CN113234705A - Acid-tolerant 1,3-1, 4-beta-glucanase mutants - Google Patents
Acid-tolerant 1,3-1, 4-beta-glucanase mutants Download PDFInfo
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Abstract
The invention discloses an acid-resistant 1,3-1, 4-beta-glucanase mutant, belonging to the field of genetic engineering and enzyme engineering. The invention respectively mutates glutamine at position 1, isoleucine at position 133 and valine at position 134 of 1,3-1, 4-beta-glucanase derived from Bacillus tequilensis (Bacillus tequilensis) into glutamic acid, leucine and alanine by overlap extension PCR to respectively obtain single mutants Q1E, I133L, V134A and double mutants Q1E/I133L. The four mutants have better stability in acid environment and catalytic activity, and are more favorable for industrial application compared with the wild enzyme.
Description
Technical Field
The invention relates to an acid-resistant 1,3-1, 4-beta-glucanase mutant, belonging to the technical field of genetic engineering and enzyme engineering.
Background
Beta-glucan is a main component of endosperm cell walls of many cereal crops (barley, sorghum, rice and the like), and the solution viscosity is increased after being dissolved in water, so that a plurality of problems are brought to industrial production. During beer brewing, a large amount of beta-glucan in malt is dissolved in wort, so that the viscosity of the wort is increased, the filtering speed is reduced, the structure of spent grains is easy to block, the filtering speed of the wort is reduced, condensate precipitation is increased during cooling, the yield of the wort is reduced, and the non-biological stability of the finished beer is reduced. In the process of livestock breeding, as the gastrointestinal tract of animals lacks enzymes for decomposing beta-glucan, the beta-glucan in grains cannot be effectively digested and absorbed, the utilization rate of feed is reduced, the beta-glucan can also increase the viscosity of intestinal liquid, interfere the action between digestive enzymes and substrates, reduce the absorption efficiency of nutrient substances, simultaneously provide nutrient conditions for intestinal harmfulness, and increase intestinal flora imbalance and intestinal diseases of livestock caused by proliferation of harmful bacteria.
1,3-1, 4-beta-glucanase, beta-glucanase for short, belongs to glycoside hydrolase family 16, breaks the beta-1, 4-glycosidic bond closely adjacent to the beta-1, 3-glycosidic bond in the beta-glucan through hydrolysis, decomposes the beta-glucan into low polydextrose with small molecular weight, and can effectively solve the problems of the beta-glucan in the industry. However, the industrial application environment of the beta-glucanase usually has the characteristics of high temperature and low pH, such as the saccharification temperature of wort (65-70 ℃) and acidic pH (about 5.0), and the acidic environment of the digestive system of livestock (pH3.0-4.0), which seriously limits the high-efficiency industrial application of the beta-glucanase.
The optimum pH value of the beta-glucanase from natural sources is concentrated at pH6.0-7.5, the optimum temperature is concentrated at 40-60 ℃, and the industrial requirements are difficult to meet, so that the improvement of the catalytic activity, the thermal stability and the acid resistance of the wild beta-glucanase is an important precondition for the high-efficiency industrial application of the beta-glucanase. The catalytic activity and thermal stability of beta-glucans are relatively well studied, while the acid resistance of beta-glucanases is relatively lacking.
Therefore, if the acid-resistant beta-glucanase can be obtained, the method has important significance for the industrial application of the beta-glucanase.
Disclosure of Invention
In order to solve the problems of low enzyme activity and poor tolerance of the beta-glucanase under the acidic condition, the invention finds potential key sites which may influence the acid resistance of the enzyme and modifies the potential key sites by carrying out structural analysis on the 1,3-1, 4-beta-glucanase from bacillus, and correspondingly modifies the acidic residues on the surface of the enzyme. The mutant with improved acid resistance is obtained by screening from a plurality of modified mutants, so that the mutant has higher tolerance between pH4.5-5.5.
The invention provides a1, 3-1, 4-beta-glucanase mutant, which takes 1,3-1, 4-beta-glucanase shown in SEQ ID NO.5 as parent enzyme and mutates the amino acid of one or more sites of 1 st, 133 th and 134 th positions of the parent enzyme.
In one embodiment, the 1 st position of the parent enzyme is mutated into glutamic acid, the obtained mutant is named as Q1E, and the amino acid sequence is shown as SEQ ID NO. 1.
In one embodiment, the 133 th site of the parent enzyme is mutated to leucine, and the obtained mutant is named as I133L, and the amino acid sequence is shown as SEQ ID NO. 2.
In one embodiment, the 134 th site of the parent enzyme is mutated to alanine, and the obtained mutant is named as V134A, and the amino acid sequence is shown as SEQ ID NO. 3.
In one embodiment, the 1 st position of the parent enzyme is mutated into glutamic acid, and the 133 th position is mutated into leucine, and the obtained mutant is named as Q1E/I133L, and the amino acid sequence is shown as SEQ ID NO. 4.
The invention provides a gene for coding the 1,3-1, 4-beta-glucanase mutant.
The present invention provides a recombinant plasmid carrying the gene of claim 3.
In one embodiment, the expression vector is any one of the pET series, Duet series, pGEX series, pHY300PLK, pPIC series.
In one embodiment, the expression vector is the pET28a (+) plasmid.
The invention provides microbial cells expressing the mutants, or carrying the genes.
In one embodiment, the microbial cells are derived from Escherichia coli.
In one embodiment, the escherichia coli is escherichia coli BL21(DE 3).
The invention also aims to provide a method for constructing the 1,3-1, 4-beta-glucanase mutant, which is obtained by using 1,3-1, 4-beta-glucanase gene from Bacillus terquilens CGX5-1 as a template and performing site-directed mutagenesis by adopting an overlap extension PCR method; the nucleotide sequence of the obtained beta-glucanase mutant gene is shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO. 9; all mutation sites were successfully mutated according to the predetermined target by gene sequencing, and then the gene fragment encoding the mutant was ligated to pET28a (+) plasmid and transformed into E.coli BL21(DE 3).
The invention provides a method for producing the mutant, which takes a microbial cell expressing the mutant or carrying the gene as a fermentation strain to ferment and produce the mutant.
In one embodiment, the microbial cells are inoculated into a TB culture medium and cultured to a growth logarithmic phase, IPTG and alpha-lactose are added for induction expression, and after the expression is finished, the supernatant is centrifugally collected, namely the crude enzyme solution containing the mutant.
The invention provides an application of the mutant, the gene or the microbial cell in hydrolyzing beta-glucan.
The invention has the beneficial effects that:
the invention provides four strains of beta-glucanCompared with wild enzyme, the xylanase mutant has higher acid resistance, catalytic activity and catalytic efficiency. The specific enzyme activity values of the four mutant enzymes are respectively improved by 30.53%, 7.91%, 44.63% and 16.92%, and simultaneously the catalytic efficiency k is improvedcat/KmThe values are respectively improved by 60.94%, 14.69%, 91.20 and 33.57%, the acid resistance is also improved to a certain extent, particularly, the relative enzyme activity of the mutant Q1E/I133L under the condition of pH4.0-8.0 is higher than that of the wild enzyme, the relative enzyme activity is 119.86% when the pH value is 4.5, and the relative enzyme activity is only 32.23% when the wild enzyme is used. Therefore, the acid resistance and catalytic activity of the four mutant enzymes are improved compared with wild enzymes, and the four mutant enzymes have extremely high application prospects in the fields of food, feed, prebiotics and biofuel.
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Description of the meanings indicated in the figures
FIG. 1 is a graph comparing the optimum temperatures of wild enzymes and four beta-glucanase mutants; □: wild enzyme,. smallcircle: Q1E, Δ: I133L +: V134A o: Q1E/I133L,
Q1E/V134A，
I133L/V134A，+：Q1E/I133L/V134A。
FIG. 2 is a graph comparing the optimum pH of wild enzyme and four beta-glucanase mutants; □: wild enzyme,. smallcircle: Q1E, Δ: I133L +: V134A o: Q1E/I133L,
Q1E/V134A，
I133L/V134A，+：Q1E/I133L/V134A。
FIG. 3 is a graph comparing the pH stability of wild enzymes and four beta-glucanase mutants; □: wild enzyme,. smallcircle: Q1E, Δ: I133L +: V134A o: Q1E/I133L,
Q1E/V134A，
I133L/V134A，+：Q1E/I133L/V134A。
Detailed Description
The media and solutions involved in the following implementation were as follows:
Luria-Bertani (LB) Medium: sodium chloride 10 g.L-1Peptone 10 g. L-1Yeast powder 5 g.L-1(ii) a 20 g.L of the culture medium is added when preparing the solid culture medium-1Agar powder; and (3) sterilization conditions: 121 ℃ for 20 min.
Terrific-Broth (TB) Medium: glycerol 14.1 g.L-12.17 g.L potassium dihydrogen phosphate-1Dipotassium hydrogen phosphate 2.74 g.L-1Tryptone 12.5 g. L-1Yeast powder 20 g.L-1(ii) a And (3) sterilization conditions: 121 ℃ for 20 min.
Reaction termination solution: potassium acetate 32.5 g.L-1Zinc sulfate 4.0 g.L-1Ethylene glycol methyl ether 80% (v/v); adjusting the pH value of the solution to 8.0 by adopting concentrated hydrochloric acid; storing at room temperature in dark place, and collecting supernatant when using.
The specific enzyme activity of the beta-glucanase is determined by the following method:
the catalytic activity of the beta-glucanase is determined by adopting a modified AZO method. Definition of unit enzyme activity: an amount of enzyme required to degrade barley beta-glucan to 1. mu. mol reducing sugar per minute at 60 ℃ and pH 6.5. Enzyme activity determination: 200 μ L of AZO blue barley beta-glucan substrate was mixed with 20 mmol.L-1And pH6.5 sodium phosphate buffer solution, preheating in a water bath at 60 deg.C, adding 100 μ L beta-glucanase, slightly blowing and sucking, and allowing the enzyme and substrate to react. After the reaction is carried out for 10min, 3mL of reaction stop solution is gently added into the reaction system, the reaction system is kept stand for 5-10min, 7000 Xg is centrifuged for 5min, the light absorption value of the supernatant at 590nm is measured, and the control group is a sample obtained by treating 100 mu L of deionized water instead of enzyme solution. The enzyme activity calculation formula is as follows: beta-glucanase enzyme activity (U.mL)-1)＝(OD590+0.0558)/(0.0012 × 180); wherein n is the dilution multiple of enzyme solution, OD590The absorbance of the supernatant at 590nm is determined.
Example 1: screening of beta-glucanase acid-resistant site and mutant
Searching eight strains of Bacillus-derived mesophilic 1,3-1,4- β -glucanases from NCBI databases and literature, VEL (Bacillus velezensis origin, optimum pH of 5.0), AMY (Bacillus amyloliquefaciens origin, optimum pH of 5.0), LIC (Bacillus amyloliquefaciens origin, optimum pH of 5.6), 3O5S (Bacillus subtilis 168 origin, optimum pH of 6.0), AMY2(Bacillus amyloliquefaciens TB2 origin, optimum pH of 6.2), SUB (Bacillus subtilis MA139 origin, optimum pH of 6.4), Bacillus terquilensis CGX5-1 origin, optimum pH of 6.5), 1GBG (Bacillus amyloliquefaciens origin, optimum pH of 7.0) and WT 2 strain source, optimum pH of 7.539; arranging the amino acid sequences according to the optimal pH value from low to high, carrying out amino acid sequence alignment, screening residues which are highly conserved in the sequence with the optimal pH value lower than pH6.0 from non-conserved residues for mutation, and obtaining 8 strains of single-point mutants of D8E, G12S, N28D, L44S, I133L, I133V, V134A and E191D on the basis of the beta-glucanase from Bacillus tequilensis CGX5-1.
The protein surface plays an important role in attracting substrates by enzymes and maintaining their conformational stability in extreme environments, and in addition, the enzyme surface with a lower optimum pH value generally has more acidic residues, which are presumed to play an important role in maintaining the structural stability and catalytic activity of the enzyme in acidic environments. The exposure degree of wild enzyme residues is calculated by a GetARea online server, glutamine and asparagine which are completely exposed (the exposure degree is more than 30 percent) are screened out, and the glutamine and the asparagine are respectively mutated into glutamic acid and aspartic acid with stronger acidity, so that 11 single-point mutants of Q1E, Q171E, N11D, N26D, N37D, N129D, N142D, N154D, N170D, N197D and N200D are obtained.
After the 19 single-point mutant enzymes obtained above were subjected to warm bath at 40 ℃ and four pHs (pH4.5, pH5.5, pH6.5 and pH7.5) for 1 hour, the relative enzyme activities thereof were measured, respectively, and the results are shown in Table 1, where the untreated enzyme activity was 100% relative enzyme activity: under the pH value of 4.5, the relative enzyme activities of Q1E, Q171E and N26D are greatly improved compared with the wild type; when the pH value reaches pH5.5, the relative enzyme activities of I133L, V134A, E191D, Q1E, Q171E, N26D and N200D are improved compared with the wild enzyme; after the heat bath treatment under the conditions of pH6.5 and pH7.5, the relative enzyme activities of the 7 single mutant enzymes I133L, V134A, E191D, Q1E, Q171E, N26D and N200D are all higher than 50%, which indicates that the stability of the 7 mutant enzymes in an acidic environment is enhanced.
In order to ensure the catalytic activity of the mutant, the specific enzyme activities of 7 single mutant enzymes are compared, and the results show that the specific enzyme activities of the I133L, V134A, Q1E and N200D mutants are 5372.22 +/-126.54U/mg, 4441.36 +/-157.49U/mg, 5952.72 +/-541.36U/mg and 5248.13 +/-296.48U/mg respectively, and are improved by 30.53%, 7.91%, 44.63% and 27.51% respectively compared with the wild enzyme, while the specific activity values of the other 4 single mutants are lower than that of the wild enzyme.
In order to ensure the temperature stability of the mutants, the relative enzyme activities of the I133L, V134A, Q1E and N200D mutants at ph6.5 and six different temperatures (45 ℃, 50 ℃, 55 ℃, 60 ℃, 55 ℃ and 70 ℃) were determined, with the maximum enzyme activity being 100% relative enzyme activity. The optimum temperature analysis result shows that the optimum temperature of the I133L mutant is 60 ℃, the temperature is increased by 5 ℃ compared with that of the wild enzyme, and the relative enzyme activity is still 87.71% at 65 ℃, and at the moment, the relative enzyme activity of the wild enzyme is only 64.70%. The optimum temperature of the V134A and Q1E mutants is not obviously different from that of the wild type, the optimum temperature of the N200D mutant is reduced by 5 ℃, the relative enzyme activity at 55-70 ℃ is lower than that of the wild enzyme, and the relative enzyme activity at 65 ℃ is reduced by 30.52%, which indicates that the thermal stability is poor.
TABLE 1 enzymatic Activity of wild enzymes and Single-Point mutant enzymes
Example 2: construction of acid-resistant beta-glucanase multi-mutant
On the basis of three single-point mutants I133L, V134A and Q1E obtained in example 1,3 double mutants Q1E/I133L, Q1E/V134A, I133L/V134A and 1 triple mutant Q1E/I133L/V134A are constructed and characterized in enzymological properties.
A recombinant plasmid pET28a (+) -BglT (Jinking Wang, Chengtuo Niu, Xialing Liu, Xi Chen, Qi Li. characteristics of a New 1,3-1, 4-beta-Glucanase Gene from Bacillus tequilensis CGX5-1.Applied Biochemistry and Biotechnology,2014,173(3)) containing a Gene of Bacillus tequilensis CGX5-1 secreting beta-Glucanase is used as a template, and a recombinant plasmid containing a mutation at a corresponding site is obtained by amplification through an overlap extension PCR method.
Primers used to introduce the Q1E mutation (lower case letters are the mutated bases):
a forward primer: 5'-TCACTTCTACTGCCTCGGCTgaaACAGGTG-3', SEQ the ID NO.10 of the said product,
reverse primer: 5'-TCAAAAAACGATCCACCTGTttcAGCCGAGGC-3', SEQ ID NO. 11;
primers used to introduce the I133L mutation (lower case letters are the mutated base):
a forward primer: 5'-AACCATGAGAAGctgGTTGATCTCGGGTTTGATGCA-3', SEQ ID NO.12, reverse primer: 5'-CGAGATCAACcagCTTCTCATGGTTTCCTGCACC-3', SEQ ID NO. 13;
primers used to introduce the V134A mutation (lower case letters are the mutated bases):
a forward primer: 5'-TGAGAAGATTgcaGATCTCGGGTTTGATGCAGCC-3', SEQ ID NO.14, reverse primer: 5'-ACCCGAGATCtgcAATCTTCTCATGGTTTCCTGC-3', SEQ ID NO. 15;
the PCR reaction system is as follows: 2 x Phanta Max Master Mix 25. mu.L, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu.L, template DNA 1. mu.L, double distilled water to make up to 50. mu.L; the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 min; then denaturation at 95 ℃ for 1min, 30s at 55 ℃ and 5min at 72 ℃ for 30 cycles; the storage temperature was 4 ℃.
After obtaining a recombinant plasmid with a single site mutation by overlap extension PCR, the PCR product was hydrolyzed with Dpn I enzyme to remove the template strand. The enzyme cutting system is as follows: PCR product 46uL, Dpn I1 uL, 10 XBuffer 5 uL. The enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 1 h.
And carrying out PCR by taking the single mutant as a template to finally obtain the recombinant plasmid corresponding to the mutation of the two sites. The PCR product was hydrolyzed with Dpn I enzyme to remove the template strand.
And carrying out PCR by taking the double mutants as templates to finally obtain the recombinant plasmids corresponding to the three site mutations. The PCR product was hydrolyzed with Dpn I enzyme to remove the template strand.
Directly transforming the enzyme digestion product into escherichia coli BL21(DE3) competent cells; coating a kanamycin-resistant plate, and screening positive clones; extracting plasmid sequencing, and respectively carrying out recombinant bacteria containing mutants Q1E/133L, Q1E/V134A, I133L/V134A and Q1E/I133L/V134A.
Example 3: expression and enzymatic property determination of beta-glucanase mutant
1. Expression and purification of beta-glucanase mutants
A single colony of the recombinant strain obtained in example 1 was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, cultured at 37 ℃ at 180rpm for 10-12 hours, transferred to TB liquid medium containing 50. mu.g/mL kanamycin at 4% (v/v) inoculum size, and cultured at 37 ℃ at 200rpm to OD6000.8-1.0, adding 0.0336mM IPTG and 10mM alpha-lactose, inducing expression at 24 deg.C and 200rpm for 12h to obtain fermentation liquid. The fermentation broth was centrifuged at 8000rpm for 20min at 4 ℃ and the supernatant was collected by discarding the cells.
And purifying the target protein by using a Ni-NTA affinity chromatography column. The 1 XBinding Buffer and imidazole solution with different concentration used in the protein purification process should be filtered in vacuum through a 0.45 μm filter membrane before use, and ultrasonic degassing is carried out for 20 min.
First, the Ni-NTA affinity chromatography column was equilibrated with 1 × Binding Buffer, and the resulting supernatant was loaded onto the column, followed by elution with 1 × Binding Buffer until OD280And (4) stably eluting the target protein by adopting imidazole solutions with final concentrations of 50mM, 100mM and 250mM respectively, and collecting the eluates.
The enzyme activity of the eluate was measured and analyzed by SDS-PAGE, and it was found that the mutant enzyme was eluted mainly at an imidazole concentration of 100mM and the band was single. After the eluate containing the target protein was concentrated by an ultrafiltration tube having a molecular weight cut-off of 3000Da, the target protein was replaced with 20mM phosphate buffer (pH6.5) by a GE PD-10 desalting column to obtain a purified mutant enzyme sample.
2. Analysis of enzymatic Properties
(1) Protein concentration determination
The protein concentration is determined by the Bradford method, and the specific steps are as follows: quickly and uniformly mixing 200 mu L of Bradford working solution and 20 mu L of sample to be detected; reacting at 25-30 deg.C for 10min, measuring absorbance at 595nm, and collecting three samples in parallel, wherein the blank sample is phosphate buffer solution with pH of 6.5. And (4) substituting the obtained light absorption value into a standard curve to obtain the protein concentration of the sample.
(2) Determination of optimum temperature
The enzyme activity of the purified mutant enzyme obtained in example 3 was measured at different reaction temperatures (45, 50, 55, 60, 65, 70 ℃) using an appropriate amount of enzyme solution, and the optimum action temperature of the enzyme was examined with the highest enzyme activity being 100% relative enzyme activity (see FIG. 1 for the results). The optimal temperature of the single mutants Q1E and V134A is the same as that of the wild enzyme, while the optimal temperature of I133L, Q1E/I133L, Q1E/I133L, I133L/V134A and Q1E/I133L/V134A is 60 ℃, which is increased by 5 ℃ compared with the wild enzyme, thereby indicating that the temperature tolerance of the mutants I133L, Q1E/I133L, Q1E/I133L, I133L/V134A and Q1E/I133L/V134A is enhanced.
(3) Determination of optimum pH
Using 20mM acetic acid/potassium acetate buffer (pH4.0, pH4.5, pH5.0 and pH5.5) and 20mM sodium phosphate buffer (pH6.0, pH6.5, pH7.0, pH7.5 and pH8.0) and barley beta-glucan substrate equal volume mixture, configuration of substrate reaction buffer, determination of the obtained purified mutant enzyme activity, with the highest enzyme activity of 100% relative enzyme activity, investigation of the enzyme optimum action pH (results are shown in figure 2). The optimum pH values of V134A, Q1E/I133L, Q1E/I133L, I133L/V134A and Q1E/I133L/V134A are pH6.0, and are the same as those of the wild-type enzyme. The optimum pH value of the single mutant I133L is reduced by 0.5, the relative enzyme activity reaches 51.17% at pH4.5, and the wild enzyme only has 24.51% at the moment, which shows that the optimum pH value of I133L moves to an acidic environment and the activity at an acidic pH is enhanced.
(4) Determination of pH stability
The obtained purified mutant enzyme was diluted in 20mM acetic acid/potassium acetate buffer (pH4.0, pH4.5, pH5.0 and pH5.5) and 20mM sodium phosphate buffer (pH6.0, pH6.5, pH7.0, pH7.5 and pH8.0), and the enzyme activity was determined after storage at 40 ℃ for 1 hour, and the enzyme activity was determined as 100% relative enzyme activity with respect to the untreated sample, and the pH stability of the enzyme was examined (see the results in FIG. 3). The relative enzyme activities of I133L, Q1E/V134A and Q1E/I133L/V134A in the pH range of 4.0-8.0 are not obviously improved compared with the wild enzyme, the relative enzyme activities of V134A and I133L/V134A in the pH range of 4.5-6.0 are slightly higher than that of the wild enzyme, the relative enzyme activities of the mutant Q1E and Q1E/I133L in the pH range of 4.0-8.0 are both higher than that of the wild enzyme, particularly, the relative enzyme activities in the pH range of 4.0-5.5 are obviously higher than that of the wild enzyme, and the relative enzyme activities after water bath treatment under the condition of pH4.0 respectively reach 76.34% and 81.28%, while the wild enzyme is only 27.25%, which shows that the acid resistance of Q1E and Q1E/I133L is greatly improved.
Example 4: kinetic parameter analysis of beta-glucanase mutants
TABLE 2 comparison of kinetic parameters of wild enzymes and beta-glucanase mutants
Barley beta-glucan substrate was mixed with 20mM, pH6.5 sodium phosphate buffer to make substrate solutions of different concentrations (0.5, 1.0, 1.25, 2.5, 4.5 and 10 mg. multidot.mL)-1) And reacting with beta-glucanase with proper concentration under the optimal condition to determine the enzyme activity. The amount of reducing sugar produced per unit time is used as the initial rate V of the enzyme reaction, and the equation V-K is determined by the Michaelis constantm·V/[S]+VmaxDrawing an Eadi-Hofstee curve according to the measured data, and obtaining K through linear fittingmAnd VmaxValue of from VmaxThe enzyme catalytic constant k can be determined from the corresponding substrate concentrationcatValue, further calculated to obtain the catalytic efficiency constant kcat/KmThe results are shown in Table 2. Compared with wild enzymes, K of I133L, V134A, Q1E, Q1E/I133L and Q1E/I133L/V134AmThe values are all reduced, which indicates that the affinity of the enzyme and the substrate is improved; the specific enzyme activities of I133L, V134A, Q1E and Q1E/I133L are respectively improved by 30.53%, 7.91%, 44.63% and 16.92%, and the catalytic efficiency is respectively improved by 60.94%, 14.69%, 91.20 and 33.57%, which indicates that I133L, V134A, Q1E and Q1E are based on the ionThe catalytic activity of I133L was improved. K of I133L, V134A, Q1E and Q1E/I133L mutantscatThe value is not lower than that of the wild enzyme, and the K of the Q1E/V134A, I133L/V134A and Q1E/I133L/V134A mutantscatThe value is significantly reduced, which is not favorable for industrial application of the enzyme.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> acid-tolerant 1,3-1, 4-beta-glucanase mutants
<130> BAA210358A
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 214
<212> PRT
<213> Artificial sequence
<400> 1
Glu Thr Gly Gly Ser Phe Phe Asp Pro Phe Asn Gly Tyr Asn Ser Gly
1 5 10 15
Phe Trp Gln Lys Ala Asp Gly Tyr Ser Asn Gly Asn Met Phe Asn Cys
20 25 30
Thr Trp Arg Ala Asn Asn Val Ser Met Thr Ser Leu Gly Glu Met Arg
35 40 45
Leu Ala Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn
50 55 60
Arg Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
65 70 75 80
Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly
85 90 95
Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly
100 105 110
Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Ala Gly
115 120 125
Asn His Glu Lys Ile Val Asp Leu Gly Phe Asp Ala Ala Asn Ala Tyr
130 135 140
His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser Ile Lys Trp Tyr Val
145 150 155 160
Asp Gly Gln Leu Ser His Thr Ala Thr Asn Gln Ile Pro Thr Thr Pro
165 170 175
Gly Lys Ile Met Met Asn Leu Trp Asn Gly Thr Gly Val Asp Glu Trp
180 185 190
Leu Gly Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp
195 200 205
Val Arg Tyr Thr Lys Lys
210
<210> 2
<211> 214
<212> PRT
<213> Artificial sequence
<400> 2
Gln Thr Gly Gly Ser Phe Phe Asp Pro Phe Asn Gly Tyr Asn Ser Gly
1 5 10 15
Phe Trp Gln Lys Ala Asp Gly Tyr Ser Asn Gly Asn Met Phe Asn Cys
20 25 30
Thr Trp Arg Ala Asn Asn Val Ser Met Thr Ser Leu Gly Glu Met Arg
35 40 45
Leu Ala Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn
50 55 60
Arg Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
65 70 75 80
Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly
85 90 95
Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly
100 105 110
Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Ala Gly
115 120 125
Asn His Glu Lys Leu Val Asp Leu Gly Phe Asp Ala Ala Asn Ala Tyr
130 135 140
His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser Ile Lys Trp Tyr Val
145 150 155 160
Asp Gly Gln Leu Ser His Thr Ala Thr Asn Gln Ile Pro Thr Thr Pro
165 170 175
Gly Lys Ile Met Met Asn Leu Trp Asn Gly Thr Gly Val Asp Glu Trp
180 185 190
Leu Gly Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp
195 200 205
Val Arg Tyr Thr Lys Lys
210
<210> 3
<211> 214
<212> PRT
<213> Artificial sequence
<400> 3
Gln Thr Gly Gly Ser Phe Phe Asp Pro Phe Asn Gly Tyr Asn Ser Gly
1 5 10 15
Phe Trp Gln Lys Ala Asp Gly Tyr Ser Asn Gly Asn Met Phe Asn Cys
20 25 30
Thr Trp Arg Ala Asn Asn Val Ser Met Thr Ser Leu Gly Glu Met Arg
35 40 45
Leu Ala Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn
50 55 60
Arg Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
65 70 75 80
Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly
85 90 95
Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly
100 105 110
Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Ala Gly
115 120 125
Asn His Glu Lys Ile Ala Asp Leu Gly Phe Asp Ala Ala Asn Ala Tyr
130 135 140
His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser Ile Lys Trp Tyr Val
145 150 155 160
Asp Gly Gln Leu Ser His Thr Ala Thr Asn Gln Ile Pro Thr Thr Pro
165 170 175
Gly Lys Ile Met Met Asn Leu Trp Asn Gly Thr Gly Val Asp Glu Trp
180 185 190
Leu Gly Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp
195 200 205
Val Arg Tyr Thr Lys Lys
210
<210> 4
<211> 214
<212> PRT
<213> Artificial sequence
<400> 4
Glu Thr Gly Gly Ser Phe Phe Asp Pro Phe Asn Gly Tyr Asn Ser Gly
1 5 10 15
Phe Trp Gln Lys Ala Asp Gly Tyr Ser Asn Gly Asn Met Phe Asn Cys
20 25 30
Thr Trp Arg Ala Asn Asn Val Ser Met Thr Ser Leu Gly Glu Met Arg
35 40 45
Leu Ala Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn
50 55 60
Arg Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
65 70 75 80
Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly
85 90 95
Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly
100 105 110
Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Ala Gly
115 120 125
Asn His Glu Lys Leu Val Asp Leu Gly Phe Asp Ala Ala Asn Ala Tyr
130 135 140
His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser Ile Lys Trp Tyr Val
145 150 155 160
Asp Gly Gln Leu Ser His Thr Ala Thr Asn Gln Ile Pro Thr Thr Pro
165 170 175
Gly Lys Ile Met Met Asn Leu Trp Asn Gly Thr Gly Val Asp Glu Trp
180 185 190
Leu Gly Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp
195 200 205
Val Arg Tyr Thr Lys Lys
210
<210> 5
<211> 214
<212> PRT
<213> Bacillus tequilensis
<400> 5
Gln Thr Gly Gly Ser Phe Phe Asp Pro Phe Asn Gly Tyr Asn Ser Gly
1 5 10 15
Phe Trp Gln Lys Ala Asp Gly Tyr Ser Asn Gly Asn Met Phe Asn Cys
20 25 30
Thr Trp Arg Ala Asn Asn Val Ser Met Thr Ser Leu Gly Glu Met Arg
35 40 45
Leu Ala Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn
50 55 60
Arg Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
65 70 75 80
Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly
85 90 95
Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly
100 105 110
Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Ala Gly
115 120 125
Asn His Glu Lys Ile Val Asp Leu Gly Phe Asp Ala Ala Asn Ala Tyr
130 135 140
His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser Ile Lys Trp Tyr Val
145 150 155 160
Asp Gly Gln Leu Ser His Thr Ala Thr Asn Gln Ile Pro Thr Thr Pro
165 170 175
Gly Lys Ile Met Met Asn Leu Trp Asn Gly Thr Gly Val Asp Glu Trp
180 185 190
Leu Gly Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp
195 200 205
Val Arg Tyr Thr Lys Lys
210
<210> 6
<211> 645
<212> DNA
<213> Artificial sequence
<400> 6
gaaacaggtg gatcgttttt tgaccctttt aacggctata actccggttt ttggcaaaaa 60
gcagatggtt attcgaatgg aaatatgttc aactgcacgt ggcgggctaa taacgtatca 120
atgacgtcat tgggtgaaat gcgtttagcg ctaacaagcc catcttataa caagtttgac 180
tgcggggaaa accgttctgt tcaaacatat ggctatggac tttatgaagt cagaatgaaa 240
ccagctaaaa acacagggat cgtttcatcg ttcttcactt acacaggtcc aacagatgga 300
actccttggg atgagattga tatcgaattt ttaggaaaag acacaacgaa ggttcaattt 360
aactattata caaatggtgc aggaaaccat gagaagattg ttgatctcgg gtttgatgca 420
gccaatgcct atcatactta tgcattcgat tggcagccaa actctattaa atggtatgtc 480
gacgggcaat taaaacatac tgcaacaaac caaattccga caacacctgg aaagatcatg 540
atgaacttgt ggaatggcac gggtgtcgat gaatggcttg gctcctacaa tggtgtaaat 600
ccgctatacg ctcattatga ctgggtgcgc tatacaaaaa aataa 645
<210> 7
<211> 645
<212> DNA
<213> Artificial sequence
<400> 7
caaacaggtg gatcgttttt tgaccctttt aacggctata actccggttt ttggcaaaaa 60
gcagatggtt attcgaatgg aaatatgttc aactgcacgt ggcgggctaa taacgtatca 120
atgacgtcat tgggtgaaat gcgtttagcg ctaacaagcc catcttataa caagtttgac 180
tgcggggaaa accgttctgt tcaaacatat ggctatggac tttatgaagt cagaatgaaa 240
ccagctaaaa acacagggat cgtttcatcg ttcttcactt acacaggtcc aacagatgga 300
actccttggg atgagattga tatcgaattt ttaggaaaag acacaacgaa ggttcaattt 360
aactattata caaatggtgc aggaaaccat gagaagctgg ttgatctcgg gtttgatgca 420
gccaatgcct atcatactta tgcattcgat tggcagccaa actctattaa atggtatgtc 480
gacgggcaat taaaacatac tgcaacaaac caaattccga caacacctgg aaagatcatg 540
atgaacttgt ggaatggcac gggtgtcgat gaatggcttg gctcctacaa tggtgtaaat 600
ccgctatacg ctcattatga ctgggtgcgc tatacaaaaa aataa 645
<210> 8
<211> 645
<212> DNA
<213> Artificial sequence
<400> 8
caaacaggtg gatcgttttt tgaccctttt aacggctata actccggttt ttggcaaaaa 60
gcagatggtt attcgaatgg aaatatgttc aactgcacgt ggcgggctaa taacgtatca 120
atgacgtcat tgggtgaaat gcgtttagcg ctaacaagcc catcttataa caagtttgac 180
tgcggggaaa accgttctgt tcaaacatat ggctatggac tttatgaagt cagaatgaaa 240
ccagctaaaa acacagggat cgtttcatcg ttcttcactt acacaggtcc aacagatgga 300
actccttggg atgagattga tatcgaattt ttaggaaaag acacaacgaa ggttcaattt 360
aactattata caaatggtgc aggaaaccat gagaagattg cagatctcgg gtttgatgca 420
gccaatgcct atcatactta tgcattcgat tggcagccaa actctattaa atggtatgtc 480
gacgggcaat taaaacatac tgcaacaaac caaattccga caacacctgg aaagatcatg 540
atgaacttgt ggaatggcac gggtgtcgat gaatggcttg gctcctacaa tggtgtaaat 600
ccgctatacg ctcattatga ctgggtgcgc tatacaaaaa aataa 645
<210> 9
<211> 645
<212> DNA
<213> Artificial sequence
<400> 9
gaaacaggtg gatcgttttt tgaccctttt aacggctata actccggttt ttggcaaaaa 60
gcagatggtt attcgaatgg aaatatgttc aactgcacgt ggcgggctaa taacgtatca 120
atgacgtcat tgggtgaaat gcgtttagcg ctaacaagcc catcttataa caagtttgac 180
tgcggggaaa accgttctgt tcaaacatat ggctatggac tttatgaagt cagaatgaaa 240
ccagctaaaa acacagggat cgtttcatcg ttcttcactt acacaggtcc aacagatgga 300
actccttggg atgagattga tatcgaattt ttaggaaaag acacaacgaa ggttcaattt 360
aactattata caaatggtgc aggaaaccat gagaagctgg ttgatctcgg gtttgatgca 420
gccaatgcct atcatactta tgcattcgat tggcagccaa actctattaa atggtatgtc 480
gacgggcaat taaaacatac tgcaacaaac caaattccga caacacctgg aaagatcatg 540
atgaacttgt ggaatggcac gggtgtcgat gaatggcttg gctcctacaa tggtgtaaat 600
ccgctatacg ctcattatga ctgggtgcgc tatacaaaaa aataa 645
<210> 10
<211> 30
<212> DNA
<213> Artificial sequence
<400> 10
tcacttctac tgcctcggct gaaacaggtg 30
<210> 11
<211> 32
<212> DNA
<213> Artificial sequence
<400> 11
tcaaaaaacg atccacctgt ttcagccgag gc 32
<210> 12
<211> 36
<212> DNA
<213> Artificial sequence
<400> 12
aaccatgaga agctggttga tctcgggttt gatgca 36
<210> 13
<211> 34
<212> DNA
<213> Artificial sequence
<400> 13
cgagatcaac cagcttctca tggtttcctg cacc 34
<210> 14
<211> 34
<212> DNA
<213> Artificial sequence
<400> 14
tgagaagatt gcagatctcg ggtttgatgc agcc 34
<210> 15
<211> 34
<212> DNA
<213> Artificial sequence
<400> 15
acccgagatc tgcaatcttc tcatggtttc ctgc 34
Claims (10)
1,3-1, 4-beta-glucanase mutant, characterized in that 1,3-1, 4-beta-glucanase shown in SEQ ID NO.5 is used as parent enzyme, and amino acid of one or more sites of 1 st, 133 th and 134 th of the parent enzyme is mutated.
2. The mutant according to claim 1, wherein position 1 is mutated to glutamic acid, position 133 is mutated to leucine, and/or position 134 is mutated to alanine.
3. A gene encoding the 1,3-1,4- β -glucanase mutant of claim 1 or 2.
4. A recombinant plasmid carrying the gene of claim 3.
5. The recombinant plasmid of claim 4, wherein the expression vector is any one of pET series, Duet series, pGEX series, pHY300PLK, pPIC series.
6. A microbial cell expressing the mutant of claim 1 or 2 or carrying the gene of claim 3.
7. The host cell of claim 6, wherein the microbial cell is derived from Escherichia coli.
8. A method for producing the mutant according to claim 1, wherein the mutant is produced by fermentation using the microbial cell according to claim 6 as a fermentation strain.
9. The method of claim 8, wherein the microbial cells are inoculated into TB culture medium and cultured to the logarithmic phase of growth, IPTG and alpha-lactose are added for induction expression, and after the expression is finished, the supernatant is collected by centrifugation, namely the crude enzyme solution containing the mutant of claim 1.
10. Use of the mutant of claim 1 or 2, or the gene of claim 2, or the microbial cell of claim 6 for the hydrolysis of β -glucan.
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| CN114657166A (en) * | 2020-12-22 | 2022-06-24 | 福尼亚生物处理股份有限公司 | Additional endoglucanase variants and methods |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| CN105671022A (en) * | 2016-03-11 | 2016-06-15 | 江南大学 | 1,3-1,4-beta-glucanase mutant |
| CN112481240A (en) * | 2020-12-10 | 2021-03-12 | 江苏科技大学 | GH16 family heat-resistant glucanase mutant and construction method and application thereof |
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| CN103045560A (en) * | 2011-10-14 | 2013-04-17 | 中国农业大学 | Method for acquiring directed mutation gene and acidic Beta-1,3-1,4-glucanase discovered on basis of method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114657166A (en) * | 2020-12-22 | 2022-06-24 | 福尼亚生物处理股份有限公司 | Additional endoglucanase variants and methods |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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|---|---|
| CN113234705B (en) | 2022-09-27 |
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