CN108048430B - Endoglucanase NfEG12A mutant and coding gene and application thereof - Google Patents
Endoglucanase NfEG12A mutant and coding gene and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of agricultural biology, in particular to an endoglucanase NfEG12A mutant and a coding gene and application thereof. The mutant is characterized in that the amino acid Y at the 7 th site and/or the 111 th site of the endoglucanase NfEG12A with the amino acid sequence shown as SEQ ID No.1 is mutated into W. The mutant has the optimum temperature and the optimum pH which are consistent with those of wild enzymes and are 65 ℃ and 5.0, the catalytic efficiency of the mutant on beta-1, 3-1, 4-glucan and xyloglucan is respectively improved by 0.5-0.8 times, and the mutant has good heat resistance and pH tolerance, so the mutant has very considerable application prospect in industry.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to an endoglucanase NfEG12A mutant and a coding gene and application thereof.
Background
Cellulose, hemicellulose and lignin are the main components of plant cell walls. Cellulose is a long-chain polysaccharide polymer formed by connecting glucopyranose with beta-1, 3 and beta-1, 4 glycosidic bonds in different proportions. The beta-glucan chains form insoluble cellulose fibrils by the close arrangement of intramolecular and intermolecular hydrogen bonds.
The NfEG12A is a high-temperature acidic enzyme, the optimum temperature is 65 ℃, the optimum pH value is 5.0, the enzyme is the enzyme with the highest enzyme activity in the family known at present, and in order to further improve the industrial application potential of the enzyme, the molecular engineering modification is carried out on the enzyme so as to improve the efficiency of the enzyme in catalyzing and hydrolyzing fiber substrates.
Disclosure of Invention
The invention aims to provide endoglucanase mutants NfEG12A-Y7W, NfEG12A-Y111W and NfEG 12A-Y7/111W.
It is still another object of the present invention to provide a gene encoding the endoglucanase mutant as described above.
Another objective of the invention is to provide a recombinant vector containing the endoglucanase mutant gene.
Another object of the present invention is to provide a recombinant strain comprising the above endoglucanase mutant gene.
Another objective of the invention is to provide a genetic engineering method for preparing the endoglucanase mutant gene.
The invention also aims to provide application of the endoglucanase mutant.
The invention carries out site-directed mutagenesis on endoglucanase NfEG12A to respectively construct mutants NfEG12A-Y7W, NfEG12A-Y111W and NfEG 12A-Y7/111W.
The amino acid sequence of the endoglucanase NfEG12A is shown in SEQ ID NO. 1: (bold amino acid sequence is signal peptide, underlined amino acids are mutation sites)
The invention constructs mutants of NfEG12A-Y7W, NfEG12A-Y111W and NfEG12A-Y7/111W at the 7 and 111 sites of NfEG12A through site-specific mutagenesis, wherein the mutants are obtained by mutating the 7 th amino acid Y and the 111 th amino acid Y of endoglucanase NfEG12A into W respectively or simultaneously, so that the catalytic efficiencies of the endoglucanase NfEG12 to beta-1, 3-1, 4-glucan and xyloglucan are respectively improved by 0.5-0.8 and 0.9-2.5 times, and the industrial application value of the endoglucanase is improved.
According to the specific embodiment of the invention, each mutation primer is designed, and the wild-type plasmid pPIC9 gamma-Nfeg 12A of the gene coding endoglucanase NfEG12A is used as a template for site-directed mutation. And purifying to obtain a plasmid containing the mutant codon, and transforming the plasmid into a pichia pastoris competent cell GS115 for expression to obtain the recombinant mutant protein.
The invention introduces mutation by a PCR mode to obtain mutant endoglucanases NfEG12A-Y7W, NfEG12A-Y111W and NfEG 12A-Y7/111W. The invention also provides a recombinant vector containing the endoglucanase gene, which is named as pPIC9 gamma-Nfeg 12A-Y7W, pPIC9 gamma-Nfeg 12A-Y111W and pPIC9 gamma-Nfeg 12A-Y7/111W.
The endoglucanase mutant gene is inserted between proper restriction enzyme cutting sites of an expression vector, so that the nucleotide sequence of the endoglucanase mutant gene is operably connected with an expression regulation sequence. As a most preferred embodiment of the present invention, it is preferred that the endoglucanase gene of the present invention is inserted between Eorr I and Not I restriction sites on the plasmid pPIC9 γ such that the nucleotide sequence is located downstream of and under the control of the AOX1 promoter.
The invention also provides a recombinant strain containing the gene, and the preferred recombinant strain is GS115/NfEG12A-Y7W, GS115/NfEG12A-Y111W, and GS115/NfEG 12A-Y7/111W.
The invention also provides a method for preparing the endoglucanase mutant, which comprises the following steps:
1) transforming host cell pichia GS115 by using the recombinant vector to obtain a recombinant strain;
2) culturing the recombinant strain, and inducing the recombinant cellulase to express;
3) recovering and purifying the expressed endoglucanase mutant.
The invention also provides application of the endoglucanase mutant.
The optimum temperature and the optimum pH of the mutants of the invention, namely NfEG12A-Y7W, NfEG12A-Y111W and NfEG12A-Y7/111W, are consistent with those of wild enzymes and are 65 ℃ and pH5.0, the catalytic efficiency of the mutants on beta-1, 3-1, 4-glucan and xyloglucan is respectively improved by 0.5-0.8 times, and the mutants have good heat resistance and pH tolerance, so the mutants have very considerable application prospect in industry.
Drawings
FIG. 1 shows the enzymatic determination of NfEG12A and its mutants;
FIG. 2 shows the determination of the specific activity of the wild enzyme and the mutant enzyme.
Detailed Description
Test materials and reagents
1. Bacterial strain and carrier: the pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.
2. Enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, the ligase was purchased from Sigma, and the others were made from general biochemicals.
3. Culture medium:
(1) neosartorya fischeri p1. medium is potato medium (1000 ml): 200g of potato cooking juice, 10g of glucose, 25g of agar, and pH 5.0.
(2) Coli culture LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).
(3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (W/V).
(4) BMMY medium: the components were identical to BMGY, pH6.0, except that 0.5% methanol was used instead of glycerol.
Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
Example 1
1. Construction of mutations
The mutants NfEG12A-Y7W, NfEG12A-Y111W and NfEG12A-Y7/111W are constructed by a site-directed mutagenesis method, the PCR result is verified by electrophoresis, and if a target band exists, 1 mu L of DMT enzyme is added to treat for 1h at 37 ℃ to remove the original plasmid template, and then TransI-T1 sequencing is transformed.
2. Transformation of Pichia pastoris GS115 by each mutant vector and screening of engineering bacteria
Preparing receptor competent cells.
Picking monoclonals from the MD plate by using a sterilization toothpick, marking the monoclonals according to the sequence, and dotting the monoclonals on the MM and then on the MD plate with the corresponding number; both plates were incubated in an incubator at 30 ℃ for 2 days. Selecting normal-growth transformants according to the number, inoculating the transformants into 3mL of BMGY medium, placing the centrifuge tube filled with the BMGY medium into a shaker at 30 ℃ for 48h, wherein the centrifuge tube is strictly sterile and is coated with eight layers of gauze; the bacterial solution cultured by shaking for 48h is placed in 3000g and centrifuged for 10min, the supernatant is removed, 1mL of BMMY culture medium containing 0.5% methanol is added into a centrifuge tube, and induction culture is carried out at 30 ℃ and 260 rpm. After induction culture for 48h, placing the bacterial liquid at 12000rpm, centrifuging for 3min, taking the supernatant to detect the activity, and screening out a transformant with the glucanase activity.
Inoculating the strain with higher endoglucanase enzyme activity into a 1L triangular flask of 300mL BMGY culture medium, and performing shake culture at 30 ℃ and 220rpm for 48 h; after this time, the culture broth was centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 100mL BMMY medium containing 0.5% methanol and again placed at 30 ℃ for induction culture at 220 rpm. 0.5mL of methanol is added every 12h, so that the concentration of the methanol in the bacterial liquid is kept at 0.5%, and meanwhile, the supernatant is taken for enzyme activity detection.
3. Determination of the Activity of the mutant endoglucanase according to the present invention
3.1 endoglucanase Activity Unit (U) definition: the amount of enzyme required to decompose dextran to produce 1. mu. molD- (+) -reducing sugars per minute under the given conditions.
3.2 determination of the optimum pH and pH stability of the recombinase reaction:
the purified recombinant endoglucanase was subjected to enzymatic reactions at 65 ℃ in substrates of different pH to determine its optimum pH. The buffers used were: McIlvaine buffer (0.2M disodium hydrogen phosphate/0.1M citric acid) at pH 2.0-8.0, Tris-HCl buffer at 0.1mol/L at pH 7.0-9.0, and Gly-NaOH buffer at pH 10.0-12.0.
And (3) measuring the pH stability: treating the concentrated pure enzyme solution in buffer solutions with different pH values at 37 deg.C for 1h, diluting with buffer solution with optimum pH, and measuring the residual enzyme activity under conditions of optimum pH and optimum temperature.
3.3 determination of the optimal temperature and temperature stability of the recombinase reaction:
the optimum temperature of the dextranase was determined by performing the enzymatic reaction in a citrate-disodium hydrogen phosphate buffer system (pH5.0) at different temperatures. The heat resistance is measured by treating dextranase at 65 deg.C and 70 deg.C for 10, 20, 30, and 60min respectively, and measuring the residual enzyme activity at the optimum temperature.
3.4 specific Activity of recombinase, KmValue and VmaxDetecting the recombinant enzyme
The first order reaction time of the reaction was determined with reference to the method of prunin (prunin, 2009). Determination of assay KmAnd VmaxThe reaction time of (3) was 5 min. Glucose (0.5, 0.4, 0.25, 0.2, 0.2, 0.175, 0.15, 0.1, 0.05%) with different concentrations was used as a substrate, the enzyme activity was measured under the optimum conditions, the corresponding reaction rate was calculated, and K was calculated using GraphPad Prism 5 softwaremValue and Vmax。
Standard curves were drawn according to the Bio-Rad kit method. The determination method comprises the following steps: the content of the target protein is calculated through a standard curve, the enzyme activity of the recombinase is measured under the optimal condition, and the specific activity of the enzyme can be obtained by dividing the enzyme activity by the concentration of the protein. Specific activity is defined as: the enzyme activity unit per mg of enzyme protein.
The results of basic property measurements of the wild-type enzyme and the mutant enzyme are shown in FIG. 1, and it can be seen from the results that the optimum temperatures and the optimum pH values of the mutant enzymes NfEG12A-Y7W, NfEG12A-Y111W and NfEG12A-Y7/111W were not changed from those of the wild-type enzyme NfEG12A, and were 65 ℃ and pH5.0, respectively. In the aspect of pH stability, the stability range of the wild enzyme and the mutant enzyme is 2-9, and about 40% -80% of relative enzyme activity remains under the conditions of pH10 and pH 11. In the aspect of thermal stability, the wild enzyme and the mutant enzyme are kept stable at 65 ℃, and the enzyme activity is lost after treatment at 70 ℃ for 20 min. These results indicate that the basic enzymatic properties of the mutants are not altered
The kinetic assay of the wild-type enzyme and the mutant enzyme is shown in Table 1 below.
TABLE 1 determination of kinetic parameters of NfEG12A and its mutants
Through the analysis of kinetic parameters of the enzyme and the mutant thereof, the K of all the mutants is found to be compared with the K of the wild enzymemAll values are reduced, kcatThe values varied to different extents, eventually increasing the catalytic efficiency of NfEG12A-Y7W, NfEG12A-Y111W, and NfEG12A-Y7/111W to 2.0, 1.8, and 1.5 times, respectively, for β -glucan. When xyloglucan is used as a substrate, all mutants show improved substrate affinity and catalytic efficiency, and the double mutant NfEG12A-Y7/111W is improved most obviously and reaches 3.5 times. These results indicate that positions 7 and 111, W, are beneficial in increasing the affinity of GH12 for substrates, especially branched xyloglucan.
Determination of specific Activity of wild enzyme and mutant enzyme
The specific activity of the NfEG12A on beta-glucan is 6587U/mg, and the specific activity on xyloglucan is 102U/mg. Compared with the wild enzyme, the specific activity of the single mutant on two types of substrates is increased, the specific activity of the NfEG12A-Y7W and NfEG12A-Y111W on beta-glucan is improved by 1.4 and 1.5 times of that of the wild enzyme, and the specific activity on xyloglucan is improved by 2 times. However, the specific activity of the double mutant NfEG12A-Y7/111W is consistent with that of the wild enzyme, but the specific activity on xyloglucan is improved by 2 times. In addition, the percentage of activity of NfEG12A on both xyloglucan and β -glucan substrates was 1.5%. When Y is replaced by W, the relative enzyme activity ratio is improved, the improvement is most remarkable in NfEG12A-Y7/111W, and the ratio respectively reaches 3.2%. This indicates that the introduction of W at either position 7 or 111 alone favors the binding of both types of substrates, whereas the introduction of W at both positions favors the hydrolysis of branched xyloglucan.
Sequence listing
<110> institute of feed of Chinese academy of agricultural sciences
<120> endoglucanase NfEG12A mutant and coding gene and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 234
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Lys Thr Phe Ala Ile Leu Gly Ala Phe Phe Ser Ser Ala Leu Ala
1 5 10 15
Gln Thr Leu Cys Asp Gln Tyr Ala Thr Tyr Ser Asn Gly Arg Tyr Thr
20 25 30
Val Asn Asn Asn Leu Trp Gly Lys Ser Ser Gly Ser Gly Ser Gln Cys
35 40 45
Thr Tyr Val Asp Ser Ile Ser Asn Ser Gly Val Ala Trp His Thr Thr
50 55 60
Trp Thr Trp Ser Gly Gly Asp Asn Gln Val Lys Ser Tyr Ala Asn Ser
65 70 75 80
Gln Val Ser Leu Thr Lys Lys Leu Val Ser Gln Ile Ser Ser Ile Pro
85 90 95
Thr Thr Val Gln Trp Ser Tyr Asp Asn Thr Asn Thr Arg Ala Asp Val
100 105 110
Ala Tyr Asp Leu Phe Thr Ala Ala Asp Ile Asn His Val Thr Tyr Ser
115 120 125
Gly Asp Tyr Glu Leu Met Ile Trp Leu Ala Arg Tyr Gly Ser Val Gln
130 135 140
Pro Ile Gly Ser Gln Ile Asp Ser Val Asn Ile Gly Gly His Thr Trp
145 150 155 160
Glu Leu Trp Tyr Gly Gly Ser Thr Gln Lys Thr Tyr Ser Phe Val Ser
165 170 175
Ala Thr Pro Ile Thr Ser Phe Ser Gly Asp Val Met Asp Phe Trp Asp
180 185 190
Tyr Leu Thr Ser Arg His Gly Tyr Pro Ala Ser Ser Gln Tyr Leu Ile
195 200 205
Asn Met Gln Phe Gly Thr Glu Pro Phe Thr Gly Gly Pro Ala Thr Leu
210 215 220
Arg Val Ser Gln Trp Thr Ala Ser Val Asn
225 230
Claims (7)
1. The mutant of endoglucanase NfEG12A is characterized in that the amino acid Y at the 7 th site and/or the 111 th site of the endoglucanase NfEG12A with the amino acid sequence shown in SEQ ID No.1 is mutated into W.
2. A mutant gene of endoglucanase NfEG12A, wherein the gene encodes the mutant endoglucanase NfEG12A of claim 1.
3. A recombinant vector comprising the endoglucanase NfEG12A mutant gene of claim 2.
4. A recombinant strain comprising the endoglucanase NfEG12A mutant gene of claim 2.
5. Use of the mutant endoglucanase NfEG12A of claim 1 for hydrolysis of β -glucan and xyloglucan.
6. Use of the endoglucanase NfEG12A mutant gene of claim 2 for hydrolysis of β -glucan and xyloglucan.
7. A method for efficiently expressing the endoglucanase NfEG12A mutant of claim 1, comprising the step of fermenting the strain with the recombinant strain of claim 4.
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| EP4021192A1 (en) * | 2019-08-30 | 2022-07-06 | AB Enzymes GmbH | Use of gh12 cellulases in preparing bakery products comprising rye-flour |
| CN111893124A (en) * | 2020-07-01 | 2020-11-06 | 深圳润康生态环境股份有限公司 | Endoglucanase gene, endoglucanase, preparation method and application thereof |
| CN112481240B (en) * | 2020-12-10 | 2021-11-09 | 江苏科技大学 | GH16 family heat-resistant glucanase mutant and construction method and application thereof |
| CN114317495A (en) * | 2022-01-10 | 2022-04-12 | 鑫缘茧丝绸集团股份有限公司 | Glucanase mutant with improved heat stability and application thereof |
| CN114752583B (en) * | 2022-03-30 | 2023-07-21 | 齐鲁工业大学 | Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof |
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