A heat-resistant alkaline protease combined mutant and its application

Abstract

The present invention belongs to the technical field of protein engineering transformation, and provides a thermostable alkaline protease combination mutant and its application. The parent protease of the alkaline protease mutant is a protease of 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 the amino acid sequence SEQ ID NO: 1. The fermentation enzyme activity of the mutant is 21493.11 U / ml, which is 38% higher than that of the wild-type strain. At the same time, the enzyme activity residual rate of the fermentation broth of the mutant strain after being treated at 80°C for 5 min is 65%, which is 29% higher than that of the wild-type enzyme activity residual rate. The thermostable alkaline protease mutant can be better applied to the industrial field, especially the detergent industry. The application of the mutant will help reduce the production cost of alkaline protease, laying a foundation for its better adaptation to industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and in particular relates to a heat-resistant alkaline protease combined mutant and application thereof. Background Art

[0002] Protease refers to a hydrolase that can degrade proteins into small peptides and amino acids, accounting for 60% of the entire industrial enzyme market. Alkaline protease is a general term for proteases that are active in the neutral to alkaline pH range. Alkaline protease hydrolyzes protein peptide bonds, ester bonds and amide bonds under neutral to alkaline conditions, and its optimal pH is generally 8~11. This enzyme was first discovered in the pancreas of pigs. Alkaline protease is widely present in microorganisms, plants and animals. The main application areas of alkaline protease include detergents, feed, medicines, leather, soybean processing, breweries, meat tenderizing, waste management, photography and diagnosis and other light industrial fields. In 1945, Jaag et al. first used alkaline protease in Bacillus licheniformis Bacillus licheniformis Alkaline proteases were discovered in the human body. Most microbial proteases are extracellular enzymes. Compared with animals and plants, alkaline proteases from microorganisms have the remarkable characteristics of simple separation and purification process, short production cycle and high yield. Alkaline proteases have strong alkali and heat resistance and protein hydrolysis activity. They are mainly used in the detergent industry, especially in the production of laundry and dishwashing detergents. The production strains and research objects of alkaline proteases are mainly Bacillus subtilis ( Bacillus subtilis ) and Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus pumilus ( Bacillus pumilus ), such as the Bacillus licheniformis currently used in my country B.licheniformis 2709, Bacillus pumilus B. pumilus 289 and 209 strains, etc. There are few actinomycetes that produce alkaline protease, mainly from Streptomyces. In addition, some molds can also produce alkaline protease. At present, the overall trend of alkaline protease research in my country is developing well, but domestic enterprises still cannot achieve industrial production of high-end alkaline protease preparations. The main reasons are the monopoly of foreign patented technologies, the lack of excellent enzyme-producing strains in China, the low unit activity of the enzyme, and the poor stability of the enzyme under washing conditions. Therefore, how to improve the activity of alkaline protease is imminent.

[0003] In the past 30 years, protein engineering has become an important tool for developing enzymes that can enhance their activity. Currently, the modification of alkaline proteases using protein engineering technology is mainly focused on improving their thermal stability, antioxidant properties, and specificity. Jaouadi et al. discovered through protein engineering that five amino acids: Leu31, Thr33, Asn99, Phe159, and Gly182 are effective in inhibiting the activity of Bacillus pumilus. B. pumilusThe enzyme activity of SAPB was significantly affected. 12 mutants were constructed by site-directed mutagenesis. The results showed that the triple mutant L31I / T33S / N99Y had the highest specific activity, which was about twice that of the wild-type enzyme. Li et al. performed site-directed mutagenesis at the -1 and -2 positions of its N-terminal sequence to increase the expression level of Streptomyces keratinase Sfp2, and finally found that the specific activity of the L(-1)F mutant (48935 U / mg) was 9 times that of the wild-type Sfp2. Shi Yawei et al. used protein engineering technology to transform PB92 subtilisin to obtain the A188P+V262I alkaline protease combination mutant. Compared with the wild-type, its thermal stability and alkali resistance were significantly improved. It was evaluated in two detergent systems (STPP system and MGDA system) and found that without adding any protective agent and stabilizer, the mutant had better stability and washing performance than the parent enzyme. Protein engineering technology has been around for nearly 40 years, and various technical means have become increasingly mature. Combined with the revelation of a large number of protease spatial structures, it has become an important means to enhance the activity of proteases by amino acid mutations at the genetic level. Summary of the invention

[0004] In view of this, the main purpose of the present invention is to provide an alkaline protease combination mutant and its application. The thermal stability of the alkaline protease combination mutant is greatly improved, which lays a foundation for its better adaptation to industrial production, thereby facilitating the widespread application of alkaline protease in the washing field.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A thermostable alkaline protease combination mutant, wherein the parent protease of the alkaline protease mutant is Bacillus nissori ( Niallia circulans ) alkaline protease NcAP, GenBank SPU21234.1, corresponding to the amino acid sequence SEQ ID NO: 1. The alkaline protease mutant corresponds to the amino acid sequence SEQ ID NO: 2, which comprises an amino acid sequence having at least 90% identity with 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 compared to SEQ ID NO: 1.

[0007] In some embodiments of the invention, the amino acid sequence of the mutant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:2.

[0008] In some more specific embodiments, the amino acid sequence of the mutant is at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identical to SEQ ID NO:2.

[0009] The present invention also provides a method for preparing an alkaline protease combination mutant, comprising the following steps:

[0010] Step 1: Obtain a DNA molecule encoding an alkaline protease mutant, wherein the alkaline protease mutant comprises an amino acid sequence having at least 90% identity with SEQ ID NO:1.

[0011] Step 2: fusing the DNA molecule obtained in step 1 with an expression vector to construct a recombinant expression vector and transforming a host cell;

[0012] Step 3: Induce host cells containing the recombinant expression vector to express recombinant protein, and separate and purify the expressed recombinant protein.

[0013] In some embodiments of the present invention, the alkaline protease mutant described in step 1 comprises substitutions of amino acids in the following group: A126T, A127V, V141I, S147A, T148Q, L191I, V230I, V259I, L301I, V310I, S364N, T365S, N366S, L367Q, Y368F.

[0014] In some embodiments of the present invention, the host cell in step 2 is Bacillus subtilis ( Bacillus subtilis ).

[0015] In some embodiments of the present invention, the host cell in step 2 is Bacillus licheniformis ( Bacillus licheniformis ).

[0016] The present invention is based on alkaline protease NcAP, and provides a combined mutant comprising 15 mutation sites in A126T, A127V, V141I, S147A, T148Q, L191I, V230I, V259I, L301I, V310I, S364N, T365S, N366S, L367Q, and Y368F, and the alkaline protease mutant is named NcAPm2. The fermentation enzyme activity of the mutant NcAPm2 is 21493.11 U / ml, which is 29% higher than that of the wild-type strain. At the same time, through a thermal stability experiment test, the fermentation broth of the mutant strain NcAPm2 has an enzyme activity residual rate of 65% after being treated at 80°C, and the thermal stability is improved by 29% compared with the wild-type strain NcAP. The alkaline protease mutant NcAPm2 has higher enzyme activity and better thermal stability than the wild type. The good performance of the alkaline protease mutant NcAPm2 helps to expand the scope of use of alkaline protease and lays the foundation for its better adaptation to industrial production. Implementation

[0017] The method of the present invention is further described below with reference to examples. The experimental methods for which specific conditions are not indicated in the examples can be operated under conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide compiled by J. Sambrook (Sambrook, 2001), or under the conditions recommended by the manufacturer. Those skilled in the art can better understand and grasp the present invention with the help of the examples. However, the method for implementing the present invention should not be limited to the specific method steps recorded in the examples of the present invention.

[0018] The following examples are provided to better illustrate the present invention, and those skilled in the art can better understand and grasp the present invention with the help of the examples. However, the protection and claims of the present invention are not limited to the examples provided.

[0019] Alkaline protease mutant annotation: "amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the alkaline protease mutant. For example, S259K means that the amino acid at position 259 is replaced by Lys (K) from Ser (S) of the original alkaline protease, and the position number corresponds to the number in SEQ ID NO: 1 in the attached sequence table.

[0020] For the culture medium involved in the embodiments of the present invention, the specific formula is as follows:

[0021] LB liquid medium: tryptone 1%, yeast powder 0.5%, NaCl 1%;

[0022] LB plate: 1% tryptone, 0.5% yeast powder, 1% NaCl, 2% agar;

[0023] Solution A: 0.4 g yeast extract, 0.08 g casein hydrolysate, dissolved in 40 mL water.

[0024] Solution B: 5 g glucose dissolved in 10 mL water.

[0025] Solution C: 4.8 g KH2PO4, 11.2 g K2HPO4, 0.16 g MgSO4•7H2O, 0.8 g trisodium citrate, 1.6 g (NH4)2SO4, dissolved in 200 mL water.

[0026] Solution D: 0.9 g MnCl2•4H2O, 1.415 g boric acid, 0.68 g FeSO4•7H2O, 13.45 mg CuCl2•2H2O, 23.5 mg ZnSO4•7H2O, 20.2 mg CoCl2•6H2O, 12.6 mg sodium permolybdate, 0.855 g sodium tartrate, dissolved in 500 mL water.

[0027] Solution E: 2.16 g MgCl2•6H2O, dissolved in 20 mL water.

[0028] Solution F: 147 mg CaCl2 dissolved in 20 mL water.

[0029] Solution G: 36.5 g sorbitol dissolved in 100 mL water.

[0030] GMⅠ: 10 mL solution A, 1.5 mL solution B, 25 mL solution C, 100 uL solution D, 25 mL solution G, add sterile water to 100 mL.

[0031] GMII: 98 mL GMⅠ, 1 mL solution E, 1 mL solution F, mix well.

[0032] GMⅢ: 9 mL GMⅡ, 1 mL glycerol.

[0033] Seed culture medium: yeast extract powder 0.5%, tryptone 0.5%, glucose 1%, K2HPO4 1.8%;

[0034] Fermentation medium: yeast powder 1~2%, soybean cake powder 2~5%, maltodextrin 5~10%, sodium citrate 0.1~0.5%, CaCl2 0.1~0.5%, MgSO4 0.1~0.5%, K2HPO4 0.5~2%.

[0035] The method for determining the activity of alkaline protease in the practice of the present invention can be as follows:

[0036] Method I for determining the activity of alkaline protease:

[0037] Alkaline protease activity determination method: refer to GB / T23527-2009 Appendix B Folin method, the specific reaction process is as follows.

[0038] Under certain temperature and pH conditions, proteases hydrolyze casein substrates to produce amino acids containing phenolic groups (such as tyrosine, tryptophan, etc.). Under alkaline conditions, Folin reagent is reduced to generate molybdenum blue and tungsten blue. The absorbance of the solution is measured at a wavelength of 680 nm using a spectrophotometer. The enzyme activity is directly proportional to the absorbance, from which the enzyme activity of the product can be calculated. The definition of protease activity, that is, protease activity is expressed in protease activity units, is defined as 1 g of solid enzyme powder (or 1 ml of liquid enzyme), under certain temperature and pH conditions, 1 min hydrolysis of casein produces 1 μg of tyrosine, that is, 1 enzyme activity unit, expressed in μ / g (μ / ml).

[0039] Reagents and solutions

[0040] (1) Folin reagent (Folin: water = 1:2); (2) 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 g / L casein solution; (6) 100 μg / mL and 1 mg / ml L-tyrosine standard solutions; (7) 6.54% trichloroacetic acid.

[0041] Enzyme activity assay

[0042] (1) Preparation of standard curve: Prepare L-tyrosine standard solutions with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / mL, respectively. Take 1.00 ml of each standard solution, add 5.00 ml of 0.4 mol / L sodium carbonate solution and 1.00 ml of Folin reagent solution, shake evenly, place in a 40℃ water bath for 20 min to develop color, take out and use a spectrophotometer at a wavelength of 680 nm, 10 mm cuvette, and use tube 0 without tyrosine as blank to measure its absorbance. Use absorbance A as the ordinate and tyrosine concentration C as the abscissa to draw a standard curve (this line should pass through the zero point).

[0043] (2) Enzyme activity determination

[0044] Take 1 ml of pre-diluted enzyme solution, then add 1 ml of 40℃ preheated 10% casein, react at 40℃ for 10 min; then add 2 ml of trichloroacetic acid (concentration 6.54%), mix well and let stand at room temperature for 10 min to terminate the reaction. Take out 1 ml of the terminated reaction solution, then add 5 ml of 42.4 g / L sodium carbonate solution, then add 1 ml of Folin reagent, color reaction at 40℃ for 20 min; finally measure OD 680 value.

[0045] (3) Calculation

[0046] Read the enzyme activity of the final dilution of the sample from the standard curve in μ / mL. The enzyme activity of the sample is calculated according to the following formula:

[0047] X=A×K×4 / 10×n=2 / 5×A×K×n

[0048] Where: X——enzyme activity of the sample (μ / g or μ / ml)

[0049] A——average absorbance of parallel samples

[0050] K——Absorption constant

[0051] 4——Total volume of reaction reagents (ml)

[0052] 10——Reaction time 10min, calculated as 1min

[0053] n——dilution multiple.

[0054] Method II for determining the activity of alkaline protease:

[0055] Proteolytic activity can be determined by a method using Suc-AAPF-PNA substrate. Suc-AAPF-PNA is an abbreviation for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and it is a blocking peptide that can be cleaved by endoproteases. After cleavage, a free PNA molecule is released and it has a yellow color and can therefore be measured by visible light spectrophotometry at a wavelength of 405 nm. Suc-AAPF-PNA substrate is manufactured by Bachem (Catalog No. L1400 dissolved in DMSO).

[0056] The protease samples to be analyzed were diluted in residual activity buffer (100 mM Tris pH 8.6). The assay was performed by transferring 30 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 μl of substrate working solution (0.72 mg / ml in 100 mM Tris pH 86). The solution was mixed at room temperature and absorption was measured at OD405nm every 20 seconds for 5 minutes.

[0057] Under a given set of conditions, the slope of the time-dependent absorbance curve (absorbance per minute) is directly proportional to the activity of the protease in question. The protease sample should be diluted to a level where the slope is linear.

[0058] Example 1: Construction and expression of wild-type alkaline protease NcAP and mutant NcAPm2: The wild-type alkaline protease NcAP and mutant NcAPm2 of the present invention can be constructed and expressed by methods well known to those skilled in the art.

[0059] The amino acid sequence of the wild-type alkaline protease NcAP is SEQ ID NO: 1. According to the amino acid sequence of SEQ ID NO: 1, the optimized nucleic acid sequence SEQ ID NO: 3 is obtained. The amino acid sequence of the alkaline protease mutant NcAPm2 is SEQ ID NO: 2. According to the amino acid sequence of SEQ ID NO: 2, the optimized nucleic acid sequence SEQ ID NO: 4 is obtained. SEQ ID NO: 3 and SEQ ID NO: 4 are synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. SEQ ID NO: 3 and SEQ ID NO: 4 are respectively connected to the pBE2R vector using the Gibson Assembly method, and are transformed into Escherichia coli DH5α by the heat shock method. The plasmids are extracted and sequenced to obtain the alkaline protease recombinant plasmids pBE2R-NcAP and pBE2R-NcAPm2.

[0060] The correctly sequenced recombinant plasmids pBE2R-NcAP and pBE2R-NcAPm2 were respectively transferred into the competent cells WB600. The specific transformation process was as follows: a single colony of WB600 grown on the LB plate was picked up with a pipette tip and placed in 2 mL GMⅠ, and cultured for 12 h; the overnight culture was added to 98 mL GMⅠ, and cultured at 37°C, 200 rpm for about 4 h; 10 mL of the culture was added to 90 mL GMII, and cultured at 37°C, 200 rpm for about 1.5 h; the bacteria were placed in an ice water bath for 30 min, centrifuged at 4000 rpm, 4°C for 30 min, and the supernatant was removed; 10 mL GMⅢ was added and mixed to obtain the competent cells WB600. Then, 5 μL of recombinant plasmid was added to 500 μL competent cells, and the competent cells were directly placed in a shaking incubator at 37°C and 200 rpm for 1.5 h, centrifuged at low speed for 3 min, and part of the supernatant was discarded and evenly spread on a skim milk powder medium plate containing 40 μg / mL kanamycin, and cultured in a constant temperature incubator at 37°C for 12 h. The single colonies on the plate the next day were recombinant strains, named WB600 / pBE2R-NcAP and WB600 / pBE2R-NcAPm2, respectively. The recombinant engineered bacteria were inoculated in 5 mL LB liquid medium (peptone 1%, NaCl 1%, yeast powder 0.5%), and cultured at 37°C and 200 rpm for 12 h. The bacterial solution was transferred to the fermentation enzyme production medium at a 2% inoculum, and cultured at 37°C and 200 rpm for 72 h.

[0061] Example 2: Isolation and purification of alkaline protease: After fermentation, the fermentation broth was centrifuged at 13000 r / min 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. Samples were taken to measure the activity of alkaline protease. WB600 / pBE2R-NcAP was used as a control strain.

[0062] The experimental results are shown in the following table:

[0063] As can be seen from the table, the enzyme activity of the recombinant strain was significantly improved. The enzyme activity of the recombinant strain WB600 / pBE2R-NcAPm2 reached 21493.11 U / mL, which was 38% higher than that of the original strain.

[0064] Example 3: Thermal stability analysis of alkaline protease mutants:

[0065] Take 5 ml of the supernatant of the alkaline protease NcAP and its mutant NcAPm2 after fermentation, place them in a water bath at 80°C for 5 minutes, and immediately determine the alkaline protease activity. The enzyme activity determination method is carried out according to method I or method II, and the test results are shown in the following table:

[0066] Strain name Alkaline protease activity (U / ml) Alkaline protease activity (after treatment) (U / ml) Residual enzyme activity WB600 / pBE2R-NcAP 15574.72 5606.90 36% WB600 / pBE2R- NcAPm2 21493.11 13970.52 65%

[0067] Without any protective agent or stabilizer, the wild-type alkaline protease NcAP and its mutant NcAPm2 were kept at 80℃ for 5 min. It can be clearly seen that the residual activity of the mutant NcAPm2 was significantly higher than that of the wild-type NcAP. The residual enzyme activity of the fermentation broth of the mutant strain WB600 / pBE2R-NcAPm2 was 65% after being treated at 80℃ for 5 min, which was 29% higher than that of the control strain WB600 / pBE2R-NcAP.

[0068] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A thermostable alkaline protease combined mutant, characterized in that: The amino acid sequence of the heat-resistant alkaline protease combination mutant is shown in SEQ ID NO:2.