CN116478970A

CN116478970A - Heat-resistant alkaline protease combined mutant and application thereof

Info

Publication number: CN116478970A
Application number: CN202310517227.2A
Authority: CN
Inventors: 汪小杰; 冯娟; 邓子新
Original assignee: Shanghai Jikaixing Biotechnology Co ltd
Current assignee: Shanghai Jikaixing Biotechnology Co ltd
Priority date: 2023-05-10
Filing date: 2023-05-10
Publication date: 2023-07-25

Abstract

The invention belongs to the technical field of protein engineering and provides a heat-resistant alkaline protease combined mutant and application thereof. The parent protease of the alkaline protease mutant is a protease of bacillus niloticus (Niallia circulans), and the alkaline protease mutant comprises the following amino acid substitutions: A126T/A127V/V141I/S147A/T148Q/L191I/V230I/V259I/L301I/V310I/S364N/T365S/N366S/L367Q/Y368F, wherein the position corresponds to the amino acid sequence SEQ ID NO:1, and a polypeptide having an amino acid position. The mutant ferment enzyme activity is 21493.11U/ml, which is improved by 38% compared with the wild strain. Meanwhile, the enzyme activity residual rate of the fermentation broth of the mutant strain is 65% after being treated for 5min at 80 ℃, and is improved by 29% compared with the wild type enzyme activity residual rate. The heat-resistant alkaline protease mutant can be better applied to the industrial field, in particular to the detergent industry. The application of the mutant is beneficial to reducing the production cost of alkaline protease enzyme, and lays a foundation for better adapting to industrial production.

Description

Heat-resistant alkaline protease combined mutant and application thereof

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to a heat-resistant alkaline protease combined mutant and application thereof.

Background

Protease refers to a hydrolase capable of degrading proteins into small peptides and amino acids, accounting for 60% of the whole industrial enzyme market. Whereas alkaline proteases are a generic term for proteases that are active in the neutral to alkaline pH range. The alkaline protease hydrolyzes protein peptide bonds, ester bonds and amide bonds under neutral to alkaline conditions, and the optimal pH is generally 8-11. The enzyme species was found in the pig pancreas at the earliest. Alkaline proteases are widely found in microorganisms, plants and animals. Alkaline protease main componentThe application fields include the fields of light industry such as detergents, feeds, medicines, leather, soybean processing, breweries, meat tenderization, waste management, photography and diagnosis. Jaag et al were earliest in Bacillus licheniformis in 1945Bacillus licheniformisAlkaline proteases were found. The microbial protease belongs to extracellular enzymes, and compared with animals and plants, the microbial alkaline protease has the remarkable characteristics of simple separation and purification process, short production period and high yield. The alkaline protease has strong alkali resistance, heat resistance and activity of hydrolyzed protein, and is mainly applied to the detergent industry, especially the production of laundry and tableware detergents. The alkaline protease producing strain and research object are mainly bacillus subtilisBacillus subtilis) And Bacillus licheniformisBacillus licheniformis) Bacillus pumilus @Bacillus pumilus) Such as Bacillus licheniformis currently used in ChinaB.licheniformis2709 Bacillus pumilusB. pumilus 289 and 209 strains, etc. Less actinomycetes produce alkaline protease and are mainly derived from streptomycete. In addition, some molds may also produce alkaline proteases. At present, the general trend of the research of alkaline protease in China is better, but domestic enterprises cannot realize the industrial production of high-end alkaline protease preparations, and the main reasons are monopoly of foreign patent technology, lack of excellent domestic enzyme-producing strains, low unit activity of enzyme, poor stability of enzyme under washing conditions and the like. Therefore, it is urgent how to improve the activity of alkaline protease.

Protein engineering has become an important tool for developing enhanced enzyme activity over the last 30 years. Currently, modification of alkaline protease by protein engineering technology is mainly focused on research for improving thermal stability, oxidation resistance, specificity and the like of alkaline protease. Jaouadi et al found 5 amino acids by protein engineering: leu31, thr33, asn99, phe159 and Gly182 against Bacillus pumilusB. pumilusThe SAPB enzyme activity has an important effect, and 12 mutants are constructed through site-directed mutagenesis, so that the triple mutant L31I/T33S/N99Y has the highest specific activity, which is about 2 times that of the wild-type enzyme. Li et al by site-directed mutagenesis of the positions at the-1 and-2 positions of the N-terminal sequenceTo increase the expression level of S.keratinase Sfp2, it was finally found that the specific activity of L (-1) F mutant (48935U/mg) was 9 times that of wild-type Sfp 2. Dan Yawei and the like are modified by utilizing a protein engineering technology to obtain an A188 P+V267I alkaline protease combined mutant, compared with a wild type, the thermal stability and the alkali resistance of the mutant are obviously improved, and the mutant has better stability and washing performance compared with a parent enzyme on the premise of not adding any protective agent and stabilizing agent when evaluated by two detergent systems (STPP system and MGDA system). In recent 40 years, protein engineering technology has become mature, and various technical means are combined with the disclosure of a large number of protease space structures, so that the improvement of the activity of protease through amino acid mutation at the gene level is an important means for improving the activity of the protease.

Disclosure of Invention

In view of the above, the main purpose of the invention is to provide an alkaline protease combination mutant and application thereof, wherein the thermal stability of the alkaline protease combination mutant is greatly improved, and a foundation is laid for better adapting to industrial production, so that the alkaline protease combination mutant is favorable for wide application in the washing field.

In order to achieve the above object, the present invention provides the following technical solutions:

a heat-resistant alkaline protease combined mutant, wherein the parent protease of the alkaline protease mutant is bacillus niloticus @ or a parent protease of the alkalineNiallia circulans) The alkaline protease NcAP of (C) and GenBank is SPU21234.1, and corresponds to the amino acid sequence SEQ ID NO. 1. The alkaline protease mutant, corresponding to the amino acid sequence SEQ ID NO. 2, comprises an amino acid sequence having at least 90% identity to SEQ ID NO. 1 and comprises an amino acid substitution at a position selected from the group consisting of: a126T, a127V, V141I, S147A, T148Q, L191I, V230I, V259I, L301I, V310I, S364N, T365S, N366S, L367Q, Y368F.

In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identity as compared to SEQ ID NO. 2.

In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or at least 99.9% identity as compared to SEQ ID NO. 2.

The invention also provides a preparation method of the alkaline protease combined mutant, which comprises the following steps:

step 1: obtaining a DNA molecule encoding an alkaline protease mutant comprising an amino acid sequence having at least 90% identity to SEQ ID No. 1.

Step 2: fusing the DNA molecule obtained in the step 1 with an expression vector, constructing a recombinant expression vector, and transforming a host cell;

step 3: inducing host cells containing the recombinant expression vector to express the recombinant protein, and separating and purifying the expressed recombinant protein.

In some embodiments of the invention, the alkaline protease mutants described in step 1 comprise amino acid substitutions in the group consisting of: a126T, a127V, V141I, S147A, T148Q, L191I, V230I, V259I, L301I, V310I, S364N, T365S, N366S, L367Q, Y368F.

In some embodiments of the present invention, the host cell of step 2 is Bacillus subtilisBacillus subtilis）。

In some embodiments of the present invention, the host cell of step 2 is Bacillus licheniformisBacillus licheniformis）。

The invention is based on alkaline protease NcAP, provides a combined mutant comprising 15 mutation sites in A126T, A127V, V141I, S147A, T148Q, L191I, V230I, V259I, L301I, V310I, S364N, T365S, N366S, L367Q, Y368F, which alkaline protease mutant is named NcAPm2. The activity of the mutant NcAPm2 fermentation enzyme is 21493.11U/ml, which is improved by 29% compared with the wild strain. Meanwhile, according to the thermal stability test, the enzyme activity residual rate of the fermentation broth of the mutant strain NcAPm2 after being treated at 80 ℃ is 65%, and compared with the wild strain NcAP, the thermal stability is improved by 29%. Compared with the wild type, the alkaline protease mutant NcAPm2 has higher enzyme activity and better thermal stability, and the good performance of the alkaline protease mutant NcAPm2 is beneficial to expanding the application range of alkaline protease, thus laying a foundation for better adapting to industrial production.

Description of the embodiments

The method of the present invention will be further described with reference to examples, in which the experimental method without specific conditions are not specified, and may be performed under conventional conditions, such as those described in molecular cloning Experimental guidelines written in J.Sambucus (Sambrook, 2001), et al, or under conditions recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art by reference to examples. However, the methods of practicing the present invention should not be limited to the specific method steps described in the examples of the present invention.

The following examples are set forth to provide a better illustration of the present invention and are intended to provide a better understanding and appreciation of the invention by those skilled in the art. However, the protection of the invention and the scope of the claims are not limited to the cases provided.

Labeling of alkaline protease mutants: "amino acid substituted at the original amino acid position" is used to denote the mutated amino acid in the alkaline protease mutant. As shown in S259K, the amino acid at position 259 is replaced by lys (K) from Ser (S) of the original alkaline protease, and the number at the position corresponds to the number in SEQ ID No. 1 of the accessory sequence Listing.

For the culture medium related in the embodiment of the invention, the specific formula is as follows:

LB liquid medium: 1% of tryptone, 0.5% of yeast powder and 1% of NaCl;

LB plate: 1% of tryptone, 0.5% of yeast powder, 1% of NaCl and 2% of agar;

solution a:0.4g of yeast extract, 0.08g of casein hydrolysate, was dissolved in 40mL of water.

Solution B:5g of glucose, dissolved in 10mL of water.

Solution C:4.8gKH ₂ PO ₄ ，11.2gK ₂ HPO ₄ ，0.16gMgSO ₄ •7H ₂ O,0.8g trisodium citrate, 1.6 gg(NH ₄ ) ₂ SO ₄ Dissolved in 200mL of water.

Solution D:0.9g MnCl ₂ •4H ₂ O,1.415g boric acid, 0.68 g FeSO ₄ •7H ₂ O，13.45 mg CuCl ₂ •2H ₂ O，23.5 mg ZnSO ₄ •7H2O，20.2mg CoCl ₂ •6H ₂ O, 12.6. 12.6 mg sodium permanganate, 0.855. 0.855 g sodium tartrate, was dissolved in 500mL of water.

Solution E:2.16g MgCl2.6H2O in 20 mL water.

Solution F:147 mg CaCl2 was dissolved in 20 mL water.

Solution G:36.5g of sorbitol, dissolved in 100mL water.

GM i: 10mL of solution a,1.5 mL solution B,25 mL solution C,100 uL solution D,25 mL solution G, and sterilized water was added to 100mL.

GMII:98 mLGMI, 1mL solution E,1mL solution F, and mix well.

GM iii: 9 mLGMII, 1mL Glycerol.

Seed culture medium: yeast extract 0.5%, tryptone 0.5%, glucose 1%, K ₂ HPO ₄ 1.8%；

Fermentation medium: 1-2% of yeast powder, 2-5% of bean cake powder, 5-10% of maltodextrin, 0.1-0.5% of sodium citrate and CaCl ₂ 0.1~0.5%，MgSO ₄ 0.1~0.5%，K ₂ HPO ₄ 0.5~2%。

The method for measuring the enzyme activity of the alkaline protease in the implementation of the invention can adopt the following method:

method for measuring enzyme activity of alkaline protease:

the method for measuring the enzyme activity of alkaline protease comprises the following steps: reference is made to the GB/T23527-2009 annex B Fulin method, the specific reaction procedure being as follows.

The protease hydrolyzes casein substrate under certain temperature and pH condition to generate amino acid containing phenolic group (such as tyrosine, tryptophan, etc.), and in alkaline condition, folin reagent (Folin) is reduced to generate molybdenum blue and tungsten blue, and the absorbance of the solution is measured by spectrophotometer at 680 and nm. The enzyme activity is directly proportional to the absorbance, whereby the enzyme activity of the product can be calculated. The protease activity is defined as 1 g solid enzyme powder (or 1ml liquid enzyme) and 1 mu g tyrosine is produced by hydrolyzing casein for 1min under the conditions of a certain temperature and a certain pH value, namely 1 enzyme activity unit is expressed in mu/g (mu/ml).

Reagents and solutions

(1) Forskolin (Folin) reagent (fos Linshui =1:2); (2) 42.4. 42.4 g/L sodium carbonate solution; (3) 0.5 mol/L sodium hydroxide solution; (4) boric acid buffer (pH 10.5); (5) 10.0. 10.0 g/L casein solution; (6) L-tyrosine standard solution of 100. Mu.g/mL and 1 mg/mL; (7) 6.54% trichloroacetic acid.

Enzyme activity assay

(1) And (3) preparing a standard curve: l-tyrosine standard solutions were prepared at concentrations of 0. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL, 40. Mu.g/mL and 50. Mu.g/mL, respectively. Taking 1.00 of standard solution and ml of standard solution respectively, adding 5.00 ml of 0.4 mol/L sodium carbonate solution and 1.00 ml of forskolin reagent use solution respectively, oscillating uniformly, placing in a water bath at 40 ℃ for color development for 20min, taking out a cuvette with the wavelength of 680 nm and 10 mm by using a spectrophotometer, and measuring the absorbance of the cuvette respectively by taking a 0 tube without tyrosine as a blank. The absorbance a is taken as the ordinate and the concentration C of tyrosine is taken as the abscissa, and a standard curve is drawn (this line should pass through the zero point).

(2) Enzyme activity assay

Taking a proper amount of pre-diluted enzyme solution 1ml, then adding 1ml of 40 ℃ preheated 10% casein, and reacting for 10min at 40 ℃; then, 2 ml trichloroacetic acid (concentration: 6.54%) was added, and after mixing, the mixture was allowed to stand at room temperature for 10 minutes to terminate the reaction. Taking out 1ml of the terminated reaction solution, adding 5ml of 42.4 g/L sodium carbonate solution, then adding 1ml Folin (Folin) reagent, and performing color development reaction at 40 ℃ for 20min; finally, OD is measured ₆₈₀ Values.

(3) Calculation of

The enzyme activity of the final dilutions of the samples was read from the standard curve in μ/mL. The enzyme activity of the sample was calculated according to the following formula:

X=A×K×4/10×n=2/5×A×K×n

wherein: x-enzyme Activity of sample (mu/g or mu/ml)

Average absorbance for parallel test of A-sample

K-absorption constant

4-total volume of reagents (ml)

10-reaction time 10min, 1min

n-dilution factor.

Method II for measuring enzyme activity of alkaline protease:

proteolytic activity can be determined by a method employing a Suc-AAPF-PNA substrate. Suc-AAPF-PNA is an abbreviation for N succinyl-alanine-proline-phenylalanine-p-nitroaniline, and it is a blocking peptide that can be cleaved by endoprotease, after cleavage, a free PNA molecule is released, and it has a yellow colour and can therefore be measured by visible spectrophotometry at wavelength 405 nm. Suc-AAPF-PNA substrates were manufactured by Baheng (Bachem) (catalog number L1400 dissolved in DMSO).

The protease sample to be analyzed is diluted in residual active buffer (100 mM Tris pH 8.6). The assay was performed by transferring 30. Mu.l of diluted enzyme sample to a 96 well microtiter plate and adding 70. Mu.l of substrate working solution (0.72 mg/mI in 100mM Tris pH 86). The solution was mixed at room temperature and absorbance was measured every 20 seconds over 5 minutes at 0D405 nm.

The slope of the time-dependent absorption curve (absorbance per minute) is directly proportional to the activity of the protease in question under a given set of conditions. The protease sample should be diluted to a level where the slope is linear.

Example 1: construction and expression of alkaline protease wild-type NcAP and mutant NcAPm 2: construction of alkaline protease wild-type NcAP and mutant NcAPm2 of the invention can be constructed and expressed by methods well known to those skilled in the art.

The amino acid sequence of the alkaline protease wild type NcAP is SEQ ID NO. 1, and the optimized nucleic acid sequence SEQ ID NO. 3 is obtained according to the amino acid sequence of the SEQ ID NO. 1. The amino acid sequence of the alkaline protease mutant NcAPm2 is SEQ ID NO. 2, and the optimized nucleic acid sequence SEQ ID NO. 4 is obtained according to the amino acid sequence of the SEQ ID NO. 2, and SEQ ID NO. 3 and SEQ ID NO. 4 are synthesized by the division of biological engineering (Shanghai) Co. SEQ ID NO. 3 and SEQ ID NO. 4 are respectively connected to a pBE2R vector by using a Gibson Assembly method, escherichia coli DH5 alpha is transferred by using a heat shock method, and plasmids are subjected to sequencing determination to obtain alkaline protease recombinant plasmids pBE2R-NcAP and pBE 2R-NcAPm 2.

The recombinant plasmids pBE2R-NcAP and pBE 2R-NcAPm 2 with correct sequence are respectively transferred into competent cells WB600, and the specific transformation process is as follows: picking WB600 single colony growing on LB plate with gun head in 2 mLGMI, culturing 12 h; the overnight cultured broth was added to 98 mLGMI and incubated at 37℃and 200 rpm for approximately 4 h; adding 10mL bacteria solution into 90 mLGMII, and culturing at 37deg.C and 200 rpm for about 1.5 h; centrifuging the thalli in ice water bath at 30 min,4000 rpm,4 ℃ for 30 min, and removing the supernatant; adding 10 mLGM III, and mixing to obtain competent cell WB600. Then, 5. Mu.L of recombinant plasmid was added to 500. Mu.L of competent cells, and the competent cells were directly subjected to shaking culture at 200 rpm at 37℃for 1.5. 1.5 h, low-speed centrifugation for 3 min, and part of the supernatant was discarded, and the resultant was uniformly spread on a skim milk powder medium plate containing 40. Mu.g/mL kanamycin, and cultured in a constant temperature incubator at 37℃for 12. 12 h. The single colonies on the next day plates were designated as recombinant strains, WB600/pBE 2R-NcAP and WB600/pBE 2R-NcAPm 2, respectively. The recombinant engineering bacteria are inoculated in 5mL LB liquid culture medium (peptone 1%, naCl 1% and yeast powder 0.5%), the culture is carried out at 37 ℃ under shaking at 200 rpm for 12 h, the bacterial solutions are respectively transferred into fermentation enzyme production culture medium according to 2% bacterial grafting amount, and the culture is carried out at 37 ℃ under shaking at 200 rpm for 72 h.

Example 2: separation and purification of alkaline protease: after the fermentation was completed, the fermentation broth 13000/r/min was centrifuged for 15 min, and then the supernatant was filtered on a positive pressure filter with a 0.22 μm membrane to remove residual bacillus subtilis. Sampling to measure the enzyme activity of alkaline protease. WB600/pBE 2R-NcAP was used as control strain.

The experimental results are shown in the following table:

as can be seen from the table, the enzyme activity of the recombinant strain is obviously improved, and the enzyme activity of the recombinant strain WB600/pBE 2R-NcAPm 2 reaches 21493.11U/mL, which is improved by 38% compared with the original strain.

Example 3: thermal stability analysis of alkaline protease mutants:

taking 5ml of each supernatant of the fermented alkaline protease NcAP and the mutant NcAPm2 thereof, placing the supernatant in a water bath kettle at 80 ℃ for reaction for 5min, and immediately measuring the activity of the alkaline protease. The enzyme activity determination method is carried out according to the method I or the method II, and the test results are shown in the following table:

strain name	Alkaline protease enzyme Activity (U/ml)	Alkaline protease enzyme Activity (after treatment) (U/ml)	Residual rate of enzyme activity
				WB600/pBE2R-NcAP	15574.72	5606.90	36%
WB600/pBE2R- NcAPm2	21493.11	13970.52	65%

On the premise of not adding any protective agent and stabilizing agent, the wild type alkaline protease NcAP and the mutant NcAPm2 thereof are kept at 80 ℃ for 5min, and the residual activity of the mutant NcAPm2 is obviously higher than that of the wild type NcAP. The enzyme activity residual rate of the fermentation broth of the mutant strain WB600/pBE 2R-NcAPm 2 after being treated at 80 ℃ for 5min is 65%, and compared with the comparison strain WB600/pBE 2R-NcAP, the enzyme activity residual rate is improved by 29%.

The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent structural changes made by the content of the present invention or direct/indirect application in other related technical fields are included in the scope of the present invention.

Claims

1. A thermostable alkaline protease combination mutant characterized by: the parent protease of the alkaline protease mutant is an alkaline protease of bacillus niloticus (Niallia circulans), and the alkaline protease mutant comprises the following amino acid substitutions: a126T, a127V, V141I, S147A, T148Q, L191I, V230I, V259I, L301I, V310I, S364N, T365S, N366S, L367Q, Y368F, wherein the position corresponds to the amino acid position of the polypeptide of amino acid sequence SEQ ID No. 1.

2. The thermostable alkaline protease combination mutant according to claim 1, characterized in that: the alkaline protease mutants further comprise a a16t+a316v+v141 i+s147a+t148q+l191i+v230i+v259i+l301i+v310i+s364n+t365s+n366s+l367q+y368F substitution combination.

3. The thermostable alkaline protease mutant according to any one of claims 1-2, characterized in that: the parent protease has an amino acid sequence with at least 95% sequence identity to the amino acid sequence SEQ ID NO. 1.

4. The thermostable alkaline protease combination mutant according to any one of claims 1-2, characterized in that: the parent protease has an amino acid sequence represented by SEQ ID NO. 2.

5. The thermostable alkaline protease combination mutant according to claim 4, wherein: the parent protease has an amino acid sequence with at least 95% sequence identity to the amino acid sequence SEQ ID NO. 2.