Low-temperature alkaline amylase and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and relates to low-temperature alkaline amylase as well as a coding gene and application thereof. Compared with the original low-temperature alkaline amylase, the amylase mutant provided by the invention has the advantages that the enzymatic property is optimized, and the optimal reaction pH is 7.5-10.5. The low-temperature alkaline amylase mutant has the advantages that the amylase can keep high activity under the low-temperature condition, the optimal reaction temperature range is 20-40 DEG C, the low-temperature alkaline amylase mutant is more suitable for a detergent environment, and the enzyme activity can be kept at 95% or above when the low-temperature alkaline amylase mutant is stirred for 40 min under the condition of 25 DEG C in a detergent containing 0.5-5 g / L of the low-temperature alkaline amylase mutant. In a preparation experiment, the average fermentation enzyme activity in a 50L fermentation tank is improved by 242% compared with the original enzyme activity, the production cost of the amylase can be further reduced, and wide application of the amylase in the detergent industry is promoted.

Description

Technical field: The invention belongs to the technical field of bioengineering and relates to a low-temperature alkaline amylase and a coding gene and application thereof. Background technology: Amylase is a biological enzyme that can hydrolyze starch or similar polysaccharides. It is divided into three categories according to the optimal pH: acid amylase, general amylase and alkaline amylase. The application pH range of alkaline amylase is pH9.0-11. It is mainly used in textile desizing, detergent addition, paper desizing, environmental management and starch processing.

[0003] Amylase can be added to detergent to remove starchy dirt. Due to the limitation of washing conditions, the amylase to be added needs to have high enzyme activity under low temperature conditions and be able to tolerate alkaline environment. Currently, there are commercial products of this type of amylase, which are all obtained through microbial fermentation. However, due to the low production efficiency of this type of amylase, the production cost is high, and amylase is not widely used as a detergent additive.

[0004] Tjalsma H (2004), Yang Mingming (2013) and others have shown that the optimization of amino acid sequences in the Bacillus subtilis expression system can improve the transcription level, thereby increasing protein expression. Ma Yingfang studied how increasing the positive charge in the amino acid sequence can improve protein secretion efficiency and enzyme production. Xia Yu (Jiangnan University, 2007) found that the modification of the Bacillus subtilis signal peptide affects protein secretion expression.

[0005] The purpose of the present invention is to provide a low-temperature alkaline amylase with superior performance through amino acid sequence modification and recombination under the existing low-temperature alkaline amylase resource conditions of the enterprise, and the low-temperature alkaline amylase is mainly used in the detergent industry. Summary of the invention: The main purpose of the present invention is to provide a low-temperature alkaline amylase and its encoding gene, a recombinant expression vector and a recombinant strain containing the gene, and the application of the amylase, aiming to solve the problem in the prior art that the low fermentation enzyme activity level leads to a high price of amylase for detergents.

[0007] To achieve the above objectives, one of the technical solutions provided by the present invention is an amylase mutant, which is obtained by deleting the amino acid residues at positions 6-8 and 16-21 based on the original amylase amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence of the mutant is shown in SEQ ID NO.2.

[0008] The present invention also provides a gene encoding the mutant; Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0009] The second technical solution provided by the present invention is the application of the amylase mutant described in the first technical solution, especially in detergents.

[0010] The third technical solution provided by the present invention is a recombinant vector or recombinant strain comprising the above-mentioned amylase mutant encoding gene; Preferably, the expression plasmid used in the recombinant vector is plasmid pHT43; Preferably, the host cell used by the recombinant strain is the commonly used model strain Bacillus subtilis WB800N.

[0011] The fourth technical solution provided by the present invention is the application of the above-mentioned recombinant expression vector or recombinant strain, especially in the preparation of the amylase mutant described in the first technical solution.

[0012] Beneficial effects: The amylase mutant provided by the present invention has optimized enzymatic properties compared to the original low-temperature alkaline amylase, and the optimal reaction pH is in the range of 7.5-10.5. The amylase can also maintain high activity under low temperature conditions, with an optimal reaction temperature range of 20-40°C, which is more suitable for the detergent environment. In a detergent containing 0.5-5g / L of the low-temperature alkaline amylase mutant (sodium alkyl aryl sulfonate (LAS) 10-30%, sodium silicate 10-25%, sodium sulfate 5-20%, sodium hexametaphosphate 5-25%), the enzyme activity is maintained at more than 95% after stirring for 40 minutes at 25°C.

[0013] The amylase mutant provided by the present invention is expressed in Bacillus subtilis WB800N via expression plasmid pHT43. In the preparation experiment, the average fermentation enzyme activity in a 50L fermenter is 242% higher than the original enzyme activity, which can further reduce the production cost of amylase and promote the widespread application of amylase in the detergent industry.

[0014] In addition, the present invention confirms the amino acid fragments PD1 and PD2 that affect the synthesis and secretion of amylase protein in the Bacillus subtilis expression system, providing a reliable reference for subsequent research on amylase. Description of the drawings: Figure 1 The optimal reaction pH curve of amylase before and after modification; Fig. 2 Optimal reaction temperature curve of amylase before and after modification; Fig. 3 Effect curve of surfactant on enzyme activity; Fig. 4 Changes in the activity of amylase in the washing solution before and after modification. Specific implementation method: The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0017] The amylase mutant of the present invention is obtained by deleting the PD1 fragment (amino acids at positions 6-8) and the PD2 fragment (amino acid residues at positions 16-21) of the amino acid sequence of the original amylase. The amino acid sequence of the amylase mutant is shown in SEQ ID NO.2.

[0018] In the present invention, YAMY represents the original amylase derived from Bacillus subtilis, yamy The gene encodes the original amylase, BAMY indicates the amylase mutant, bamy It is the coding gene of amylase mutant. The specific information is as follows:

[0019] The amino acid sequence of the original amylase is shown in SEQ ID NO.1: MLFNLKRSTRTRLSTETGFALLGGHPCFLTTQDIHLGVNESLTDTARVLSSMTDAVLARVYKQSDLDRILLNNAAFRNGHNFVVRNFWCGQPLQNKVQLKGRDLLTLKNFTGEEIKYMLWLSADLKFRINPIINGLSDLYHPIQIDYLTLQEHYSSQGKTERRVFAFISLGMIFET LAKEASILKGLTLSWIGDGNNILHSIMMSAAKFGMHLQAATPKGYEPDASITKLAEQYAKENGTKLLLTNDPLEAAHGGNVLITDTWISMGQEEEKKKRLQAFQGYQVTMKTAKVAASDWTFLHCLPRKPEEVDDEVFYSPRSLVFPEAENRKWTIMAVMVSLLTDYSPQLQKPKF* The amino acid sequence of the amylase mutant is shown in SEQ ID NO.2: MLFNLTRTRLSTLGGHPCFLTTQDIHLGVNESLTDTARVLSSMTDAVLARVYKQSDLDRILLNNAAFRNGHNFVVRNFWCGQPLQNKVQLKGRDLLTLKNFTGEEIKYMLWLSADLKFRINPIINGLSDLYHPIQIDYLTLQEHYSSQGKTERRVFAFISLGMIFETLAKEA SILKGLTLSWIGDGNNILHSIMMSAAKFGMHLQAATPKGYEPDASITKLAEQYAKENGTKLLLTNDPLEAAHGGNVLITDTWISMGQEEEKKKRLQAFQGYQVTMKTAKVAASDWTFLHCLPRKPEEVDDEVFYSPRSLVFPEAENRKWTIMAVMVSLLTDYSPQLQKPKF* The vector construction primers and amino acid sequence directed modification primers used in this study were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0020] Bacillus subtilis WB800N and plasmid pHT43 were purchased from Shanghai Shenggong Biotechnology Co., Ltd.

[0021] The amylase activity determination method adopts the alkaline amylase activity detection method in CN202211662114.3 previously disclosed by the applicant to detect the enzyme activity, wherein the temperature for detecting the enzyme activity is 37°C, the pH is 9.5, and the standard curve equation is: Y (glucose concentration) = 0.512*A550 measured absorbance value - 0.34.

[0022] The present invention is further explained below by means of specific examples.

[0023] Example 1 Obtaining modified amylase gene, expression vector and recombinant strain 1.1 Obtaining modified amylase genes The original amino acid sequence of amylase and the corresponding nucleotide sequence were analyzed and constructed with the help of bioinformatics software to construct an electronic library of amylase hypothetical protein sequences. Combined with the analysis of the transmembrane secretion mechanism of Bacillus subtilis protein, unstable options were removed. Finally, it was determined that the mutant obtained by deleting the amino acid sequence fragments (PD1, PD2) at positions 6-8 and 16-21 in the original amylase amino acid sequence could be secreted by Bacillus subtilis, especially Bacillus subtilis WB800N.

[0024] After the nucleotide sequence of the mutant was determined based on the amino acid sequence, the DNA sequence was synthesized with the help of Sangon Biotechnology Co., Ltd. to obtain the amylase mutant encoding gene Bamy (SEQ ID NO.4).

[0025] The amylase mutant encoding gene Bamy (SEQ ID NO. 4) obtained above was connected to a T vector for storage and named as T-Bamy vector.

[0026] The present invention connects the modified amylase gene to the expression vector pHT43 to obtain pHT43-Bamy, and then expresses the amylase mutant in Bacillus subtilis WB800N through the expression vector pHT43-Bamy, as follows: 1.2 Obtaining the recombinant expression vector 1.2.1 Enzyme digestion The recombinant T-vector T-Bamy containing the modified amylase gene and the expression vector pHT43 were respectively EcoR Ⅰ and XB Ⅰ Enzyme digestion, the enzyme digestion system is as follows:

[0027] The above reaction system was digested overnight in a 37°C water bath to obtain the amylase mutant encoding gene Bamy and the linear fragment of the linearized vector pHT43.

[0028] 1.2.2 Connection The above linear fragments were identified by gel electrophoresis, purified and recovered using a gel recovery kit, and then ligated using T4 DNA ligase. The ligation system is as follows:

[0029] The above reaction was carried out at 16°C for 16 h.

[0030] Transform Escherichia coli DH5α with the ligation product: Take 100 μL of competent E. coli and add 20 μL of the above ligation product. Mix thoroughly and place in an ice bath for 30 minutes, then place in a 42°C water bath for 90 seconds. After another 5-minute ice bath, add 500 μL of resistance-free LB medium, culture at 37°C, 150 rpm shaking for 1 hour, spread on an LB plate containing chloramphenicol (final concentration 15 μg / mL), and culture at 37°C for 12 hours.

[0031] Screening of the recombinant expression vector pHT43-Bamy: single colonies on the plate were selected and cultured in LB liquid culture medium (containing chloramphenicol). After 12 hours, the recombinant expression vector pHT43-Bamy was screened by colony PCR.

[0032] Extraction of recombinant expression vector pHT43-Bamy: For the positive clones with correct sequencing, inoculate them into a test tube containing LB liquid culture medium and culture them at 37°C and 150 rpm for 10 h until OD 600About 1.0, and then use a plasmid extraction kit to extract the recombinant expression vector pHT43-Bamy.

[0033] The same method as above was used to obtain the recombinant expression vector pHT43-Yamy of the original amylase encoding gene (SEQ ID NO. 3).

[0034] 1.3 Obtaining recombinant strains 1.3.1. Plasmid linearization Recombinant expression vector pHT43-Bamy or pHT43-Yamy was cleaved with restriction endonucleases Sac I linearization, 37 ℃ overnight enzyme digestion. Linearization enzyme digestion reaction system is:

[0035] The above reaction system was reacted at 37°C for 2 h, and the plasmid after enzyme digestion was purified using a DNA purification kit for use.

[0036] 1.3.2 Preparation of competent cells of Bacillus subtilis WB800N Reagents: Reagent GM: LB + 0.5M sorbitol; ETM: 0.5 M sorbitol, 0.5 M mannitol, 10% glycerol; RM: LB + 0.5 M sorbitol + 0.38 M mannitol.

[0037] 1) Pick a single colony and inoculate it into a 5 mL LB medium shake tube and culture it overnight. 2) Measure the OD in the shake tube 600 , and then transfer to a shake flask to make the OD of the culture medium between 0.19 and 0.2 after inoculation. The culture medium is 50 mL GM, and culture at 37°C and 200 rpm until the OD 600 =about 1.0.

[0038] 3) Place the entire bacterial solution in an ice water bath for 10 min, then centrifuge at 5000 rpm, 8 min, 4°C to collect the bacteria. 4) Wash the cells with 40 ml of pre-cooled electroporation buffer ETM, centrifuge at 5000 rpm, 8 min, 4°C to remove the supernatant, and rinse three times. 5) Resuspend the washed cells in 500 μL of ETM and dispense 60 mL into each tube.

[0039] 1.3.3. Electroconversion Add 1-6 μL of plasmid pHT43-Bamy or pHT43-Yamy to 60 μL of competent cells, incubate on ice for 5 min, add to a pre-cooled electroporation cup (1 mm), and electroporate once. Electroporation instrument settings: 2.0 kV, 25 μF, 200 Ω, 1 mm, 1 electroporation (duration 4.5 ms-5 ms).

[0040] After the electroporation, 1 ml of recovery medium RM was added immediately, and the culture was incubated at 37°C, 200 rpm, and after 3 hours of recovery, the plates were coated and incubated upside down at 37°C. The resulting strains were WB800N / pHT43-Bamy Bacillus subtilis engineered bacteria and WB800N / pHT43-Yamy Bacillus subtilis engineered bacteria.

[0041] Example 2 Preparation of amylase 2.1 Preparation of amylase The recombinant strains WB800N / pHT43-Bamy and WB800N / pHT43-Yamy constructed in Example 1 were fermented and prepared by the following method: Seed culture in 50L seed tank: 2% inoculation, temperature 37°C, tank pressure 0.04-0.08Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 40% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, ammonia was added to control the pH at 6.0, and the culture was carried out for 15h; Seed culture medium composition: 5% glucose, 3% soybean meal, 5% potassium dihydrogen phosphate, 2.5% magnesium sulfate, pH 6.0; Fermentation culture in a 50L fermenter: 30L of feed was added to the fermenter, 5% of the volume of the fermenter medium was inoculated, the culture temperature was 30°C, the tank pressure was 0.04-0.08MPa, the initial speed was 100rpm, and the initial air volume was 2.0m 3 / h, maximum speed 600rpm, maximum ventilation volume 6.0 m 3 / h; Feed control: when the fermentation reaches the wet OD of the bacteria 600 When the temperature reaches 15, start feeding 20% ​​liquefied starch. When the dissolved oxygen drops to 40%, increase the speed and air volume alternately. When the speed and air volume are at their maximum, feed to control the dissolved oxygen at 30%-35%. Ammonia is added to control the pH at 6.0. When the autolysis of the bacteria is severe, the fermentation is terminated. The culture period is 80 hours.

[0042] The fermentation medium composition is: 5% corn starch, 3% soybean cake powder, 5% potassium dihydrogen phosphate, 2.5% magnesium sulfate, pH 6.0.

[0043] The results of three batches of fermentation are as follows: the average fermentation enzyme activity of the mutant is 66402 U / mL, and the average fermentation enzyme activity of the original enzyme is 19116 U / mL.

[0044]

[0045] 2.2 Results Analysis It can be seen that the modified amylase mutant recombinant strain has a more efficient enzyme production efficiency, and the enzyme activity is increased by 247% within the same fermentation time, which greatly reduces the fermentation cost and improves the competitiveness of this product in the industry.

[0046] Example 3 Enzymatic properties of amylase mutants The amylase mutant provided by the present invention has an optimum reaction temperature of 30°C and good low-temperature enzyme activity. At 25°C, the enzyme activity remains above 95%, and at 20-40°C, the enzyme activity still remains above 90%. The optimum reaction pH is 9.0, and the relative enzyme activity remains above 90% under pH 7.0-10.5. The modified amylase has strong resistance to the surfactant LAS, and the enzyme activity is maintained at up to 95% in a 35% LAS content solution. The details are as follows: 3.1 Optimal reaction pH The supernatant obtained by centrifugation of the fermentation liquid of batch 1 in Example 2.1 was taken as the crude enzyme liquid of the mutant and the original enzyme, and the amylase activity was measured at pH 6.0-12.0 (interval 0.5) at a temperature of 30°C, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The measurement results are as follows: Figure 1 As shown, the modified amylase has the highest enzyme activity at pH 9.0, and the enzyme activity still remains above 90% in the pH range of 7.0-10.5.

[0047] 3.2 Optimum reaction temperature The supernatant obtained by centrifugation of the fermentation liquid of batch 1 in Example 2.1 was used as the crude enzyme liquid of the mutant and the original enzyme. The amylase activity was measured at 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and 90° C. under pH 9.0, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The results are shown in FIG. Figure 2 As shown, the optimal reaction temperature of the modified amylase is 30°C, and the enzyme activity remains above 90% in the range of 20-40°C.

[0048] 3.3 Effect of surfactant on enzyme activity The supernatant obtained by centrifugation of the fermentation liquid of batch 1 in Example 2.1 was used as the crude enzyme liquid of the mutant and the original enzyme, and the enzyme liquid was placed in a solution containing 0, 5, 10, 15, 20, 25, 30, 35, and 40% LAS surfactant, respectively. After being incubated at 25°C for 30 minutes, the enzyme activity was measured and the relative enzyme activity was calculated, with the original enzyme activity as 100%. The experimental results are shown in Figure 2. Figure 3 As shown in Figure 2, the enzymatic activity of the modified amylase was maintained at 95% in a 35% LAS solution. Example 4 Changes in low temperature activity of original amylase and modified amylase in washing liquid This example uses the change in activity of the original amylase and the modified amylase in the washing liquid with temperature to analyze the effect of the washing liquid and temperature on the enzyme activity.

[0049] 4.1 Experimental methods Detergent composition: 30% sodium alkyl aryl sulfonate, 25% sodium silicate, 20% sodium sulfate, 25% sodium hexametaphosphate; 5 g / L amylase was added to the above detergent, stirred at pH 9.0 and different temperatures, and the activity of amylase was detected after keeping warm for 40 min.

[0050] 4.2 Results Analysis According to the above method, the present invention measured the changes in the enzymatic activity of amylase before and after modification after being incubated in detergent at different temperatures for 40 minutes. The results are as follows Figure 4 As shown, within 40°C, the activity of the mutant enzyme in the detergent remains above 85%, while that of the original enzyme is only 80%. As the insulation temperature increases, the activity of amylase in the detergent decreases. At 60°C, the activity of the mutant enzyme in the detergent still remains above 65% after 40 minutes of insulation, while that of the original enzyme is only 55%.

[0051] Example 5 Washing experiment with addition of amylase The standardized Ubud was used as the washing object, and the detergent containing the amylase mutant of the present invention (washing 1) and the detergent without amylase (washing 2) were used as experimental detergents. A commercially available enzyme-free detergent (Hongchang Chemical) (with enzyme as washing 3, without enzyme as washing 4) was used as a comparative experiment.

[0052] The detergent described in Example 4 was added with 5 g / L amylase, 5 mL was used, 1 L of water was added, pH 9.0, temperature 25 ° C, and the Ubu was added and washed for 15 minutes. Before and after washing, the whiteness of the Ubu was detected by a whiteness detector, and the whiteness change was compared and analyzed. The effect of the detergent was evaluated by the difference in whiteness change. The results showed that the washing effect of the detergent with the amylase mutant of the present invention was better than that of the detergent without addition under the same conditions, and the stain removal effect was better after adding amylase.

[0053]

[0054] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. An amylase mutant, characterized in that The amino acid sequence of the amylase mutant is shown in SEQ ID No.

2.

2. A gene encoding the amylase mutant according to claim 1.

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

4.

4. A recombinant vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression plasmid used was pHT43.

6. The recombinant strain according to claim 4, characterized in that The host bacteria used was Bacillus subtilis WB800N.

7. Use of the recombinant vector or recombinant strain according to claim 4 in producing the amylase mutant according to claim 1.

8. Use of the amylase mutant according to claim 1.

9. The use according to claim 8, characterized in that It is used in the detergent industry.

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