A neutral protease mutant and its application

Abstract

The present invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically discloses a neutral protease mutant and its application. The present invention uses DNA shuffling technology to perform directed evolution on neutral metalloprotease empr obtained by amplifying the genome of Bacillus LCB14, obtains a high-activity neutral protease mutant gene after screening, constructs a recombinant vector with the mutant gene, and successfully expresses it in Pichia pastoris to obtain a recombinant strain with improved enzyme activity. The obtained neutral protease mutant emps can be applied to the textile leather industry, food industry, feed industry and medical field, and when applied to the depilatory process of leather making, it has excellent depilatory performance and significantly improves depilatory efficiency, which will have a good application prospect in the leather industry.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and specifically relates to a neutral protease mutant and application thereof. Background Art

[0002] Protease is one of the three major industrial enzymes, accounting for about 60% of the enzyme market share, and is widely used in food, medicine, washing and feed. Compared with traditional methods, the method of using protease to hydrolyze protein has many conveniences and has gradually become the main way of protein processing. The protease preparation industry can bring considerable economic benefits every year and has a very broad development prospect. The main sources of proteases currently produced are animals, plants and microorganisms. Microbial proteases have received widespread attention due to the advantages of fast growth and reproduction, artificial cultivation, short growth cycle and strong biodiversity. Proteases can be divided into acidic proteases, neutral proteases and alkaline proteases according to their optimal pH. Neutral proteases are a type of protease that acts on protein peptide bonds under neutral pH conditions. The pH is generally between 6.0-7.5. Proteins can be hydrolyzed into amino acids, polypeptides and free amino acids. They can be widely used in the hydrolysis of animal and plant proteins, the preparation and production of condiments and food nutritional enhancers, and can also be used for leather dehairing, softening, wool and silk degumming and other processing.

[0003] Enzymatic depilation is a depilation method that uses the catalytic action of proteases to hydrolyze proteins at the junction of hair and skin in raw hides, destroy the bond between hair and skin, and separate the hair and skin. Enzymatic depilation avoids the use of a large number of harmful chemicals, is environmentally friendly, and the hair structure after depilation is intact and can be reused, so it has broad application prospects. Neutral protease is a commonly used enzyme for leather depilation. The activity of neutral protease directly affects the depilation effect. At present, research on improving the activity of neutral protease is mostly focused on optimizing the fermentation conditions of neutral protease-producing strains. After optimization, the activity of neutral protease can be increased by 1-2 times, but neutral protease has poor thermal stability and is easy to self-dissolve in adverse environments. It is the most unstable enzyme among microbial proteases and is sensitive to temperature during the depilation process, which seriously affects the depilation effect. The neutral protease empr derived from heterologous expression microorganisms has a stable conformation, improved temperature stability and pH stability, an optimum temperature of 50°C and an optimum pH of 6.5. It is used in the dehairing process of the leather making process and has excellent dehairing performance. However, when the temperature exceeds 60°C, the enzyme activity is significantly reduced and the high temperature resistance is poor, it is easily inactivated, and cannot be used in high temperature production conditions.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] Based on the above reasons, the object of the present invention is to provide a neutral protease mutant with improved thermal stability.

[0006] The present invention is achieved through the following technical solutions:

[0007] A neutral protease mutant, the amino acid sequence of the mutant is shown as SEQ ID NO.1.

[0008] The present invention also provides a gene, which encodes the neutral protease mutant.

[0009] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0010] The present invention also provides a recombinant expression vector, which carries the gene encoding the neutral protease mutant.

[0011] The present invention also provides a recombinant host cell, wherein the recombinant host cell comprises the above-mentioned recombinant expression vector.

[0012] Preferably, the host cell is Pichia pastoris.

[0013] The invention also provides the application of the neutral protease mutant in the degradation of proteins and peptide chains.

[0014] The present invention also provides the application of the neutral protease mutant in the textile and leather industry, food industry, feed industry and medical field.

[0015] Furthermore, it is used in the dehairing process of leather making.

[0016] The present invention also provides an enzyme preparation, which comprises the above-mentioned neutral protease mutant.

[0017] The present invention has the following beneficial effects:

[0018] The present invention uses DNA shuffling technology to perform directed evolution on neutral metalloprotease empr obtained by amplifying the genome of Bacillus LCB14, obtains high-activity neutral protease mutant genes after screening, constructs a recombinant vector with the mutant genes, and successfully expresses it in Pichia pastoris to obtain a recombinant strain with improved enzyme activity. The thermal stability of the transformed neutral protease mutant emps is significantly improved. Under 70°C, the enzyme activity is 2.7 times that of the original protease empr. After 60 minutes of heat preservation, the enzyme activity retention rate is 4 times that of the original protease empr. It can be applied to the textile and leather industry, food industry, feed industry and medical fields. When applied to the depilation process in the leather making process, it has excellent depilation performance and significantly shortens the depilation time, which will have a good application prospect in the leather industry. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.

[0020] The terms involved in the present invention are explained as follows:

[0021] 1. Parent protease: refers to the protease that can produce the mutant of the present invention after undergoing the mutation described in the present invention, namely the neutral protease empr.

[0022] 2. Protease mutant: refers to a mutated protease having one or more mutations in the sequence compared to the amino acid sequence of the parent protease. The mutant is expressed in a host cell by transferring the mutant gene into a plasmid.

[0023] 3. Host cell: refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a vector containing a nucleic acid of the present invention; it covers any progeny of a parent cell that is not completely identical to the parent cell due to mutations that occur during replication.

[0024] 4. Protease activity is defined as: the amount of protease required to hydrolyze casein to produce 1 μg of tyrosine in 1 min at 30°C and pH = 7.5, which is 1 protease activity unit U.

[0025] 5. The identification of the neutral protease mutant uses "amino acid replaced by the parent amino acid position" to indicate the mutated amino acid in the neutral protease mutant. For example, K13M means that the amino acid at position 13 is replaced by M from K of the parent neutral protease empr, and the position number corresponds to the number in SEQ ID NO: 4 in the attached sequence table.

[0026] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] The embodiment of the present invention provides a neutral protease mutant, and the amino acid sequence of the neutral protease mutant is shown in SEQ ID NO. 1. The neutral protease mutant is mainly prepared by DNA shuffling technology, using DNase I to digest the neutral protease gene empr from Bacillus LCB14, purifying the target gene fragment, using overlap extension PCR technology, designing primers with overlapping sequences, so that different DNA fragments are recombined during the PCR process, constructing a neutral protease mutant library, transforming the recombined DNA into a host cell, and screening to obtain mutants with significantly improved thermal stability. The mutation sites include K13M, S57Q, H75L, Y111N, E167I, L198M, N218K, and V260K, i.e., lysine, serine, histidine, tyrosine, glutamic acid, leucine, asparagine, and valine at amino acid positions 13, 57, 75, 111, 167, 198, 218, and 260 of the neutral protease empr mutate to methionine, glutamine, leucine, asparagine, isoleucine, methionine, lysine, and valine, respectively.

[0028] The embodiment of the present invention also provides the gene coding of the neutral protease mutant and a recombinant expression vector carrying the gene coding; the nucleotide sequence of the coding gene is shown in SEQ ID NO.2.

[0029] It should be noted that the vector backbone of the recombinant expression vector can be selected by those skilled in the art according to the implementation needs, and can be any vector with expression ability disclosed or undisclosed in the art, including but not limited to eukaryotic vectors and prokaryotic vectors. Preferably, it is a pMETB plasmid, and the protease gene of the present invention is inserted into a suitable site of the expression vector so that its nucleotide sequence can be operably connected to the expression regulatory sequence.

[0030] The present invention also provides a host cell comprising the above-mentioned recombinant expression vector, wherein the host cell is Pichia pastoris. The base cell of the host cell can be any cell type with expression ability disclosed or undisclosed in the art, including but not limited to eukaryotic cells or prokaryotic cells; preferably Pichia pastoris, more preferably Pichia pastoris KM71.

[0031] The embodiment of the present invention also provides the application of the neutral protease mutant in the textile and leather industry, food industry, feed industry and medical field.

[0032] The high-temperature resistant neutral protease mutant obtained by the present invention can be applied to the textile and leather industry, specifically can be used for textile dyeing, leather depilation, softening, etc., can effectively decompose keratin and elastin, effectively improve dyeing and depilation efficiency, thereby improving leather quality, and the softening process can also improve the ductility and toughness of the leather, and has huge application potential; in the food industry, it can be used in baked foods, and the high-temperature resistant neutral protease can hydrolyze the protein of the dough into peptones, peptides or amino acids, weaken the dough gluten strength, make the dough have good plasticity and extensibility, and improve the gloss and taste of the finished product; in the feed industry, adding protease to poultry feed can promote the digestion and absorption of plant protein in the feed, and improve the nutritional value of the feed; in the medical field, protein is dissolved in the pharmaceutical process and drug development process, which is conducive to improving digestion efficiency.

[0033] In some specific embodiments, the neutral protease mutant of the present invention can be applied to the dehairing process of the leather making process. The neutral protease degrades the sticky protein around the hair root, loosens the hair root and makes it fall off, and the hair falls off with the help of mechanical force, thereby achieving the purpose of dehairing. The neutral protease mutant of the present invention has higher enzyme activity and high temperature stability, and is suitable for leather dehairing in a high temperature environment.

[0034] The present invention also provides an enzyme preparation comprising the neutral protease mutant.

[0035] The present invention will be described in more detail below through specific examples. Unless otherwise specified, in the following examples,

[0036] The culture medium and related determination methods used in the present invention are as follows:

[0037] 1. Preparation of culture medium

[0038] LB medium: 1% tryptone, 0.5% yeast powder, 1% NaCl, pH 7.2±0.2;

[0039] LB plate: 1% tryptone, 0.5% yeast powder, 1% NaCl, 2% agar powder, pH 7.2±0.2;

[0040] YPD medium: 1% yeast extract, 2% tryptone, 2% glucose;

[0041] YPD plate: 1% yeast extract, 2% tryptone, 2% glucose, 2% agar powder;

[0042] MD medium: 1.34% YNB, 10-5% biotin, 2% glucose;

[0043] MD plate: 1.34% YNB, 10-5% biotin, 2% glucose, 2% agar powder;

[0044] BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 4×10-5% Biotin, 1% glycerol (v / v), 100 mmol / L potassium phosphate buffer (pH 6.0);

[0045] BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 4×10-5% Biotin, 3% methanol, 100 mmol / L potassium phosphate buffer (pH 6.0).

[0046] 2. Protease activity determination method: refer to the Folin method for the determination of protease activity in the national standard "GB / T 23527.1-2023 Quality requirements for enzyme preparations Part 1: Protease preparations"; place the casein solution in a 30℃ constant temperature water bath for preheating for 5 minutes, take 1mL of protease solution (preheated at 30℃ for 2 minutes) and mix it with 1mL of casein substrate (10.0g / L, pH=7.5), react at 30℃ for 10 minutes, then add 2mL of trichloroacetic acid solution (0.4mol / L) to terminate the reaction, centrifuge the suspension to take 1mL of supernatant, add 5mL of sodium carbonate solution (0.4mol / L) and 1mL of Folin solution for color development at 30℃ for 20 minutes, and then determine the protease activity according to the absorbance at 680nm.

[0047] The following will disclose specific embodiments for implementing the present application, as well as corresponding comparative examples to demonstrate the relevant technical effects of the present application.

[0048] Example 1 Construction of neutral protease expression vector

[0049] (1) Synthesis and amplification of the neutral protease gene empr

[0050] The protease gene sequence from Bacillus sp. LCB14 was obtained according to Genbank ID: MN480449. After codon optimization, the optimized neutral protease empr gene sequence (as shown in SEQ ID NO: 3) and its amino acid sequence (as shown in SEQ ID NO: 4) were obtained. Subsequently, a biological company was commissioned to synthesize the above gene sequence and amplify it by PCR. The primer sequences are as follows:

[0051] empr upstream primer F: 5′-CTCGGGGAATTTATCTTGTAGCCATAACAGTTCTTGATCTGTCCGTCCTTCCTTTTTTC-3′;

[0052] empr downstream primer R: 5′-GTTGCTAGTAACATCTGACCGAGATTTTTTTGAGCAACTTCAGTTTTCATTTGGAATGG-3′;

[0053] The above and downstream primers are used to amplify the target gene.

[0054] The amplification reaction system was: DNA template 0.5-1 μL, 10 μM upstream primer 2.5 μL, 10 μM downstream primer 2.5 μL, 2×Phanta Max Buffer 25 μL, 2.5 mM dNTP Mix 1 μL, and Taq DNA Polymerase 1 μL.

[0055] The amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 55°C for 60 s, extension at 72°C for 15 s, 30 cycles; and extension at 72°C for 10 min.

[0056] The PCR amplification product was subjected to 1% agarose gel electrophoresis to obtain a 1480 bp band, which was then cut and recovered to obtain the complete target gene fragment.

[0057] (2) Construction of neutral protease mutants

[0058] The above complete target gene fragment was digested with DNase I, subjected to agarose gel electrophoresis, and the digested fragment was recovered by a DNA gel recovery kit; the DNase I digestion system was: 3 μL DNA, 1 μL 1U / μL DNase I, 20 μL ddH2O; the reaction conditions were: digestion at 15°C for 15 min, and then heating at 90°C for 10 min to terminate the reaction.

[0059] Neutral protease empr was used as a parent, and point mutations were performed one by one, such that K at position 13 was mutated to M, S at position 57 was mutated to Q, H at position 75 was mutated to L, Y at position 111 was mutated to N, E at position 167 was mutated to I, L at position 198 was mutated to M, N at position 218 was mutated to K, and V at position 260 was mutated to K. The primers used for the above mutation points are as follows:

[0060] K13M forward primer: 5′-CTCTTAAAGGAATGACGGTCTCATTAAATATTTC-3′;

[0061] K13M reverse primer: 5′-GAAATATTTAATGAGACCGTCATTCCTTTAAGAG-3′;

[0062] S57Q forward primer: 5′-GGGCACGCTCGTACAAAGCACTACAAAC-3′;

[0063] S57Q reverse primer: 5′-GTTTGTAGTGCTTTGTACGAGCGTGCCC-3′;

[0064] H75L forward primer: 5′-GCCGTTGATGCGCTCTACAATCTCGGC-3′;

[0065] H75L reverse primer: 5′-GCCGAGATTGTAGAGCGCATCAACGGC-3′;

[0066] Y111N forward primer: 5′-CATTACGGCAGCAAAAACAACAACGCGGCC-3′;

[0067] Y111N reverse primer: 5′-GGCCGCGTTGTTGTTTTTGCTGCCGTAATG-3′;

[0068] E167I forward primer: 5′-GGTGCTTTAAACATCTCCTTCTCTGATGTATTC-3′;

[0069] E167I reverse primer: 5′-GAATACATCAGAGAAGGAGATGTTTAAAGCACC-3′;

[0070] L198M forward primer: 5′-GCTCTCCGCAGTATGTCCAATCCGACAAAATAC-3′;

[0071] L198M reverse primer: 5′-GTATTTTGTCGGATTGGACATACTGCGGAGAGC-3′;

[0072] N218K forward primer: 5′-CGAAACCTTCCGAAAACTGATGCCGGCGAC-3′;

[0073] N218K reverse primer: 5′-GTCGCCGGCATCAGTTTTCGGAAGGTTTCG-3′;

[0074] V260K forward primer: 5′-CCGTGCACTGACGAAATATCTCACTCCGTCATC-3′;

[0075] V260K reverse primer: 5′-GATGACGGAGTGAGATATTTCGTCAGTGCACGG-3′.

[0076] Taking the point mutation of K13M as an example, use K13M forward primer and K13M reverse primer, take the original neutral protease gene empr as template, add reagents according to the system in the point mutation kit, and perform PCR amplification. After the amplification, take 10μL of the above mutation PCR amplification product and perform 1% agarose gel electrophoresis detection. After the band is correct, add 1μL of digestion enzyme to the mutation product, mix well, and incubate at 37℃ for 60min.

[0077] Extract the pMETB plasmid, and refer to the kit's operating manual for the extraction process. After digestion with Sal I and Spe I, perform agarose gel electrophoresis, and then use a DNA gel recovery kit to recover the product to obtain a linearized expression vector. The neutral protease mutant genes obtained were cloned into the expression vector pMETB to obtain several recombinant plasmids, which were transformed into yeast KM71 competent cells by electroporation and cultured at 30°C for 2-3 days until single colonies appeared. Pick the transformants grown on the MD plate for further expression experiments.

[0078] Example 2 Screening and expression of neutral protease mutants

[0079] (1) Screening of neutral protease mutant transformants

[0080] Pick a single colony from the MD plate with transformants, spot it on the YPD plate according to the number, and place the YPD plate in a 30±2℃ incubator for 2-3 days until colonies appear. Pick transformants from the YPD plate according to the number and inoculate them into a centrifuge tube containing 3mL BMGY medium, and culture them in a shaking incubator at 30±2℃ and 200rpm for 48h; centrifuge the bacterial solution cultured in the shaking incubator for 48h at 3000×g for 15min, remove the supernatant, add 1mL of BMMY medium containing 0.5% methanol to the centrifuge tube, and induce culture at 30±2℃ and 220rpm; after induced culture for 48h, centrifuge at 3000×g for 5min, take the supernatant for enzyme activity detection, and screen out transformants with high protease activity, and finally screen out a strain with significantly improved neutral protease activity.

[0081] The neutral protease gene sequence of the screened recombinant strain was extracted and sequenced (Shanghai Biotechnology Co., Ltd.), and the coding gene was named emps (as shown in SEQ ID NO: 2).

[0082] (2) Expression of neutral protease mutants

[0083] The strain was inoculated into a 1L Erlenmeyer flask with 300mL of BMGY liquid culture medium, and cultured on a shaker at 30±2℃ and 220rpm for 48h; centrifuged at 5000rpm for 5min, and the supernatant was gently discarded. Then 100mL of BMMY liquid culture medium containing 0.5% methanol was added to the bacteria, and induced culture was carried out at 30±2℃ and 200rpm for 72h. During the induction culture, methanol solution was added once every 24h to compensate for the loss of methanol, so that the methanol concentration was maintained at about 0.5%. Centrifuged at 12000×g for 10min, the supernatant fermentation liquid was collected, and the enzyme activity was detected.

[0084] Example 3 Determination of thermal stability of neutral protease mutant emps

[0085] Determination of the optimal temperature of neutral protease reaction: The enzyme solutions of the parent protease empr and its neutral protease mutant emps were placed in a water bath at 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ for heat treatment for 5 minutes, and the activity of each neutral protease was determined. The results are shown in Table 1. The parent neutral protease empr has the highest enzyme activity at 50℃, and the optimal reaction temperature of the mutant emps is 55℃. The enzyme activities of the two enzymes at the optimal temperature increased by 7.14%. At each temperature, the enzyme activity of the mutant emps was improved compared with that of empr. At 80℃, the parent neutral protease empr had 10.98% residual enzyme activity, and the mutant emps had 41.47% residual enzyme activity. It can be seen that compared with the parent neutral protease empr, the mutant emps not only has significantly improved enzyme activity, but also significantly improved high temperature resistance.

[0086] Table 1 Determination results of optimal temperature of enzyme reaction

[0087] .

[0088] Neutral protease high temperature stability determination: Take the parent protease empr and its neutral protease mutant emps enzyme solution, place it in a 70°C water bath for heat treatment, sample the enzyme solution at 5min, 10min, 20min, 30min, 40min, 50min, and 60min, and determine the residual enzyme activity of each neutral protease. The results are shown in Table 2. Compared with the parent neutral protease empr, the half-life of the mutant emps is significantly prolonged, and the enzyme activity after 60min of incubation is higher than that of the parent neutral protease empr when incubated for 5min, indicating that the stability of the neutral protease mutant of the present invention in a high temperature environment is significantly improved.

[0089] Table 2 High temperature stability test results

[0090] .

[0091] Example 4 Study on the dehairing performance of neutral protease

[0092] The sheepskin was soaked in water, defleshed and weighed, added to a drum, and water was added to make a solid-liquid ratio of 1:1. The temperature was 50°C, and the parent neutral protease empr was added at 200 IU / g sheepskin. The drum was rotated for 30 minutes and stopped for 30 minutes until all the wool was taken off. Another group was added with a neutral protease mutant emps at a temperature of 55°C at 200 IU / g sheepskin. The drum was rotated for 30 minutes and stopped for 30 minutes until all the wool was taken off.

[0093] The dehairing time of the parent neutral protease empr was 120 minutes, and the dehairing time of the neutral protease mutant emps was 60 minutes. The results showed that when the same enzyme activity units of the parent protease empr and the mutant emps were added and both were at the optimal reaction temperature, the dehairing time of the neutral protease mutant emps was 60 minutes shorter than that of the parent protease empr. The neutral protease mutant emps has a good application prospect in the dehairing process of the leather industry.

[0094] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A neutral protease mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

2. A gene, characterized in that The gene encodes the neutral protease mutant as described in claim 1.

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

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector carries the gene according to claim 2.

5. A recombinant host cell, characterized in that The recombinant host cell comprises the recombinant expression vector according to claim 4.

6. The recombinant host cell according to claim 5, characterized in that The host cell is Pichia pastoris.

7. Use of the neutral protease mutant according to claim 1 in the degradation of proteins and peptide chains.

8. Use of the neutral protease mutant according to claim 1 in the dehairing process of leather making.

9. An enzyme preparation, characterized in that The enzyme preparation comprises the neutral protease mutant according to claim 1.