Trypsin as well as coding gene and application thereof

Abstract

The invention discloses trypsin as well as a coding gene and application thereof, and relates to the technical field of biology. The trypsin is any one of (1)-(3): (1) Try3 protein, the amino acid sequence of which is as shown in SEQ ID NO.2; (2) carrying out substitution, deletion and / or addition of one or more amino acid residues on the amino acid sequence of the Try3 protein to obtain a protein which has more than 90% of identity with the Try3 protein; and (3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (1) or (2). The trypsin provided by the invention is wide in pH application range, good in thermal stability and strong in tolerance to various metal ions, and can well meet the requirements of industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a trypsin, a coding gene and application thereof. BACKGROUND

[0002] Trypsin is a proteolytic enzyme secreted by the pancreas. It plays a crucial role in the digestive tract of animals, mainly participating in the process of protein digestion and decomposition in the small intestine. When food enters the small intestine, trypsin is converted from its precursor trypsinogen under the activation of enteropeptidase. It specifically cleaves the peptide bond on the carboxyl side of basic amino acids (such as lysine and arginine) in protein molecules, decomposing complex proteins into smaller peptide segments, which are then further decomposed into amino acids by other enzymes, providing raw materials for the synthesis of self-proteins and other physiological activities of animal bodies, ensuring the smooth progress of a series of life activities such as normal growth, development and metabolism of animals.

[0003] Trypsin is a serine protease with the property of specifically hydrolyzing the peptide bond on the carboxyl side of basic amino acids (such as lysine and arginine) in proteins. In the food industry, trypsin can be used for meat tenderization, low-allergy food production, and the development of bioactive peptides. In leather production, it is often used in the soaking and unhairing processes, which can make the raw leather absorb water uniformly, remove the intercellular substance, promote the penetration of lime, and make the leather swell uniformly. At the same time, it effectively hydrolyzes the protein at the root of the hair during unhairing, maintaining the integrity of the leather. In the feed production industry, trypsin improves the utilization rate of protein in feed by hydrolyzing it, especially for the feed production of young animals. Therefore, the development of new trypsin is of great significance to the papermaking, food, feed and other industries. SUMMARY

[0004] The purpose of the present application is to provide a trypsin, a coding gene and application thereof, to solve the problems existing in the prior art. The trypsin has a wide pH adaptation range and good thermal stability, which can well meet the needs of industrial production.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The present application provides a trypsin, which is any one of (1)-(3):

[0007] (1) Try3 protein, the amino acid sequence of which is shown in SEQ ID NO. 2;

[0008] (2) a protein having 90% or more identity with the Try3 protein obtained by substituting, deleting and / or adding one or more amino acid residues to the amino acid sequence of the Try3 protein;

[0009] (3) the fusion protein obtained by connecting a protein tag at the N terminal or / and C terminal of the protein of (1) or (2).

[0010] The present application also provides a gene encoding the trypsin.

[0011] Further, the nucleotide sequence of the gene encoding is shown as SEQ ID NO. 2.

[0012] The present application also provides a gene expression cassette comprising the gene encoding.

[0013] The present application also provides a recombinant expression vector comprising the gene expression cassette.

[0014] The present application also provides a recombinant host cell comprising the recombinant expression vector.

[0015] Further, the recombinant host cell is a recombinant yeast.

[0016] The present application also provides the use of the gene encoding, the gene expression cassette, the recombinant expression vector or the recombinant host cell in the preparation of the trypsin.

[0017] The present application also provides a preparation method of the trypsin, comprising the step of fermenting the recombinant host cell to prepare the trypsin.

[0018] The present application also provides the use of the trypsin in catalyzing the hydrolysis of proteins.

[0019] The present application discloses the following technical effects:

[0020] The present application discloses a trypsin gene Try3 cloned from a hot spring metagenomic library, and a product of the gene, i.e., a recombinant Try3 trypsin, expressed in Pichia pastoris, can hydrolyze Nα-benzoyl-L-arginine ethyl ester (BAEE). The trypsin activity of the recombinant Try3 trypsin under the optimal conditions of pH 8.5 and 70 DEG C is 734.2 U / mL. The enzymatic property research shows that the enzyme has good tolerance between pH 2.0-12.0, and the enzyme activity is maintained above 70% after incubation at 60 DEG C for 60 min. The enzyme has a wide pH adaptation range and good thermal stability, and can well meet the needs of industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0022] Figure 1 PCR identification results of recombinant Pichia pastoris; wherein, lane M is Takara DL2000 Marker, lane P1 is positive clone;

[0023] Figure 2 SDS-PAGE electrophoresis diagram of trypsin purification product; wherein, lane M is electrophoresis result of protein Marker; lane P1 and P2 are crude enzyme liquid; lane P3 and P4 are trypsin purification product;

[0024] Figure 3 Statistical diagram of trypsin enzyme activity under different pH conditions;

[0025] Figure 4 Statistical diagram of trypsin enzyme activity under different pH conditions after standing for 1h at 25℃;

[0026] Figure 5 Statistical diagram of trypsin enzyme activity under different temperature conditions;

[0027] Figure 6 Statistical diagram of trypsin enzyme activity after incubation for 1h under different temperature conditions. DETAILED DESCRIPTION

[0028] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is made for the purpose of illustrating the certain aspects, features and embodiments of the present application and is not intended to limit the scope of the application, which is defined solely by the appended claims.

[0029] It should be understood that the terms used herein are merely descriptive, but are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within the range of the intermediate value of any stated value or stated range, and any other stated value or intermediate value within the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the state of the art.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Terminology Explanation:

[0034] Metagenomics, also known as microbial environmental genomics or metagenomics, offers the advantage of bypassing traditional pure cultures by obtaining genetic information from all uncultured microorganisms in the environment. This allows for the analysis of microbial genetic information and the screening of functional genes. Through the analysis of metagenomic data and the screening of metagenomic libraries, a large number of uncultured microorganisms in extreme environments and their functional genes can be more easily discovered.

[0035] The term "protein" is intended to refer to a polymer of amino acid residues comprising at least nine amino acids linked by peptide bonds. The polymer can be linear, branched, or cyclic. The polymer can contain native amino acids and / or amino acid analogs, and it can be interrupted by non-amino acid residues.

[0036] The term "coding gene" refers to a segment of DNA sequence that ultimately directs protein synthesis through transcription and translation. These genes are the direct carriers of genetic information in an organism, expressing it as functional proteins.

[0037] The term "expression vector" is a man-made and modified DNA molecule (usually a plasmid or viral vector) whose core function is to introduce a foreign gene (usually a coding gene) into a specific host cell (such as bacteria, yeast, insect cells, or mammalian cells) and to guide the efficient and accurate transcription and translation of that gene, ultimately producing the target protein (usually a recombinant protein).

[0038] As used herein, "plasmid" refers to a reproducible DNA construct. Typically, plasmid vectors contain a selective marker gene that allows for the identification and / or selection of host cells carrying the plasmid as positive or negative in the presence of a compound corresponding to the selective marker. A variety of positive and negative selective marker genes are known in the art. By way of example, an antibiotic resistance gene can be used as a positive selective marker gene to select host cells in the presence of the corresponding antibiotic.

[0039] The term "host cell" is intended to mean a cell comprising a nucleic acid molecule of the application. The host cell can consist of a single type of cell or a group of different types of cells. The host cell can also be a hybrid cell, that is to say a cell resulting from the fusion of at least two different types of cells. The host cell can belong to a cultured cell line, a primary cell, a stem cell or a proliferating cell. The "host cell" according to the application is a recombinant host cell, that is to say a cell which harbours exogenous genetic material. The host cell is thus not a naturally occurring wild-type cell, but a molecular biology tool obtained by genetic manipulation techniques.

[0040] "Percent (%) sequence identity" with respect to a reference amino acid sequence is intended to mean the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference amino acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, but not considering any conservative substitutions as part of the sequence identity. For purposes of determining percent amino acid sequence identity, the sequences can be compared using a variety of ways well within the skill in the art, for example, using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0041] The present disclosure also relates to amino acid sequence variants, which can be made by introducing appropriate modifications into the nucleotide sequence encoding the molecules, or by peptide synthesis. Such modifications include, for example, deletions from, insertions into, and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final constructs possess the desired characteristics, for example, antigen binding activity. Sites of interest for substitutional mutagenesis include the HVRs and framework (FR) regions. Possible substitutions include conservative substitutions of amino acids as taught in the art. Conservative substitution tables commonly are used as a starting point. Conservative substitution amino acid groups include: Ala (A) conservative substitutions include Val, Leu, lie, preferably Val; Arg (R) conservative substitutions include Lys, Gin, Asn, preferably Lys; Asn (N) conservative substitutions include Gin, His, Asp, Lys, Arg, preferably Gin; Asp (D) conservative substitutions include Glu, Asn, preferably Glu; Cys (C) conservative substitutions include Ser, Ala, preferably Ser; Gin (Q) conservative substitutions include Asn, Glu, preferably Asn; Glu (E) conservative substitutions include Asp, Gin, preferably Asp; Gly (G) conservative substitutions include Ala; His (H) conservative substitutions include Asn, Gin, Lys, Arg, preferably Arg; lie (I) conservative substitutions include Leu, Val, Met, Ala, Phe, Nle, preferably Leu; Leu (L) conservative substitutions include Nle, lie, Val, Met, Ala, Phe, preferably lie; Lys (K) conservative substitutions include Arg, Gin, Asn, preferably Arg; Met (M) conservative substitutions include Leu, Phe, lie, preferably Leu; Phe (F) conservative substitutions include Trp, Leu, Val, lie, Ala, Tyr; Tyr; Pro (P) conservative substitutions include Ala; Ser (S) conservative substitutions include Thr; Thr (T) conservative substitutions include Val, Ser, preferably Ser; Trp (W) conservative substitutions include Tyr, Phe, preferably Tyr; Tyr (Y) conservative substitutions include Trp, Phe, Thr, Ser, preferably Phe; Val (V) conservative substitutions include lie, Leu, Met, Phe, Ala, Nle, preferably Leu.

[0042] The term "enzyme activity" is intended to mean the efficiency of an enzyme in converting a substrate into a product in a given environment. The efficiency of an enzyme is considered herein both the rate at which the enzyme converts the substrate into the product and the extent to which the enzyme converts the substrate into the product. The expression "the extent to which the enzyme converts the substrate into the product" is intended here to mean the ratio between the amount of final product obtained and the initial amount of substrate for a defined amount of enzyme.

[0043] Example 1 Bioinformatic analysis of the trypsin gene

[0044] By analyzing the metagenomes of microorganisms from hot springs at 42-90°C (from the JGI-IMG microbial functional genomics database), a 837 bp gene Try3 encoding trypsin was screened, and the gene sequence was synthesized by Shanghai Shengong Biotechnology Co., Ltd.

[0045] The nucleotide sequence of the gene Try3 is shown as SEQ ID NO. 1, and the amino acid sequence of the trypsin encoded thereby is shown as SEQ ID NO. 2.

[0046] SEQ ID NO. 1:

[0047] caaatggatgatccaactcatgctagaccaactagaccagctttgtttccaactcaaccatctgatggtatgcaaccacaaattattggtggtaatccagctgatgatggtgaatacccatggcaagttgctttggttgattctacttacattaatccatttgatggtcaattttgtggtggttctattattgctccaggttgggttttgactgctgctcactgtgttgttgatgatgactgtttctaacccagctactttggatgttgttgctggtgttaacttgctttcttctggtccaacttctggttctcaaggtcaaagaagaaaggttgctcaaattattgtttacccatctttttactatactaatggaaagattccagaaaacgatattgctttgttgagattggctgctccattgaacttgaaccaaaaggctcaaccaatttccttggctactcaagctgattctcctagatttgctccaggtgttactgctactgtttctggttggggtagaatgaatcctcctccacaagaacctattttgtttccagatgctttgatggaagttcaagtcccaattgttgatcaaactatttgttctgctgcttatcctggtgaaatcactcctaatatgttgtgtgctggttacgctgctggtggatacgattcttgtcaaggagattctggtggtccattgattgttcctgatggtaacggtggttggttgcaagctggaattgtttcttggggaaagggttgtgctcaaccaaacaagtacggtgtttacactagattggctaactacactgaatggattaactct.

[0048] SEQ ID NO. 2:

[0049] QMDDPTHARPTRPALFPTQPSDGMQPQIIGGNPADDGEYPWQVALVDSTYINPFDGQFCGGSIIAPGWVLTAAHCVVDDDGTVSNPATLDVVAGVNLLSSGPTSGSQGQRRKVAQIIVYPSFYYTNGKIPENDIALLRLAAPLNLNQKAQPISLATQADSPRFAPGVTATVSGWGRMNPPPQEPILFPDALMEVQVPIVDQTICSAAYPGEITPNMLCAGYAAGGYDSCQGDSGGPLIVPDGNGGWLQAGIVSWGKGCAQPNKYGVYTRLANYTEWINS.

[0050] Example 2 Expression of Trypsin Gene Try3 in Pichia pastoris

[0051] 1. Construction of Recombinant Expression Vector in Host Pichia pastoris

[0052] The Try3 gene and the pPIC9 vector were connected by homologous recombination, the forward primers used were GAAGAAGGGGTATCTCTCGAGAAAAG (SEQ ID NO. 3) and CTCTCGAGAAAAGAGAGGCTGAAGCTCAAATGGATGAT (SEQ ID NO. 4), the reverse primer was GTGGTGACTCGCCGGCGCTTAATTAAGCG (SEQ ID NO. 5), and the pPIC9 vector was treated with restriction endonuclease Xho I and Not I. The PCR product of Try3 and the linearized vector were recombined using the Novagen kit ClonExpress Ultra OneStep Cloning Kit V2 to obtain the recombinant plasmid pPIC9-Try3.

[0053] 2. Transformation and verification of the recombinant plasmid in E. coli

[0054] The recombinant plasmid pPIC9-Try3 constructed above was transformed into E. coli TOP10 competent cells. The plasmid was extracted from the colonies grown on the LB plate containing ampicillin.

[0055] 3. Transformation, expression of Try3 gene in Pichia pastoris and purification of the expression product

[0056] The extracted plasmid was linearized with restriction endonuclease Sac I, and concentrated with isopropanol. Pichia pastoris GS115 was cultured to OD 600about 0.6, and the bacteria were washed with pre-cooled pure water and sorbitol, respectively. The concentrated plasmid was electroporated into the sorbitol-dissolved competent cells.

[0057] Genomic templates were extracted from the colonies grown on MD plates, and the templates were amplified by PCR using the primer pair of SEQ ID NO. 6 / SEQ ID NO. 7 to identify positive clones. The results are shown in Figure 1 Figure 2, and the AOX gene carried by the yeast genome and the sequence obtained by transformation were both amplified, proving that it was a positive clone.

[0058] SEQ ID NO. 6: CTGGTTCCAATTGACAAGC;

[0059] SEQ ID NO. 7: TGGCATTCTGACATCCTC.

[0060] The positive clone was picked into BMGY medium and grown to OD 600 2, and the bacteria were resuspended and washed with sterile normal saline. The yeast was resuspended in BMMY medium to OD 600 1. Methanol was added to the final concentration of 1% every 24 h after the yeast was transferred to the BMMY medium to induce the expression of the exogenous protein.

[0061] The supernatant obtained after 6 days of culture was the crude trypsin solution, which was purified by a nickel column from Shanghai Biosciences. The eluent was 50 mM imidazole, 20 mM Tris-HCL, 500 mM NaCl, pH 8.

[0062] The SDS-PAGE electrophoresis results of the crude enzyme solution and the purified solution are shown in Figure 2 Figure 3. As can be seen from Figure 2 the electrophoresis results after elution with the eluent, a single band was obtained, indicating that high-purity Try3 target protein can be obtained under this condition. All the eluents containing the target protein were combined, concentrated and dialyzed by a 10 kDa ultrafiltration tube (Merck, Millipore, USA), and the buffer was replaced with 20 mM NaH2PO4 at pH 7.4 to remove imidazole and high-concentration NaCl in the enzyme solution and adjust the pH, to obtain the purified recombinant Try3 trypsin.

[0063] Example 3 Analysis of the properties of the recombinant Try3 trypsin

[0064] 1. Enzyme activity determination of trypsin

[0065] The enzyme activity of the recombinant Try3 trypsin prepared in Example 2 was determined by the BAEE method, and the specific method is as follows:

[0066] In a 3.2 mL reaction system, 2.7 mL of phosphate buffer (a mixture of 13 mL of 0.067 mol / L potassium dihydrogen phosphate and 87 mL of 0.067 mol / L disodium hydrogen phosphate, pH 7.6), 0.3 mL of substrate stock solution (85.7 mg of Nα-benzoyl-L-arginine ethyl ester hydrochloride dissolved in water to a final volume of 100 mL) and 0.2 mL of enzyme solution were mixed. The absorbance at 253 nm was measured using a UV spectrophotometer (with an inactivated enzyme solution as a blank control). Absorbance values ​​were read every 30 seconds for a total of 5 minutes. A graph was plotted with absorbance on the ordinate and time on the abscissa; the absorbance change should remain constant between 0.015 and 0.018 every 30 seconds, and the linear change should last for at least 3 minutes. The enzyme activity units (P / mL) were calculated using a standard curve after subtracting the control from the sample measurement.

[0067] P = (A1 - A2) / 0.003 / T / W;

[0068] Where P is the amount of trypsin in 1 mL of enzyme solution, in U / mL; A1 is the absorbance value at which the line terminates; A2 is the absorbance value at the beginning of the line; T is the time from A1 to A2, in minutes; W is the volume of enzyme solution, in mL; enzyme activity unit (U) is defined as: 1 U is the amount of enzyme required to catalyze a 0.003 change in the absorbance value of the substrate solution per minute.

[0069] The results showed that the specific activity of the recombinant Try3 trypsin was 734.2 U / mL under optimal conditions of pH 8.5 and 70℃.

[0070] 3. Determination of optimal pH and pH stability

[0071] (1) Determination of optimal pH

[0072] The recombinant Try3 trypsin prepared in Example 2 was tested for enzyme activity under different pH conditions. The pH corresponding to the highest enzyme activity is the optimal pH of the enzyme.

[0073] The optimal pH for recombinant Try3 trypsin was determined within the range of 2-12, with each increment being 0.5. The buffer was Britton-Robinson buffer (buffer range: pH 2-12). The reaction was performed and the enzyme activity of trypsin was determined using the same method as described in "1. Determination of trypsin activity" above, except that the pH during the reaction was changed. The highest enzyme activity was defined as 100%, and the relative activity under other conditions was calculated (repeated three times).

[0074] Figure 3 The results showed that the optimal pH for this recombinant Try3 trypsin was 8.5.

[0075] (2) pH stability analysis

[0076] The recombinant Try3 trypsin prepared in Example 2 was incubated at different pH at 25°C for 1 h, and then the substrate was added for reaction at the optimum pH to determine the enzyme activity of the trypsin.

[0077] The pH stability was determined by incubating the enzyme solution at the corresponding pH at 25°C for 1 h, and then adding the enzyme solution to the substrate for reaction at the optimum pH, with other reaction conditions being the same as those in the aforementioned "1. Enzyme activity determination of trypsin". The enzyme activity of the trypsin was determined according to the method in the aforementioned "1. Enzyme activity determination of trypsin", with the highest enzyme activity being defined as 100%, and the relative activity under other conditions being calculated (triplicate).

[0078] Figure 4 The results shown in Table 1 indicate that the recombinant Try3 trypsin has a wide pH tolerance range.

[0079] 4. Optimum temperature and thermal stability determination

[0080] (1) Optimum temperature determination

[0081] The recombinant Try3 trypsin prepared in Example 2 was used to detect the enzyme activity at different temperatures, and the temperature corresponding to the highest enzyme activity was the optimum temperature of the enzyme.

[0082] The optimum temperature range of the recombinant Try3 trypsin was set as 20-85°C, with every 5°C as a temperature gradient. The optimum temperature was determined by first incubating the substrate solution at different temperatures for 5 min, then adding the enzyme solution and mixing rapidly, and then reacting at the corresponding temperature, with other reaction conditions being the same as those in the aforementioned "1. Enzyme activity determination of trypsin", and then determining the enzyme activity according to the method in the aforementioned "1. Enzyme activity determination of trypsin".

[0083] Figure 5 The results shown in Table 2 indicate that the optimum temperature of the recombinant Try3 trypsin is 70°C.

[0084] (2) Temperature stability analysis

[0085] The recombinant Try3 trypsin prepared in Example 2 was incubated at different temperatures for 1 h, and then the reaction substrate was added to determine the enzyme activity at the optimum temperature.

[0086] The temperature stability was determined by selecting 30-65°C, incubating the enzyme solution at different temperatures for 1 h, and then adding the enzyme solution to the reaction substrate for reaction at the optimum temperature, with other reaction conditions being the same as those in the aforementioned "1. Enzyme activity determination of trypsin". Then the enzyme activity of the trypsin was determined according to the aforementioned method. The highest enzyme activity was defined as 100%, and the relative activity under other conditions was calculated (triplicate).

[0087] Figure 6 The results show that the recombinant Try3 trypsin has good thermal stability.

[0088] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A trypsin, characterized in that, The trypsin is any one of (1)-(3): (1) a Try3 protein, the amino acid sequence of which is shown in SEQ ID NO. 2; (2) a protein having more than 90% identity with the Try3 protein obtained by substitution, deletion and / or addition of one or several amino acid residues to the amino acid sequence of the Try3 protein; (3) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of (1) or (2).

2. A coding gene of the trypsin according to claim 1.

3. The genetic code according to claim 2, wherein, The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

4. A gene expression cassette, characterized in that, The coding gene according to claim 2 or 3.

5. A recombinant expression vector, characterized in that, The gene expression cassette according to claim 4.

6. A recombinant host cell, characterized in that, The recombinant expression vector according to claim 5.

7. The recombinant host cell of claim 6, wherein, The recombinant host cell is a recombinant yeast.

8. Use of the coding gene according to claim 2 or 3, the gene expression cassette according to claim 4, the recombinant expression vector according to claim 5, or the recombinant host cell according to claim 6 or 7 in the preparation of the trypsin according to claim 1.

9. A method for preparing trypsin, characterized by, The step of preparing the trypsin by fermentative cultivation of the recombinant host cell according to claim 6 or 7.

10. Use of the trypsin according to claim 1 in catalyzing the hydrolysis of proteins.

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