Alkaline pectinase mutant and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an alkaline pectinase mutant as well as a coding gene and application thereof. The alkaline pectinase mutant is obtained by performing T10I mutation, L113P mutation, W251S mutation and D328E mutation on original alkaline pectinase, and the amino acid sequence of the alkaline pectinase mutant is as shown in SEQ ID NO. 3. The optimal reaction pH of the mutated alkaline pectinase is 9.4, the relative enzyme activity is still kept at 95% or above after the mutated alkaline pectinase stands at room temperature for 24 hours under the condition that the pH is 5.5-10.0, the optimal reaction temperature is 60 DEG C, and the mutated alkaline pectinase can still keep 90% or above of enzyme activity after the mutated alkaline pectinase is kept at the temperature of 60 DEG C or below for 4 hours under the condition that the pH is 8.0. According to the alkaline pectinase mutant provided by the invention, the expression plasmid pPICZ alpha A is expressed in pichia pastoris X33, the average fermentation enzyme activity in a 60m < 3 > fermentation tank is 5216U / mL, the enzyme activity level is improved by 1.8 times or more than that before mutation, the production cost of the alkaline pectinase can be further reduced, and wide application of the alkaline pectinase in an enzymatic degumming process in the textile industry is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an alkaline pectinase mutant and a coding gene and application thereof. Background Art

[0002] In the traditional cotton and linen refining process, a high-concentration strong alkaline solution is generally used to treat the desized cotton and linen fabric to remove the non-fiber components on the plant surface. In this process, not only will the fiber components of the cotton and linen be damaged, affecting the quality of downstream products, but a large number of chemical components will also be introduced into the process, increasing the difficulty of wastewater treatment and the production cost of the product.

[0003] Alkaline pectinase generally refers to pectate lyase, which can break the α-1,4 glycosidic bond of polygalacturonic acid chain through trans-elimination and generate 4,5-unsaturated oligosaccharide galacturonic acid salt at the non-reducing end. The optimal reaction pH of alkaline pectinase is 8.0-10.0. Under alkaline conditions, cellulose, the main body of cotton fiber, tends to swell. Alkaline pectinase uses its specificity to effectively decompose and remove pectin on the surface of cotton fiber, which acts as a bond to hydrophobic symbionts such as cotton wax, so that hydrophobic substances fall off from the surface of cotton fiber, thereby achieving a refining effect without damaging the fiber itself.

[0004] The enzymatic degumming process is generally carried out at a relatively high temperature (50-60°C), and the degumming process generally lasts 4-8 hours. However, the enzymatic activity of the commonly used alkaline pectinase products will drop sharply when the temperature rises above 45°C.

[0005] Under actual production process conditions, alkaline pectinase is also required to have good pH stability. Bacterial alkaline pectinase is generally reported to have relatively good stability within a small range before and after its optimal reaction pH range, but the enzyme maintains stable enzyme activity for a relatively short time.

[0006] Since the first discovery of alkaline pectinase in 1972, heterologous expression and application of alkaline pectinase has gradually become a research hotspot. At present, the hosts for expressing alkaline pectinase mainly include Escherichia coli, Pichia pastoris and Bacillus subtilis. Among them, Pichia pastoris has low nutritional requirements, high proliferation rate, high bacterial density, and the protein expressed by Pichia pastoris is easy to purify and has high yield, which is suitable for large-scale production. However, overexpression of heterologous proteins will lead to growth stress, cause unfolded protein effect or fail to be effectively modified, resulting in low fermentation enzyme activity of alkaline pectinase, which limits the industrial production and application of alkaline pectinase.

[0007] Finding alkaline pectinase that is stable in a wide pH range and can maintain thermal stability for a long time is of great significance for expanding the practical application of alkaline pectinase in industry. Therefore, the problems that need to be solved in the industrial application of alkaline pectinase at this stage include but are not limited to the following: 1. High temperature resistance; 2. Acid and alkali resistance; 3. High fermentation enzyme activity level. Summary of the invention

[0008] The main purpose of the present invention is to provide an alkaline pectinase mutant and its gene, a recombinant expression vector and a recombinant strain containing the gene, and a method for preparing alkaline pectinase by the recombinant bacteria, aiming to solve the problems of poor acid, alkali and heat resistance and low fermentation enzyme activity level of alkaline pectinase in the prior art.

[0009] To achieve the above-mentioned purpose, one of the technical solutions provided by the present invention is an alkaline pectinase mutant, which is obtained by mutating T, L, W, and D at positions 10, 113, 251, and 328 in the amino acid sequence of the wild-type alkaline pectinase shown in SEQ ID NO.1 to I, P, S, and E, respectively. The amino acid sequence of the mutant is shown in the sequence listing SEQ ID NO.3.

[0010] The present invention also provides a gene encoding the above alkaline pectinase mutant; Furthermore, the nucleotide sequence of the gene encoding the alkaline pectinase mutant is shown in the sequence listing SEQ ID NO.4.

[0011] Furthermore, the nucleotide sequence of the gene encoding the alkaline pectinase mutant heterologously expressed in yeast is shown in the sequence listing SEQ ID NO.5.

[0012] The second technical solution provided by the present invention is the application of the alkaline pectinase mutant described in the first technical solution, especially the application in hydrolyzing the α-1,4 glycosidic bonds of polygalacturonic acid chains, and more particularly the application in the textile industry, such as the application in degrading pectin on the surface of cotton fibers in an alkaline environment.

[0013] The third technical solution provided by the present invention is a recombinant vector or a recombinant strain comprising the above-mentioned alkaline pectinase mutation encoding gene; Preferably, the expression plasmid used in the recombinant vector is pPICZαA; Preferably, the host cell used by the recombinant strain is Pichia pastoris X33.

[0014] The fourth technical solution provided by the present invention is the application of the recombinant expression vector or recombinant strain described in the third technical solution, especially in the preparation of the alkaline pectinase mutant described in the first technical solution; Furthermore, the method for preparing an alkaline pectinase mutant using the above recombinant strain is as follows: Culture in fermenter: inoculate at 6-8% (v / v) of the fermenter medium volume, culture temperature 28.5-30.5°C, tank pressure 0.05-0.09MPa, initial speed 100rpm, initial air volume 200m 3 / h, maximum speed 600rpm, maximum ventilation volume 4000m 3 / h; Feed control: When the wet weight of the bacteria reaches 80-95g / L, start to feed 40-45% glycerol at a rate of 610-730kg / h. When the wet weight of the bacteria reaches 170-180g / L, stop feeding glycerol, starve for 0.5-1.0h, and then start to feed methanol. In the first 0.5-1.5h, the flow rate of methanol is 80-100kg / h. After 0.5-1.5h, the flow rate of methanol increases by 25-45kg per hour until the maximum feeding rate is 320-360kg / h. Maintain this feeding rate until the end of fermentation. Control of dissolved oxygen: Before starting to add methanol, the dissolved oxygen is controlled to 33-42% by adjusting the speed and air volume. After starting to add methanol, the dissolved oxygen is controlled to 10%-20%. After reaching the maximum speed and ventilation volume, the dissolved oxygen is no longer controlled. Ammonia supplementation controls pH at 5.5-5.7; When the autolysis of the bacteria is serious, the fermentation is terminated, and the culture cycle is 190-210h.

[0015] Furthermore, the culture conditions in the seed tank are: 6-9% (v / v) inoculation amount, temperature 29-30°C, tank pressure 0.04-0.08Mpa, initial speed 100rpm, initial air volume 25m 3 / h, the dissolved oxygen is controlled to be greater than 35% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, add ammonia to control pH at 5.5-5.7, and culture for 49-55h; Further, the composition of the seed culture medium is (w / v): 4-6% glycerol, 3-5.2% monoammonium phosphate, 0.18-0.35% disodium hydrogen phosphate, 2.1-2.7% magnesium sulfate, 1.1-1.5% potassium sulfate, 0.2-0.35% calcium sulfate, 0.18-0.29% potassium hydroxide, 0.03-0.06% phosphoric acid, and the rest is water, with a pH of 5.5-5.7; Furthermore, the fermentation medium composition is (w / v): 4.5-5.6% glycerol, 5.6-6.7% monoammonium phosphate, 0.35-0.57% disodium hydrogen phosphate, 1.5-2.5% magnesium sulfate, 1.9-2.6% potassium sulfate, 0.18-0.34% calcium sulfate, 0.18-0.25% potassium hydroxide, 0.05-0.18% phosphoric acid, and the rest is water, with a pH of 5.5-5.7.

[0016] Beneficial effects: The alkaline pectinase mutant provided by the present invention has an optimum reaction pH of 9.4. Under the condition of pH 5.5-10.0, after standing at room temperature for 24 hours, the relative enzyme activity is still maintained at more than 95%. The optimum reaction temperature is 60°C. Under the condition of pH 8.0, the enzyme activity can still be retained at more than 90% after being kept below 60°C for 4 hours.

[0017] The codon-optimized alkaline pectinase mutant gene provided by the present invention is expressed in Pichia pastoris X33 via the expression plasmid pPICZαA. 3 The average fermentation enzyme activity in the fermentation tank was 5216U / mL, and the enzyme activity level was more than 1.8 times higher than before the mutation, which can further reduce the production cost of alkaline pectinase and promote the widespread application of alkaline pectinase in degumming in the textile industry.

[0018] In addition, the present invention confirms four amino acid sites in alkaline pectinase that are related to acid-base resistance and heat resistance, and proves the importance of these sites to the acid-base resistance and heat resistance of the enzyme, which provides important clues for studying the acid-base resistance and heat resistance of alkaline pectinase, and provides a reliable reference for the research of other alkaline pectinases. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 Optimal reaction pH curve of alkaline pectinase before and after mutation; Fig. 2 pH stability curve of alkaline pectinase before and after mutation; Fig. 3 Optimal reaction temperature curve of alkaline pectinase before and after mutation; Fig. 4 Temperature stability curves of alkaline pectinase before and after mutation. DETAILED DESCRIPTION The present invention is described in detail below through specific implementation cases. The specific implementation cases are only for illustration and are not intended to limit the scope of implementation of the present invention.

[0021] Identification of the alkaline pectinase mutant used in the present invention: The mutated amino acid in the mutant is represented by "original amino acid + position + replaced amino acid". For example, T10I means that the amino acid at position 10 is mutated from T to I, and the position number corresponds to the amino acid sequence number of the wild-type alkaline pectinase in SEQ ID NO.1, as shown in the following table.

[0022]

[0023] The mutant of the present invention is obtained by subjecting the amino acid sequence of the wild-type alkaline pectinase shown in SEQ ID NO.1 to point mutations of T10I, L113P, W251S and D328E. The amino acid sequence of the mutant is shown in SEQ ID NO.3.

[0024] The specific sequence information is as follows: The amino acid sequence of wild-type alkaline pectinase is shown in SEQ ID NO.1: MKRTILMPTTLCSVWVIHPPFMFSSALVMVGWRNKTNHVWPKLHKSHGEALYHGMPEIWKPQRIVEDVLSSTTHLRSMEPQAMLRYWRLHPLLVSEQTIEWNCYTVLLKREDLTLTIMGISNDALFSGALTNAYGLKCTAWQLTSRKPRSIHNDRNVLQLLPEGEHRMVNAVPLKDHN KVNNNLLYYGSGTNAAGSNKWNYNPLTFLTHLTLPPRPAAVWTAAHHPWQSNISQAFSSLQQDNVKVRPKREWRWCDQCYGWLEEDTGNHSSPMNPISKAWWSRCHPHWWHAKNVVLGAPAIATSVQVSWRSSGLGHLAAPQVSHDDPLDRRQHEEWNVSSGGLTPAGAKEKGIKWEG* The amino acid sequence of the alkaline pectinase mutant is shown in SEQ ID NO.3: MKRTILMPTILCSVWVIHPPFMFSSALVMVGWRNKTNHVWPKLHKSHGEALYHGMPEIWKPQRIVEDVLSSTTHLRSMEPQAMLRYWRLHPLLVSEQTIEWNCYTVLLKREDPTLTIMGISNDALFSGALTNAYGLKCTAWQLTSRKPRSIHNDRNVLQLLPEGEHRMVNAVPLKDHN KVNNNLLYYGSGTNAAGSNKWNYNPLTFLTHLTLPPRPAAVWTAAHHPWQSNISQAFSSLQQDNVKVRPKRESRWCDQCYGWLEEDTGNHSSPMNPISKAWWSRCHPHWWHAKNVVLGAPAIATSVQVSWRSSGLGHLAAPQVSHDDPLERRQHEEWNVSSGGLTPAGAKEKGIKWEG* The present invention will be further explained below in conjunction with specific implementation modes.

[0025] Example 1 Obtaining codon-optimized alkaline pectinase mutant gene, expression vector and recombinant strain 1.1 Obtaining the mutant gene of alkaline pectinase With the help of FoldX, Rosetta and YASARA software, an electronic library of acid-base resistant and heat-stable mutants of alkaline pectinase was constructed. At the same time, a semi-rational design method was used to analyze the catalytic mechanism, and it was finally determined that the alkaline pectinase mutant of the present invention was obtained by mutating the threonine, leucine, tryptophan and aspartic acid at the 10th, 113th, 251st and 328th positions in the amino acid sequence of the wild-type alkaline pectinase from Bacillus licheniformis JR-101 (strain collection number CGMCC NO.18643) to isoleucine, proline, serine and glutamic acid, respectively.

[0026] 100 mL of logarithmic phase seed solution of Bacillus licheniformis JR-101 was placed in two 50 mL centrifuge tubes and centrifuged at 8000 rpm for 5 min. The supernatant was discarded and the precipitated bacteria were washed twice with 200 mL of saline solution. The genome was then sent to Shanghai Biotech for genome sequencing to obtain the coding gene sequence and upstream and downstream sequence information of the wild-type alkaline pectinase. Based on the obtained genome, primers Y1 (containing EcoR Ⅰ restriction site) and Y2 (containing Xba Ⅰ restriction site) to amplify the alkaline pectinase gene pel (SEQ ID NO.2), and connected to the T vector for preservation, and the recombinant vector was named T- pelThe site-directed mutagenesis kit used was the kit from Shanghai Bioengineering, model B639281. The specific operation steps are as follows: (1) Primers for amplifying the wild-type alkaline pectinase gene and various point mutations.

[0027] Table 1 The primers used for the above point mutation are as follows

[0028] (2) Point mutation: Taking the point mutation of T10I as an example, two primers, T10I-1 and T10I-2, were used to clone the wild-type alkaline pectinase gene. pel Recombinant T vector T- pel As a template, add other reagents according to the system in the point mutation kit to perform PCR amplification. After the amplification, take 10 μL of the above mutation PCR amplification product and perform 0.8% agarose gel electrophoresis detection. After the band is correct, add 1 μL of digestion enzyme to the mutation product, mix well, and incubate at 37℃ for 60 min. (3) Transformation: Add 5 µL of the mutated product to 50 µL of DH5α competent cells, mix well, and place on ice for 30 min; heat shock at 42°C for 90 s and immediately cool on ice for 10 min; add 500 µL of LB medium, incubate at 180 rpm and 37°C for 1 h; then centrifuge the culture at 10,000 rpm for 5 min, discard part of the supernatant, retain 100-150 µL of the supernatant, flick to suspend the bacteria, take all the bacterial solution to plate, and incubate at 37°C overnight.

[0029] (4) Verification of positive clones: Pick a single colony and place it in 500 μL LB medium containing ampicillin resistance. Culture it at 200 r / min for 2-3 h, then perform PCR identification. Send the screened positive clones for sequencing. Compare the sequencing results and select the positive clones that have the mutation of C to T at position 29 in the sequence shown in nucleotide sequence SEQ ID NO.3. This positive clone is the mutant containing the recombinant plasmid with the correct mutation.

[0030] (5) Mutation of other sites: Using the correctly screened mutant as a template, the primer pairs corresponding to the mutation sites in Table 1 were used one by one, and the same method as above was used to mutate each site, and finally the alkaline pectinase mutant gene shown in SEQ ID NO.4 was screened out.

[0031] The alkaline pectinase gene obtained after the above point mutations Bpel (SEQ ID NO.4) was connected to a T vector and stored, named T- Bpel carrier.

[0032] The present invention connects the mutated alkaline pectinase gene to the expression vector pPICZαA to obtain pPICZαA- Bpel Then, in Pichia pastoris X33, the expression vector pPICZαA- Bpel , to achieve the expression of alkaline pectinase mutants, as follows: 1.2 Obtaining the recombinant expression vector 1.2.1 Enzyme digestion The recombinant T vector containing the alkaline pectinase mutant gene was Bpel and expression vector pPICZαA, respectively, using restriction endonucleases EcoR Ⅰ and Xba Ⅰ Enzyme digestion, the enzyme digestion system is as follows:

[0033] The above reaction system was digested overnight in a 37°C water bath to obtain the alkaline pectinase mutant gene. Bpel , and the linear fragment of the linearized vector pPICZαA.

[0034] 1.2.2 Connection The above linear fragments were identified by 1% agarose gel electrophoresis, purified and recovered using a gel recovery kit, and then ligated using T4 DNA ligase. The ligation system is as follows:

[0035] The above reaction was carried out at 16°C for 16 h.

[0036] 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 bleomycin (final concentration 25 μg / ml), and culture at 37°C for 12 hours.

[0037] Screening of the recombinant expression vector pPICZαA-Bpel: Select a single colony on the plate and culture it in LB liquid medium (containing bleomycin). After 12 hours, use primers Y1 and Y2 to screen the recombinant expression vector pPICZαA-Bpel by colony PCR. Bpel .

[0038] Recombinant expression vector pPICZαA- Bpel Extraction: 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 150rpm for 10h until OD 600 About 1.0, and then use a plasmid extraction kit to extract the recombinant expression vector pPICZαA-Bpel .

[0039] 1.3 Obtaining recombinant strains Recombinant expression vector pPICZαA- Bpel Restriction endonucleases Sac I linearization, 37 ℃ overnight enzyme digestion. Linearization enzyme digestion reaction system is:

[0040] The above reaction system was reacted at 37°C for 2h. The plasmid after enzyme digestion was purified with a DNA purification kit and transferred into Pichia pastoris X33 competent cells by electroporation (voltage 2.0k V, capacitance 25μF, resistance 200Ω, and electroporation time 5ms). 200μL was spread on YPDS solid medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose, 1mol / L sorbitol, 20g / L agar, autoclaved at 115°C for 20min) containing a final concentration of Zeocin of 100ug / ml, and inverted cultured at 30°C. The resulting strain was Pichia pastoris engineered bacteria X33 / pPICZαA- Bpel .

[0041] 1.4 Yeast recombinant strain X33 / pPICZαA- Bpel Inducible expression of Pick the yeast recombinant strain X33 / pPICZαA- from the YPD (100ug / mL zeocin) plate Bpel A single colony was placed in a 50 mL YPD shake flask and cultured at 30°C for about 30 hours. It was inoculated into 100 mL BMGY medium, with an initial OD600 of 0.1, and cultured at 30°C in a shaker until OD600 was about 6, ready to enter the induction stage. The bacteria in BMGY were centrifuged under sterile conditions, washed once with sterile water, transferred to BMMY medium, cultured at 30°C, and 1 mL of methanol was added every 24 hours. After continuous induction for 3 days, the fermentation liquid was centrifuged and the supernatant was taken as a crude enzyme liquid to determine the enzyme activity. The enzyme activity of alkaline pectinase was 1109U / mL.

[0042] 1.5 Codon optimization of alkaline pectinase mutant gene and construction and expression of recombinant plasmid Based on the mutated alkaline pectinase gene sequence, the nucleotide sequence of the alkaline pectinase mutant gene was artificially redesigned using the bioinformatics software DNA2.0; specifically, the alkaline pectinase gene sequence was adapted to the expression logic and codon usage preference of the host cell yeast strain X33, and codons with high usage frequency in the host cell were used to replace codons with low frequency; at the same time, the complexity of the secondary structure of the mRNA encoded by the alkaline pectinase gene was reduced, which helped to reduce the inhibitory effect of the secondary structure on the translation of alkaline pectinase, and the gene sequence of the alkaline pectinase mutant gene with codon optimization was obtained, named Bpel-X (SEQ ID NO.5). The above codon-optimized sequence was added at both ends EcoR Ⅰ and Xba The restriction site of Ⅰ was synthesized by BGI full sequence and connected to the T vector, named T-Bpel-X.

[0043] The same technical means as in Examples 1.2 and 1.3 were used to separate the recombinant T-vector T-Bpel-X and the expression vector pPICZαA using restriction endonucleases. EcoR Ⅰ and Xba I digested and ligated to obtain the expression vector pPICZαA-Bpel-X expressing the codon-optimized alkaline pectinase mutant gene. Sac After linearization, I was transformed into Pichia pastoris X33 competent cells to obtain the recombinant strain X33 / pPICZαA-Bpel-X.

[0044] 1.6 Inducible expression of recombinant yeast strain X33 / pPICZαA-Bpel-X Pick a single colony of the yeast recombinant strain X33 / pPICZαA-Bpel-X from the YPD (100ug / mL zeocin) plate and place it in a 50mL YPD shake flask and culture it at 30℃ for about 30h. Inoculate it in 100mL BMGY medium, make the initial OD600 = 0.1, and culture it in a 30℃ shaker until OD600 = about 6, ready to enter the induction stage. Centrifuge the bacteria in BMGY under sterile conditions, wash it with sterile water, transfer it to BMMY medium, culture it at 30℃, add 1 mL of methanol every 24 hours, and continue to induce for 3 days. The enzyme activity of alkaline pectinase is 2106U / mL.

[0045] 1.7 Obtaining wild-type alkaline pectinase gene expression strain Using the same technical means as in Examples 1.2 and 1.3 above, the recombinant T-vector T-pel containing the original gene of alkaline pectinase and the expression vector pPICZαA were respectively cleaved with restriction endonucleases EcoR Ⅰ and XbaI digestion and ligation were performed to obtain the expression vector pPICZαA-pel expressing the wild-type alkaline pectinase gene. Sac After linearization, it was transferred into Pichia pastoris X33 competent cells to obtain the engineered strain X33 / pPICZαA-pel expressing the original gene of alkaline pectinase. Using the same technical means as in the above Examples 1.4 and 1.6, the enzyme activity of the wild-type alkaline pectinase was 616 U / mL.

[0046] Example 2 Preparation of alkaline pectinase 2.1 Preparation of mutated alkaline pectinase The recombinant strain X33 / pPICZαA-Bpel-X constructed in Example 1 was used for fermentation preparation, and the method was as follows: 3m 3 Seed culture in seed tank: 8% inoculation, temperature 29.5℃, tank pressure 0.04-0.08Mpa, initial speed 100rpm, initial air volume 25m 3 / h, the dissolved oxygen is controlled to be greater than 35% 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 5.6, and the culture was carried out for 50h; Seed culture medium composition (w / v): 6% glycerol, 4% monoammonium phosphate, 0.2% disodium hydrogen phosphate, 2.2% magnesium sulfate, 1.3% potassium sulfate, 0.26% calcium sulfate, 0.25% potassium hydroxide, 0.04% phosphoric acid, and the rest is water, pH 5.6; 60m 3 Fermentation in the fermenter: Inoculate 7% of the volume of the fermenter medium, culture temperature 30°C, tank pressure 0.05-0.09MPa, initial speed 100rpm, initial air volume 200m 3 / h, maximum speed 600rpm, maximum ventilation volume 4000m 3 / h; control of feeding: when the wet weight of the bacteria reaches 95g / L, start to feed 40% glycerol with a flow acceleration of 650kg / h. When the wet weight of the bacteria reaches 175g / L, stop feeding glycerol, starve for 1.0h, and then start to feed methanol. In the first hour, the flow rate of methanol is 85kg / h. After 1h, the flow acceleration of methanol increases by 30kg per hour until the maximum feeding rate of 325kg / h is reached. The feeding rate is maintained until the end of fermentation; control of dissolved oxygen: before starting to feed methanol, the dissolved oxygen is controlled to 33% by adjusting the rotation speed and air volume. After starting to feed methanol, the dissolved oxygen is controlled at 16%. After reaching the maximum rotation speed and ventilation volume, the dissolved oxygen is no longer controlled; ammonia is added to control the pH at 5.6; fermentation is terminated when the bacteria are seriously autolyzed, and the culture period is 198h.

[0047] The composition of the fermentation medium (w / v): 4.5% glycerol, 6.7% monoammonium phosphate, 0.57% disodium hydrogen phosphate, 2.5% magnesium sulfate, 1.9% potassium sulfate, 0.25% calcium sulfate, 0.21% potassium hydroxide, 0.18% phosphoric acid, and the rest is water, with a pH of 5.6.

[0048] The fermentation results of three batches are as follows, and the average fermentation enzyme activity is: 5216 U / mL.

[0049]

[0050] 2.2 Preparation of wild-type alkaline pectinase The same fermentation method as in Example 2.1 was used to carry out three batches of fermentation using the X33 / pPICZαA-pel recombinant bacteria. The results were as follows: the average fermentation enzyme activity was 1802 U / mL.

[0051]

[0052] Example 3 Determination of alkaline pectinase activity The alkaline pectinase activity determination method involved in the present invention is the same as the standard QB / T 4482-2013.

[0053] Example 4 Enzymatic properties of alkaline pectinase mutants The alkaline pectinase mutant provided by the present invention has an optimal reaction pH of 9.4. Under the condition of pH 5.5-10.0, after standing at room temperature for 24 hours, the relative enzyme activity is still maintained at more than 95%. The optimal reaction temperature is 60°C. Under the condition of pH 8.0, the enzyme activity can still be retained at more than 90% after being kept below 60°C for 4 hours. The details are as follows: (1) Optimal reaction pH The supernatant obtained by centrifuging the fermentation broth of batch 1 in Example 2.1 and batch 1 in Example 2.2 was taken as a crude enzyme solution, and the alkaline pectinase activity was measured at pH values ​​of 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, and 10.0 at a temperature of 45°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 mutated alkaline pectinase has the highest enzyme activity at pH 9.4.

[0054] (2) Acid and alkali resistance The supernatant obtained by centrifugation of the fermentation broth of batch 1 of Example 2.1 and batch 1 of Example 2.2 was taken as a crude enzyme solution, and the pH of the enzyme solution was adjusted to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5 with 0.1M NaOH or 0.1M HCl, respectively. After standing at room temperature for 24 hours, the enzyme activity was measured at 45°C and pH 9.0, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The measurement results are as follows: Figure 2 As shown, the relative enzyme activity of the mutated alkaline pectinase still remained above 95% after 24 hours at pH 5.5-10.0.

[0055] (3) Optimum reaction temperature The supernatant obtained by centrifugation of the fermentation broth of batch 1 in Example 2.1 and batch 1 in Example 2.2 was taken as the crude enzyme solution, and the alkaline pectinase activity was measured at 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75°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 3 As shown, the optimal reaction temperature of the mutated alkaline pectinase is 60℃.

[0056] (4) Thermal stability The supernatant obtained by centrifuging the fermentation broth of batch 1 of Example 2.1 and batch 1 of Example 2.2 was taken as the crude enzyme solution. Under the condition of pH 8.0, the enzyme solution was placed at 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80°C for 4 hours. After the insulation, the enzyme activity was measured at 45°C and pH 9.0, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The experimental results are shown in Figure 4 The mutated alkaline pectinase can still maintain more than 90% enzyme activity when kept below 60℃ for 4h.

[0057] 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 alkaline pectinase mutant, characterized in that: The amino acid sequence of the alkaline pectinase mutant is shown in SEQ ID NO.

3.

2. The encoding gene of the alkaline pectinase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown as SEQ ID NO.4 or SEQ ID NO.

5.

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 pPICZαA.

6. The recombinant strain according to claim 4, characterized in that The host bacteria used was Pichia pastoris X33.

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

8. Use of the alkaline pectinase mutant according to claim 1 in hydrolyzing polygalacturonic acid.

9. The use according to claim 8, characterized in that It is used in the enzymatic degumming process in the textile industry.

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