Alkaline serine protease and use thereof

Abstract

The application relates to the fields of marine technology and enzyme technology, and particularly discloses a kind of alkaline serine protease and application thereof.A kind of brand-new alkaline serine protease is successfully mined by using marine-derived microorganism metagenome database, the limitation of conventional microorganism separation culture is overcome, and the source selection range of alkaline serine protease is greatly widened.Moreover, the alkaline serine protease has excellent protease activity and detergent tolerance, and can be effectively used for protein degradation or used as a detergent component.

Description

Technical Field

[0001] This invention relates to the fields of marine technology and enzyme technology, and in particular to an alkaline serine protease and its applications. Background Technology

[0002] Alkaline serine proteases are widely distributed in animals, plants, and microorganisms, and are an important group of industrial enzymes. They can hydrolyze peptide bonds in ternary structures using the classic Ser / His / Asp catalysis. Due to the species and chemical diversity of microbial alkaline proteases, these enzymes have been widely used in many fields, including silk processing, leather manufacturing, food industry, pharmaceutical formulation, silver recovery, detergents, and waste treatment.

[0003] In the detergent industry, alkaline serine protease is one of the earliest industrial enzyme preparations used. It can degrade large protein molecules in stains such as blood, milk stains, egg yolk, fruit juice, sweat stains, and coffee into smaller molecular fragments, thus facilitating stain removal. With the increasing prevalence of biological enzyme preparations in cleaning products, alkaline serine protease has been gradually introduced into laundry detergents, washing powders, laundry pods, industrial cleaning agents, and medical device cleaning agents. However, the surfactants commonly found in detergents easily inactivate proteases, severely limiting their further application in detergents. Therefore, developing novel alkaline serine proteases with high stability in detergent systems has become a pressing technical problem for the industry.

[0004] Currently, there are two main approaches to improving the stability of alkaline serine proteases in detergents: one is to perform molecular modification and optimization of known proteases; the other is to screen new protease-producing microbial strains from the natural environment.

[0005] However, both approaches have significant limitations. While modifying known enzymes can yield mutants with improved stability, the effects of these mutants vary, and consistently effective mutants cannot be obtained. Screening from environmental samples using traditional microbial isolation and culture methods fails to fully utilize the resource potential of uncultured or difficult-to-culture microorganisms because the proportion of culturable microorganisms is less than 1%, and the stability of the target proteases obtained in detergent environments often fails to meet practical application requirements. Therefore, the industry still needs to seek new technological means to obtain novel basic serine proteases with good stability and high activity in detergent systems. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide an alkaline serine protease and its applications. This invention successfully mined a novel alkaline serine protease using a marine microbial metagenomic database, overcoming the limitations of conventional microbial isolation and culture, and greatly broadening the range of sources for alkaline serine proteases. Moreover, this alkaline serine protease exhibits excellent protease activity and detergent tolerance, and can be effectively used for protein degradation or as a detergent component.

[0007] A first aspect of the present invention provides an alkaline serine protease, said alkaline serine protease comprising:

[0008] (1) An alkaline serine protease having the sequence shown in SEQ ID NO:1; or

[0009] (2) A basic serine protease variant that has the same function as the sequence shown in SEQ ID NO:1, based on the sequence shown in SEQ ID NO:1, by substitution and / or deletion and / or addition of one or more amino acids.

[0010] In some embodiments of the present invention, the basic serine protease comprises a functional fragment obtained by the deletion of one or more amino acids.

[0011] In this invention, the term "functional fragment" refers to the smallest sequence fragment capable of maintaining at least one function of the alkaline serine protease. "Maintaining function" means not losing that function, including: increasing or decreasing the function, but not completely losing it. Generally, such maintenance of function is based on conserved amino acid substitutions (such as amino acid substitutions achieved through conventional substitution methods, such as alanine scanning). In this invention, "maintaining function" includes maintaining at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the original function (or activity). In this invention, the function includes: protein degradation, environmental (such as temperature, pH, and detergent environment) adaptability and tolerance.

[0012] In some embodiments of the present invention, the basic serine protease has the amino acid sequence shown in SEQ ID NO:1.

[0013] In some embodiments of the present invention, the basic serine protease variant described in (2) has at least 70% identity with the sequence shown in SEQ ID NO:1.

[0014] In this invention, the term "sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and (if necessary) vacancies are introduced to achieve maximum sequence identity, and no conserved substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed by various methods known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art can determine appropriate parameters for determining the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0015] In some embodiments of the present invention, the sequence identity can reach 70%, 71%, 72%, 73%, 74%, 75%, 76%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0016] In some embodiments of the present invention, the alkaline serine protease variant described in (2) contains the sequence shown in SEQ ID NO:1, i.e., the sequence identity is 100%.

[0017] In some embodiments of the present invention, the basic serine protease variant is a variant obtained by adding at least one amino acid residue to SEQ ID NO:1.

[0018] In some embodiments of the present invention, the basic serine protease variant comprises an amino acid sequence obtained by further adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues to SEQ ID NO:1.

[0019] In this invention, the terms "alkaline serine protease," "alkaline serine protease variant," "variant," and "alkaline serine protease" are used interchangeably and refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be dispersed with non-amino acids. These terms also cover amino acid polymers that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipoylation, acetylation, phosphorylation, or any other operation or modification. This definition also includes, for example, polypeptides containing one or more amino acid analogs, including but not limited to non-natural amino acids, and other modifications known in the art.

[0020] In some embodiments of the present invention, the alkaline serine protease or alkaline serine protease variant is further provided with functional elements or chemically modified.

[0021] In some embodiments of the present invention, the functional element or chemical modification can improve at least one of the following aspects: selectivity, stability, solubility, binding activity, and cytotoxicity.

[0022] In some embodiments of the present invention, the functional elements or chemical modifications may also include: tag sequences (such as His-tag, GST-tag, maltose-binding protein tag, Strep-tag, c-Myc tag, HA tag, FLAG tag, V5 tag, AviTag, SNAP-tag, SUMO tag), targeting peptides, dyes, biotin, and affinity ligands.

[0023] In some embodiments of the present invention, the functional elements include, but are not limited to: cyclic peptide structures, fatty acid groups, cell-penetrating peptides, PEG, albumin, etc.

[0024] In some embodiments of the present invention, the chemical modification includes: phosphorylation, acetylation, methylation, ubiquitination or ubiquitination-like processes, glycosylation, esterification, cyclization, enzymatic cleavage, formation of disulfide bonds, and hydroxylation.

[0025] In some embodiments of the present invention, the alkaline serine protease is derived from marine animals or marine microorganisms.

[0026] In some embodiments of the present invention, the method of obtaining the alkaline serine protease is not limited, and it can be obtained by any conventional method in the art, including but not limited to: solid-phase synthesis, bioextraction, biosynthesis, etc.

[0027] In some embodiments of the present invention, the biosynthesis includes high-density fermentation production achieved through a heterologous expression system.

[0028] A second aspect of the invention provides a nucleic acid molecule encoding the basic serine protease described in the above aspect.

[0029] In some embodiments of the present invention, the nucleic acid molecule includes:

[0030] (1) A nucleic acid molecule having the sequence shown in SEQ ID NO:2;

[0031] (2) Nucleic acid molecules with sequences different from those in (1) obtained based on degenerate codons;

[0032] (3) Nucleic acid molecules obtained by sequence optimization based on the nucleic acid molecules in (1) and (2).

[0033] In this invention, the sequence optimization can be aimed at expression level, stability, translation efficiency, or for the purpose of facilitating chemical synthesis and cloning.

[0034] In some embodiments of the present invention, the sequence optimization includes at least one of: species-based codon optimization, GC content adjustment, secondary structure elimination, chemical modification, and introduction of regulatory elements or linkers.

[0035] In this invention, the term "codon optimization" refers to the process of replacing at least a portion of codons with the host's optimal codons based on the different usage frequencies (codon preference) of different organisms for codons encoding the same amino acid. This optimization can greatly improve translation speed and accuracy.

[0036] In some embodiments of the present invention, the sequence optimization is species-based codon optimization.

[0037] In some embodiments of the present invention, the sequence optimization is based on codon optimization of Bacillus subtilis. It should be understood that the species selection can be determined based on the expression vector or expression system used, without requiring additional inventive effort. In this invention, the Bacillus subtilis secretory expression system is used only as an example and does not imply that the scope of protection is limited to this.

[0038] In some embodiments of the present invention, the nucleic acid molecule is a nucleic acid molecule with the sequence shown in SEQ ID NO:2.

[0039] A third aspect of the present invention provides a biomaterial comprising at least one of the following:

[0040] (1) An expression vector containing the nucleic acid molecules described above;

[0041] (2) Transformants containing the nucleic acid molecules described above;

[0042] (3) Transformants containing the expression vector in (1).

[0043] In some embodiments of the present invention, the expression vector includes a plasmid.

[0044] In this invention, there are no restrictions on the type of plasmid, and any conventional plasmid vector in the art can be used.

[0045] In some embodiments of the present invention, the transformants include bacteria, fungi, viruses, plant cells, or animal cells.

[0046] In some embodiments of the present invention, the plant or animal cells do not involve reproductive material.

[0047] In some embodiments of the present invention, the biomaterial further includes processed products based on the above-mentioned transformants, including but not limited to: live products of the transformants, inactivated transformants, attenuated transformants, cultures of transformants, metabolites, concentrated or dried products, or compositions containing them.

[0048] In some embodiments of the present invention, the culture includes culture supernatant or fermentation product.

[0049] In some embodiments of the invention, the product is provided in liquid or solid form.

[0050] In some embodiments of the present invention, the product may be in the form of powder, solution, paste, gel, tablet, block, capsule / bead / sphere, spray, foam, etc.

[0051] In some embodiments of the present invention, the product further comprises one or more excipients or carriers.

[0052] In some embodiments of the present invention, the excipients include, but are not limited to, at least one of the following: detergent builders / chelating agents (such as phosphates (e.g., sodium tripolyphosphate), zeolites, sodium citrate, ethylenediaminetetraacetic acid, layered sodium silicate, etc.), bleaching agents (sodium perborate, sodium percarbonate, sodium hypochlorite, tetraacetylethylenediamine, etc.), fluorescent whitening agents, softeners / antistatic agents (such as quaternary ammonium compounds), fragrances, thickeners / rheology modifiers (sodium chloride, polyacrylic acid polymers, cellulose derivatives, etc.), solvents (water, ethanol, propylene glycol, etc.), fillers (sodium sulfate, sodium carbonate, etc.), anti-redeposition agents (sodium carboxymethyl cellulose, polyvinylpyrrolidone, polyacrylic acid polymers, etc.), pH adjusters / buffers (citric acid, sodium carbonate, sodium silicate, etc.), and preservatives (isothiazolinones, sodium benzoate, etc.).

[0053] A fourth aspect of the present invention provides a composition comprising the alkaline serine protease and / or biological material described in the above aspects.

[0054] In some embodiments of the present invention, the composition further contains excipients.

[0055] In some embodiments of the invention, the excipients are as defined above.

[0056] In some embodiments of the present invention, the composition further contains a second active substance.

[0057] In some embodiments of the present invention, the second active substance further includes an active substance used in at least one of the following fields: detergent industry, food industry, leather and textile industry, pharmaceutical industry, biosynthesis, feed preparation, and environmental recycling.

[0058] In some embodiments of the present invention, the second active substance is an enzyme or a surfactant.

[0059] In some embodiments of the present invention, the enzyme includes at least one of a protease (a protease other than the basic serine protease of the present invention), a lipase, an amylase, or a cellulase.

[0060] A fifth aspect of the invention provides the use of the basic serine protease, biomaterials, and compositions described above in protein degradation.

[0061] A sixth aspect of the invention provides the use of the basic serine protease, biomaterials, and compositions described above in at least one of the following aspects: detergent industry, food industry, leather and textile industry, pharmaceutical industry, biosynthesis, feed preparation, and environmental recycling.

[0062] The beneficial effects of this invention are:

[0063] 1. This invention, based on a marine microbial metagenomic database, discovered a novel alkaline serine protease, 25363. This enzyme has an optimal temperature of 50°C, but retains approximately 50% of its activity at 20°C, demonstrating good low-temperature adaptability. Furthermore, this enzyme exhibits a wide optimal pH range, maintaining over 95% relative activity within the pH range of 8-12, and retaining approximately 50% activity at pH 6. Its pH adaptability is significantly higher than that of commercially available alkaline proteases.

[0064] 2. The alkaline serine protease 25363 of this invention exhibits a 28% significantly increased activity under detergent conditions. In contrast, under the same conditions, commercially available alkaline proteases are affected by other active ingredients in detergents, resulting in a significant decrease in enzyme activity. This demonstrates that the novel marine-derived alkaline serine protease 25363 of this invention possesses extremely high detergent tolerance and exhibits better detergent application potential compared to other alkaline proteases.

[0065] 3. In this invention, the Bacillus subtilis secretory expression system is used to screen new enzymes for activity using a high-throughput screening platform based on 96-well plates, with detergent tolerance as the metric. This method can rapidly and efficiently obtain enzymes with detergent tolerance, providing an effective technical means for the development of new enzymes. Attached Figure Description

[0066] Figure 1 The results show the clear zone screening results for alkaline serine proteases.

[0067] Figure 2 The results are experimental findings on the temperature adaptability of alkaline serine protease-25363.

[0068] Figure 3 The results show the pH adaptation of alkaline serine protease-25363 and commercially available alkaline protease.

[0069] Figure 4 This is a comparison of detergent tolerance between alkaline serine protease-25363 and commercially available alkaline protease. Detailed Implementation

[0070] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0071] Example 1

[0072] In this embodiment, a method for preparing an alkaline serine protease and a transformant containing its encoding gene is provided.

[0073] In this invention, the inventors obtained a novel basic serine protease sequence based on data from the global marine microbial metagenomic database, the full-length amino acid sequence of which is shown in SEQ ID NO:1.

[0074] TPQTSVIIGFKGPPDAGLVKSHGGVIKYVYHHIPAIAASLSTQAIDALKKNPRVTYIEQDYKVYAIGHGHGKNKTSSPQPNQTIPWGIDKINAPMVWSEGYVGIDVKIAVLDTGIDIDHPDLNVAGGVTFVRGTKTYDDDNGHGTHVAGIIAALNNAIGVVGVAPDASLYAVKVLNRRGWG YVSDIIAGIEWSINNDMQIIMSFGSTSDSTSLHDECDKAYYTYNITLVAAAGNEGPGTDTITYPARYSSVIAVGATDENDVVASWSSTGPELELTAPGVDIYSTYKDGTYETLSGTSMACPHVTGTIALILSKNSSLTPTEVRSILQNTAIDLGDNGTDPVYGYGMIDAYAAVSSTP (SEQ ID NO:1).

[0075] The inventors, without altering the active site and disulfide bond arrangement of its natural enzyme protein, based on Bacillus subtilis (… Bacillus subtilis The coding sequence of the above-mentioned basic serine protease was optimized using a high-frequency codon table to obtain an artificially designed nucleotide sequence as shown in SEQ ID NO:2.

[0076]

[0077] The sequence shown in SEQ ID NO:2 was subjected to 5'- BamH I and 3'- Hind After triple enzyme digestion, it was directionally inserted into the commercially available E. coli-Bacillus subtilis shuttle vector pWB980 (located downstream of the P43 promoter, within the SacB signal peptide coding region, and similarly processed). BamH I and Hind (III) Double enzyme digestion was performed to construct the recombinant expression plasmid pWB980-25363.

[0078] The constructed pWB980-25363 was transformed into a protease-deficient host bacterium using the Spizizen chemocompetent cell method. Bacillus subtilis WB600 ( ΔaprE ΔnprE ΔnprB Δbpr Δvpr ΔwprA Then the converted Bacillus subtilis WB600 bacterial culture was plated on LB agar plates containing 25 μg / mL kanamycin and incubated at 37 ℃ for 12–16 h. Single clones were randomly selected for colony PCR and plasmid digestion to verify the correctness of the introduced sequence. Positive transformants were obtained and named WB600 / pWB980-25363.

[0079] The positive transformants obtained in the above examples were plated on LB agar plates containing 1% (w / v) skim milk powder and 25 μg / mL kanamycin and incubated at 37 °C for 48 h. The transformant introduced with the blank plasmid (pWB980) was used as a control (named pWB980-ori).

[0080] The results are as follows Figure 1 As shown.

[0081] It can be observed that the transformants expressing alkaline serine protease-25363 exhibited significantly larger clear zones of milk (skim milk powder) hydrolysis compared to the empty vector control, indicating that alkaline serine protease-25363 has significant protease activity.

[0082] Transformants that form a clear hydrolysis zone around the colony were screened and further activated for cloning. Specifically, single colonies capable of forming a clear hydrolysis zone were picked up with a sterile toothpick and inoculated into 96-well deep-well plates. Each well was incubated with 600 μL of LB liquid medium (Kan+) at 37 ℃ and 900 r / min for 16 h to complete activation.

[0083] The activated transformants were transferred to new 96-well plates at an inoculum rate of 1% (v / v), and cultured under the same conditions as described above for another 24 h. After the culture was completed, the plates were centrifuged at 3500 ×g for 10 min at 4 °C, and the supernatant was collected as the crude enzyme solution of alkaline serine protease-25363.

[0084] Example 2

[0085] In this embodiment, the effect of alkaline serine protease-25363 was verified.

[0086] (1) Enzyme activity assay at different temperatures:

[0087] The activity of basic serine protease-25363 was determined using the fluorescent substrate (Suc-Ala-Ala-Pro-Phe-AMC (Suc-AAPF-AMC, SEQ ID NO: 3)) method.

[0088] Specifically, the reaction system (100 μL) shown in the table below was constructed.

[0089] Table 1. Basic serine protease-substrate reaction system

[0090] .

[0091] After oscillating at 900 r / min for 30 min at 30 ℃, the fluorescence increment (excitation 380 nm, emission 450 nm) was immediately measured using a multi-functional microplate reader.

[0092] Meanwhile, other temperature groups (20℃, 40℃, 50℃, and 60℃) were set up to investigate the effect of temperature on enzyme activity. The pre-equilibration time for each temperature point was set to 5 min.

[0093] The relative enzyme activity was calculated with the highest activity temperature as 100%, and a temperature-activity curve was plotted to determine the optimal temperature.

[0094] The results are as follows Figure 2 As shown.

[0095] It can be observed that the optimal temperature for alkaline serine protease-25363 using Suc-AAPF-AMC as a substrate is 50℃, but it can still maintain about 50% of its activity at 20℃, indicating a good temperature adaptability range.

[0096] (2) Enzyme activity assay under different pH systems:

[0097] The effect of pH on alkaline serine protease-25363 was tested according to the fluorescent substrate method shown in (1).

[0098] The specific measurement steps are the same as in (1), except that the pH values ​​in the reaction system are adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively using sodium hydroxide and disodium bicarbonate. The pre-equilibration time for each pH value is set to 5 min. After shaking the reaction at 900 r / min for 30 min at 30 ℃, the fluorescence increment (excitation 380 nm, emission 450 nm) is immediately measured using a multi-functional microplate reader. The relative enzyme activity is calculated with the highest activity pH value as 100%, and a pH-activity curve is plotted to determine the optimal pH value.

[0099] Meanwhile, a commercially available alkaline serine protease (purchased from Solarbio, catalog number B8360) was used as a control.

[0100] The results are as follows Figure 3 As shown.

[0101] It can be observed that, using Suc-AAPF-AMC as a substrate, alkaline serine protease-25363 exhibits a wider pH stability range, with an optimal pH range of 8-12. It maintains over 80% activity at pH 7 and approximately 45% activity at pH 6 (compared to only 18% for commercially available conventional alkaline proteases). In contrast, the optimal pH range for commercially available conventional alkaline proteases is only 10-12, and their activity drops to less than 80% at pH 8. Therefore, alkaline serine protease-25363 demonstrates better pH adaptability compared to commercially available conventional alkaline proteases.

[0102] (3) Detergent tolerance test:

[0103] In the examples, the tolerance of alkaline serine protease-25363 to commercially available alkaline serine protease to commercial detergents was further compared.

[0104] The specific experimental method is as follows:

[0105] Preparation of detergent working solution: OMO commercial detergent (components: surfactant, phosphate-free water softener, enzyme preparation, fragrance, preservative (containing isothiazolinone compounds), colorant) was used as the experimental subject. Water was added to prepare a solution with a concentration of 1.4 g / L, and the solution was boiled at 100℃ for 10 min to inactivate the enzyme components and make it an enzyme-free detergent to avoid interference from its enzyme components.

[0106] Six groups were set up in a 96-well plate, with a final volume of 100 μL per well. The specific configurations for each group are as follows:

[0107] Group 1: 1 μL Suc-AAPF-AMC + remaining Tris-HCl buffer.

[0108] Group 2: 1 μL Suc-AAPF-AMC + the remainder enzyme-free detergent.

[0109] Group 3: 1 μL Suc-AAPF-AMC + 50U alkaline serine protease-25363 (final content) + remaining amount of enzyme-free detergent.

[0110] Group 4: 1 μL Suc-AAPF-AMC + 50U commercially available alkaline serine protease (final content) + remaining amount of enzyme-free detergent.

[0111] Group 5: 1 μL Suc-AAPF-AMC + 50U alkaline serine protease-25363 (final content) + Tris-HCl buffer (remaining).

[0112] Group 6: 1 μL Suc-AAPF-AMC + 50U commercially available alkaline serine protease + balance Tris-HCl buffer.

[0113] After oscillating at 900 r / min for 30 min at 30 ℃, the fluorescence increment (excitation 380 nm, emission 450 nm) was immediately measured using a multi-functional microplate reader.

[0114] The results are as follows Figure 4 As shown.

[0115] It can be observed that the activity of alkaline serine protease-25363 increases by 28% under detergent conditions, while that of commercially available alkaline serine protease decreases by 10% under the same conditions. This indicates that the alkaline serine protease-25363 in this invention has higher detergent tolerance than commercially available alkaline serine protease and can be used as a detergent ingredient.

[0116] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A basic serine protease, characterized in that, The alkaline serine protease is an alkaline serine protease with an amino acid sequence as shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the basic serine protease of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecules include: (1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:2; (2) Nucleic acid molecules with different sequences from those in (1) obtained based on degenerate codons.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleic acid molecule has undergone sequence optimization, and the nucleic acid molecule includes: At least one of the following: species-based codon optimization, GC content adjustment, and introduction of linkers.

5. A biomaterial, characterized in that, The biomaterial includes at least one of the following: (1) An expression vector containing the nucleic acid molecule of claim 3 or 4; (2) A transformant containing the nucleic acid molecule of claim 3 or 4; (3) Transformants containing the expression vector in (1).

6. A composition, characterized in that, The composition contains the alkaline serine protease of claim 1 and / or the biomaterial of claim 5.

7. The composition according to claim 6, characterized in that, The composition also contains excipients.

8. The composition according to claim 7, characterized in that, The excipients include at least one of the following: detergent builder, bleaching agent, optical brightener, fabric softener, fragrance, thickener, solvent, filler, anti-redeposition agent, pH adjuster, and preservative.

9. The composition according to claim 8, characterized in that, The detergent aid includes a chelating agent.

10. The composition according to claim 8, characterized in that, The softener includes an antistatic agent.

11. The composition according to claim 8, characterized in that, The thickener includes a rheology modifier.

12. The composition according to claim 8, characterized in that, The pH adjuster includes a buffer.

13. Use of at least one of the basic serine protease of claim 1, the biomaterial of claim 5, and the composition of any one of claims 6-12 in protein degradation.

14. Use of at least one of the alkaline serine protease of claim 1, the biomaterial of claim 5, and the composition of any one of claims 6-12 in at least one aspect of the detergent industry, food industry, leather and textile industry, pharmaceutical industry, biosynthesis, feed preparation, and environmental recycling.

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