein as a substrate. APRE-Y enzyme activity was measured at 40℃ and pH 10.5, and APRE-Y enzyme activity and protein denaturation temperature (Tm) were simultaneously measured at pH 7.4. The pH 7.4 / pH 10.5 activity ratio was calculated. Wild-type alkaline protease was used as a control, and each treatment was repeated at least three times.

[0104] The results are as follows Figure 2 As shown in Table 1, at 40℃ and pH 10.5, the enzyme activity of APRE-Y was 22158 U / mL, which was 35.7% higher than that of wild-type alkaline protease (16327 U / mL). Under neutral conditions at pH 7.4, the enzyme activity of APRE-Y was 8365 U / mL, which was 327.2% higher than that of wild-type alkaline protease (1958 U / mL), and its pH 7.4 / pH 10.5 activity ratio was approximately 3.15 times that of the wild type. In addition, the Tm of APRE-Y was 61℃, which was 6℃ higher than that of wild-type alkaline protease.

[0105] In summary, APRE-Y not only exhibits significantly higher enzyme activity under alkaline conditions than wild-type alkaline protease, but also maintains high enzyme activity under neutral conditions. Furthermore, APRE-Y has a higher denaturation temperature, making it suitable for reaction conditions requiring higher temperatures. Table 1 Performance comparison between wild type and APRE-Y Example 3: pH and temperature characteristics of APRE-Y 3.1 pH characteristics At 40°C, the pH of the reaction system was adjusted to 7.0-11.5 with buffer solution, and the relative enzyme activities of wild-type alkaline protease and APRE-Y were measured (with the enzyme activity at pH 10.5 as 100%).

[0106] The results are shown in Table 2. At pH 7.0 and pH 11.5, the relative enzyme activity of APRE-Y was significantly higher than that of wild type, and the difference was more obvious under neutral conditions, indicating that APRE-Y has a wider pH tolerance range.

[0107] Table 2. Relative enzyme activities (%) of wild type and APRE-Y under different pH conditions 3.2 Temperature characteristics The relative enzyme activities of wild-type alkaline protease and APRE-Y at different temperatures (40-75℃) were determined at pH 10.5 (with the enzyme activity at 40℃ as 100%).

[0108] The results are as follows Figure 3 As shown, at temperatures ranging from 40 to 70°C, the relative enzyme activity of APRE-Y was higher than that of the wild type. Specifically, at 70°C, APRE-Y still retained 42% of its enzyme activity, while the wild type only retained 28%.

[0109] Example 4: Determination of kinetic parameters of APRE-Y Using AAPF as a substrate, its concentrations were set to 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 1.0, 1.5, and 2.0 mM, respectively. The enzymatic kinetic parameters of wild-type alkaline protease and APRE-Y were determined at 40℃ and pH 10.5.

[0110] The results are shown in Table 3. The Km of APRE-Y was 0.28 mM, which was 20% lower than that of the wild type, indicating that APRE-Y had a stronger affinity for the substrate. The kcat of APRE-Y was 155 s. -1 The performance improved by 40.9%; kcat / Km was 554 s. -1 ·mM -1 It is about 0.8 times higher than the wild type.

[0111] In summary, the increase in APRE-Y enzyme activity is mainly based on improved affinity for substrates and improved catalytic efficiency, with catalytic efficiency being the most critical factor.

[0112] Table 3 Kinetic parameters of wild type and APRE-Y Example 5: Homology alignment of APRE-Y with known sequences The amino acid sequence of APRE-Y (SEQ ID NO:1) was submitted to the NCBI BLASTp program and compared with known protein sequences in the nr database.

[0113] The comparison results are as follows Figure 4 As shown, the sequences with the highest similarity to APRE-Y all belong to the S8 family of serine proteases (subtilisin class). The amino acid identity of the first eight sequences is 50.8%-53.2%, with sequence coverage all above 97% and E values ​​all below 1×10⁻⁶. -83 The one with the highest similarity is Halalkalibacter okhensis The S8 family peptidase from the source (NCBI accession number WP_034632645) showed a consistency rate of 53.2%. Next was... Lederbergia lenta (Original Bacillus clausii The identity of the subtilisin BL (NCBI accession number P29599) source was also 53.2%, with a sequence coverage of 97.8%.

[0114] Based on the above results, APRE-Y has an amino acid similarity of less than 53.2% with known sequences in the database, indicating a significant difference. It is a newly designed sequence rather than a simple mutation of a known protein.

[0115] Example 6: APRE-Y domain partitioning N-terminal sequencing and / or LC-MS analysis of APRE-Y were performed to further verify the processing and excision of the region 1-30 of SEQ ID NO:1 during expression / maturation, confirming the amino acid sequence of the actual mature active protein as shown in SEQ ID NO:2 (31-269 of SEQ ID NO:1, 239 aa).

[0116] Furthermore, based on the sequence characteristics and structural predictions of APRE-Y, it was divided into an N-terminal basic region (domain I) and a subtilisin-like catalytic core (domain II, containing subdomains IIa-IIe), as detailed in Table 4 and [other tables]. Figure 5 As shown.

[0117] Among them, domain I (N-terminal basic region) is located at positions 1-30. This region may affect expression, folding, secretion, processing or local electrical properties and is cleaved in mature active proteins.

[0118] Subdomain IIb is the catalytic core region (positions 71-120, 50 aa), containing the catalytic triplet histidine H62, adjacent histidines H65 / H66, and W111, which may participate in substrate aromatic stacking. Subdomain IIc S1 is the pocket region (positions 121-180, 60 aa), containing the S1 pocket aromatic residue W160 (related to substrate P1 site recognition) and proline residues P157, P162, and P166 distributed in the surface loop. Subdomains IIb and IIc S1 together constitute the catalytic and substrate recognition core region of APRE-Y.

[0119] Subdomain IIa (positions 31-70, 40 aa) is the N-terminal β-sheet region, containing the catalytic triplet aspartic acid D32, and positions 61-65 are the GHGTH motif (a marker of the subtilisin family). Subdomain IId (positions 181-230, 50 aa) is the C-terminal helical region, containing the catalytic triplet serine S215. Subdomain IIe (positions 231-269, 39 aa) is the C-terminal tail region.

[0120] Table 4 APRE-Y domain partitioning All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An isolated polypeptide, characterized in that, The polypeptide is selected from the following group: (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO:1 or 2; (2) A polypeptide formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:1 or 2, or formed by adding a signal peptide sequence, and having alkaline protease activity. (3) A derivative polypeptide whose sequence contains the polypeptide sequence described in (1) or (2); and (4) A polypeptide whose amino acid sequence is ≥85% homologous to the amino acid sequence shown in SEQ ID NO:1 or 2 and has alkaline protease activity.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or 2.

3. The polypeptide according to claim 1, characterized in that, Under pH conditions of 6.5-7.5, the enzyme activity of the polypeptide is ≥8000 U / mL.

4. An isolated polynucleotide, characterized in that, The polynucleotide encodes the polypeptide as described in claim 1.

5. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 4.

6. A genetically engineered host cell, characterized in that, The host cell contains the vector as described in claim 5, or its genome is integrated with the polynucleotide as described in claim 4.

7. The method for preparing the polypeptide according to claim 1, characterized in that, Includes the following steps: (1) Under conditions suitable for protein expression, the genetically engineered host cells as described in claim 6 are cultured to obtain a culture containing the polypeptide; and (2) Optionally, isolate and / or purify the polypeptide from the culture.

8. The use of the polypeptide as described in claim 1, characterized in that, Enzyme preparations used for hydrolyzing proteins or for preparing hydrolyzed proteins.

9. An enzyme preparation, characterized in that, The enzyme preparation comprises the polypeptide as described in claim 1.

10. A method for hydrolyzing proteins, characterized in that, Including the following steps: The protein is hydrolyzed in the presence of the polypeptide as described in claim 1 or the enzyme preparation as described in claim 9.

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