CN116218801B - Heat-resistant glucose oxidase mutant and application thereof - Google Patents
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Abstract
一种耐热葡萄糖氧化酶突变体及其应用，其特征在于，包括以葡萄糖氧化酶PaGOD‑1GPE为母本对A263和K424两个位点突变后得到的突变体，命名为PaGOD‑1GPE_A263P和PaGOD‑1GPE_K424F；所述PaGOD‑1GPE_A263P的氨基酸序列如SEQ ID NO.1所示；所述PaGOD‑1GPE_K424F的氨基酸序列如SEQ ID NO.2所示。相对于盲目筛菌或人工（自然）诱变等手段，理性设计缩短了酶学性质改造时间。因此，将本发明的耐热葡萄糖氧化酶突变体应用于饲料添加产业中，具有广阔的应用前景。
A thermostable glucose oxidase mutant and its application, characterized in that it comprises mutants obtained by mutating the two sites A263 and K424 with glucose oxidase PaGOD-1GPE as the parent, named PaGOD-1GPE_A263P and PaGOD-1GPE_K424F; the amino acid sequence of the PaGOD-1GPE_A263P is shown in SEQ ID NO.1; the amino acid sequence of the PaGOD-1GPE_K424F is shown in SEQ ID NO.2. Compared with blind screening of bacteria or artificial (natural) mutagenesis and other means, rational design shortens the time for enzymatic property modification. Therefore, the thermostable glucose oxidase mutant of the present invention is applied to the feed additive industry, which has broad application prospects.
Description
Technical Field
The invention relates to the fields of genetic engineering and protein engineering, and relates to a glucose oxidase PaGOD-1GPE mutant derived from fungus P.amagayakenase and application thereof.
Background
Glucose Oxidase (GOD) is an aerobic dehydrogenase that specifically oxidizes beta-D-glucose to gluconic acid and hydrogen peroxide. Glucose oxidase is widely distributed in animals, plants and microorganisms, which are the main sources of the glucose oxidase, and the main production strains are aspergillus niger and penicillium. The glucose oxidase is used as a novel feed enzyme preparation for replacing antibiotics, and has various functions of protecting animal intestinal tracts, promoting digestion and absorption, improving organism immunity and the like. Glucose oxidase produced from penicillium specifically and aspergillus niger has been listed in the general class 4 enzyme preparation of the department of agriculture, feed additive variety catalogue (2013).
Pen-GOD exists as a homodimer, and each subunit can be non-covalently bound to one FAD. The specificity for beta-D-glucose is relatively strong, usually the catalytic efficiency of GOD from Penicillium is higher on the substrate than that of GOD from Aspergillus, most GOD shows maximum enzyme activity at 30℃and pH 6.0, but the stability is poor. Currently, most GODs remain stable at pH 4.0.7.0, with substantially no enzyme activity at pH values less than 3.0 or greater than 10.0. GOD from Penicillium is generally stable below 50 ℃.
Glucose oxidase is used in a wide variety of applications, including mainly feed, food, medicine, textile and biofuel cells. In industrial applications, most of application environments are high temperature environments, so that obtaining glucose oxidase with excellent thermal stability is important for industrial production.
Currently, protein engineering is widely used in enzyme molecule improvement, i.e., modification of the enzyme function is achieved by modifying or modifying the gene or the protein itself to change the structure of the protein. Protein engineering is mainly used for the design and modification of enzyme properties such as thermal stability, catalytic efficiency, substrate specificity and extreme environmental tolerance of enzymes. The main related methods are directed evolution, rational design and semi-rational design. The rational design is a rapid and effective modification means, for example, by the method, GOD genes from A. Niger and P. Amagasaine are replaced with each other, so that GOD mutants with improved catalytic efficiency and stability are successfully obtained.
Disclosure of Invention
The invention provides a glucose oxidase mutant and application thereof, wherein the mutant is obtained by screening PaGOD-1GPE key amino acid sites A263 and K424 from Penicillium amagasakiense sources after mutation, and particularly 2 glucose oxidase mutants with improved thermal stability are obtained, so that the glucose oxidase mutant has a wide prospect in the industries of feed mildew prevention and antibiotic substitution.
The high temperature resistant glucose oxidase mutant comprises mutants obtained by mutating two positions A263 and K424 by taking glucose oxidase PaGOD-1GPE as a female parent, wherein the mutants are named PaGOD-1GPE_A263P and PaGOD-1GPE_K424F, the amino acid sequence of PaGOD-1GPE_A263P is shown as SEQ ID NO.1, and the amino acid sequence of PaGOD-1GPE_K424F is shown as SEQ ID NO. 2.
Translating the nucleotide of the glucose oxidase mutant, wherein the nucleotide sequence of PaGOD-1GPE_A263P is shown as SEQ ID NO.3, and the nucleotide sequence of PaGOD-1GPE_K424F is shown as SEQ ID NO.4
A recombinant vector comprising the nucleotide sequence described above.
A recombinant strain comprising the above recombinant vector.
The application of the recombinant strain in preparing feed additive.
The recombinant strain is applied to inhibiting pathogenic microorganisms.
The invention has the beneficial effects that two mutants obtained by taking Penicillium amagasakiense derived glucose oxidase PaGOD-1GPE as a female parent to mutate A263 site and K424 site are specifically constructed by constructing a recombinant strain containing the mutants, and two glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F with improved thermal stability are screened after induced culture. In terms of thermostability, the half lives (t 1/2) of the mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F at 60 ℃ are respectively 9min and 10min which are respectively 1.8 times and 2 times that of the wild-type enzyme PaGOD-1GPE (5 min), the specific activities of the mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F in terms of catalytic activity are respectively 78.32U/mg and 75.94U/mg, the difference is small compared with the wild-type PaGOD-1GPE (74.93U/mg), and the optimal pH value and the optimal temperature are basically consistent with the wild-type. Compared with the means of blind screening bacteria or artificial (natural) mutagenesis and the like, the rational design shortens the modification time of the enzymatic properties. Therefore, the heat-resistant glucose oxidase mutant provided by the invention has a wide application prospect when being applied to the feed additive industry.
Drawings
FIG. 1 is an optimum pH assay for wild-type glucose oxidase and two glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F;
FIG. 2 is an optimal temperature measurement of wild-type glucose oxidase and two glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F;
FIG. 3 is a graph showing the results of pH stability assays for wild-type glucose oxidase and two glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F.
FIG. 4 is a measurement of half-life t 1/2 of wild-type glucose oxidase and two glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F at 60 ℃.
FIG. 5 shows the results of zone diameter measurements of wild-type glucose oxidase and two glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F.
Detailed Description
Test materials used in the following examples:
1. The strain and the vector are an expression host Pichia pastoris GS, an expression plasmid vector pPIC9r laboratory self-provided;
2. Enzymes and other biochemical reagents are Taq enzyme from full gold company, endonuclease from full gold company, o-dianisidine from Sigma company, peroxidase from leaf company, and other reagents which are all domestic analytical pure reagents (all purchased from national drug group);
3. Culture medium:
(1) LB medium, 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;
(2) YPD medium 1% yeast extract, 2% peptone, 2% glucose;
(3) MD solid medium, 2% glucose, 1.5% agarose, 1.34% YNB,0.00004% Biotin;
(4) MM solid medium, 1.5% agarose, 1.34% YNB,0.00004% Biotin,0.5% methanol;
(5) BMGY medium 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB,0.00004% Biotin;
(6) BMMY medium 1% yeast extract, 2% peptone, 1.34% YNB,0.00004% Biotin,0.5% methanol (V/V).
EXAMPLE 1 obtaining of genes encoding thermostable glucose oxidase mutants
Recombinant expression vector pic9r-PaGOD-1GPE of glucose oxidase gene PaGOD-1GPE (nucleotide sequence shown as SEQ ID NO. 5) from Penicillium amagasakiense is used as a template, site-directed mutagenesis is carried out on sites A263 and K424 by adopting a site-directed mutagenesis method, and the primer design is shown in table 1, the mutation method and cloning method reference (Exploiting the activity-stability trade-off of glucose oxidase from Aspergillus niger using a simple approach to calculate thermostability of mutants.Jiang,et al.,2021).
TABLE 1 site-directed mutagenesis primer in glucose oxidase PaGOD-1GPE
EXAMPLE 2 preparation of thermostable glucose oxidase mutant
The linear recombinant expression vector obtained by PCR in example 1 is directly transformed into DMT competent, colony PCR is verified, a nucleic acid sequence of a target site mutant is obtained, the recombinant plasmid is linearized and then is transformed into pichia pastoris GS115, and recombinant yeast strains GS115/PaGOD-1GPE_A263P and GS115/PaGOD-1GPE_K424F are obtained.
GS115 strain containing recombinant plasmid is inoculated into 10mL test tube of 2mL BMGY culture medium, placed at 30 ℃, shake cultured at 220rpm for 48h, 3000g of culture solution is centrifuged for 5min, supernatant is discarded, precipitate is resuspended in 2mL BMMY culture medium containing 0.5% methanol, and placed again at 30 ℃ and induced culture at 220rpm for 48h. The supernatant is used for enzyme activity detection, and mutants PaGOD-1GPE_A263P (the amino acid sequence is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO. 3) and PaGOD-1GPE_K424F (the amino acid sequence is shown as SEQ ID NO.2, the nucleotide sequence is shown as SEQ ID NO. 4) with thermal stability improved than that of the wild enzyme are screened.
The wild type GS115/PaGOD-1GPE and two mutants GS115/PaGOD-1GPE_A263P and GS115/PaGOD-1GPE_K424F were amplified and fermented, first inoculated in YPD medium to obtain seed culture broth, inoculated in 1L flasks of 300mL BMGY medium at 1% inoculum size, placed in shaking culture at 30℃and 220rpm for 48h, after which 3000g of the broth was centrifuged for 5min, the supernatant was discarded, the pellet was resuspended in 100mL BMMY medium containing 0.5% methanol, and again placed under conditions of 30℃and 220rpm for induction culture. 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 the supernatant is taken for enzyme activity detection. Finally, the supernatant was concentrated to 20mL for enzymatic property determination and comparison.
Example 3 comparative analysis of enzymatic Properties of recombinant thermostable glucose oxidase mutant and wild-type
1. O-dianisidine assay
The specific method is that under standard conditions (pH 6.0,30 ℃), a 3mL reaction system comprises 2.5mL o-dianisidine buffer, 300 mu L substrate, 100 mu L peroxidase (90U/mL), 100 mu L diluted enzyme solution, and the reaction is carried out for 3min, and 2mL 2M H 2SO4 is added to terminate the reaction. OD was measured at 540 nm. 1 enzyme activity unit (U) is defined as the amount of enzyme required to decompose 1. Mu. MoL of substrate per minute under standard conditions to produce hydrogen peroxide.
2. Property determination of recombinant thermostable glucose oxidase mutant and wild type
1. Recombinant thermostable glucose oxidase mutant and wild-type optimum pH determination method
The glucose oxidase mutant of example 2 and the wild-type glucose oxidase were subjected to enzymatic reactions at different pH (1.0-11.0) to determine their optimal pH. The substrate beta-D-glucose was assayed for glucose oxidase activity at 30℃using 0.1mol/L citric acid-disodium hydrogen phosphate buffer at different pH (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0).
As a result, the optimal reaction pH of the wild-type glucose oxidase and the glucose oxidase mutant was 6.0 as shown in FIG. 1.
2. Recombinant thermostable glucose oxidase mutant and method for determining optimum temperature of wild type glucose oxidase mutant
The optimal temperatures of the recombinant thermostable glucose oxidase mutant and the wild-type glucose oxidase were determined by enzymatic reactions performed at different temperatures (25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃) in a 0.1mol/L buffer system of citric acid-disodium hydrogen phosphate buffer (pH 6).
The results are shown in FIG. 2, which shows that the optimum temperature of the recombinant wild-type glucose oxidase is 45 ℃, the optimum temperature of the two thermostable glucose oxidase mutants is 40 ℃, and the relative enzyme activity at high temperature (65 ℃) is significantly improved compared with the wild-type enzyme.
3. Method for measuring pH stability of wild glucose oxidase and mutant
The glucose oxidase mutant and the wild-type glucose oxidase were diluted with 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution of different pH (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0) and then placed in a 37 ℃ constant temperature water bath kettle for treatment for 1 hour, and then the relative residual enzyme activities thereof were measured at pH 6.0 and 30 ℃ without treatment, and the enzyme activities were 100% as a control.
The results are shown in FIG. 3, which demonstrate that the pH stability of the glucose oxidase mutant PaGOD-1GPE_A263P was superior to that of the recombinant wild-type glucose oxidase in an acidic environment.
4. Method for measuring thermal stability of wild glucose oxidase and mutant
Half-life at 60 ℃ (t 1/2) mutants were treated with wild type at 60 ℃ for a maximum of 30min and their respective residual enzyme activities were examined.
The half-life measurement results at 60 ℃ are shown in FIG. 4, and indicate that t 1/2 of the glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F are 9min and 10min respectively, and are prolonged by 1.8 times and 2 times respectively compared with that of the wild-type glucose oxidase (5 min), namely the mutant PaGOD-1GPE_K424F has the best heat stability.
5. Recombinant thermostable glucose oxidase mutant and dynamic parameter determination method of wild type glucose oxidase mutant
Detection method is referred to in the literature (Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution.Tu,et al.,2019).
Glucose solutions of different concentrations (3.125 mM, 6.25mM, 12.5mM, 25mM, 50mM, 62.5mM, 100mM, 125mM, 200mM, 250mM, 500mM,1000 mM) were prepared as substrates with pH 6.0,0.1mol/L citric acid-disodium hydrogen phosphate buffer, and the enzyme activities were measured under standard conditions of 30℃ C, pH 6.0.0. The measured enzyme activity data were analyzed using GRAPHPAD PRISM 5.01.01 software to obtain K m values and V max for the wild type and each mutant of the recombinant thermostable glucose oxidase.
Under standard conditions, the catalytic efficiencies (k cat/Km) of the recombinant thermostable glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F were 13.07mM -1min-1 and 38.01mM -1min-1, respectively, and the catalytic efficiency (k cat/Km) of the mutant PaGOD-1GPE_K424F was 1.47 times that of the wild type (25.74 mM -1min-1), respectively, and the specific activities of the recombinant thermostable glucose oxidase mutants were 78.32U/mg and 75.94U/mg, respectively, which were not significantly changed compared to the wild type (74.93U/mg) (see Table 2).
TABLE 2 comparison of specific Activity and kinetic parameters of recombinant thermostable glucose oxidase mutants with wild type
Example 4 in vitro antibacterial effect analysis of recombinant thermostable glucose oxidase mutant and wild-type
The Pseudomonas aeruginosa liquid cultured at 37 ℃ for 12 hours was diluted to 1x10 7 CFU·mL-1, 100 μl was sucked and coated on LB solid plates, and holes were punched equidistantly using a puncher (6 mm).
The glucose oxidase mutant and the wild-type glucose oxidase of example 2 were mixed with 10% of the β -D-glucose substrate in equal volumes, respectively, and 200 μl of the mixture was added to the wells prepared in advance. The culture was carried out at 37℃for 24 hours to determine the diameter of the zone of inhibition.
The results are shown in FIG. 5, which shows that the diameter of the inhibition zone of the recombinant wild-type glucose oxidase is 12.5mm, and the diameters of the inhibition zones of the recombinant thermostable glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F are 13mm and 16.25mm respectively. The diameters of the inhibition zones of the glucose oxidase mutants PaGOD-1GPE_A263P and PaGOD-1GPE_K424F are 1.04 times and 1.3 times that of the recombinant wild glucose oxidase respectively, namely the inhibition effect of the mutants PaGOD-1GPE_K424F on pseudomonas aeruginosa is best.
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Effective date of registration: 20251103 Address after: 530000 floors 1-5, building A-2, Nanning bioengineering technology center, No. 18 Xinji Road, Nanning, Guangxi Zhuang Autonomous Region Patentee after: NANNING HARWORLD BIOTECHNOLOGY Co.,Ltd. Country or region after: China Address before: 212100 Jiangsu Province Zhenjiang City Dantu District Changhui Road 666.NO Jiangsu University of Science and Technology Office of Science and Technology Patentee before: JIANGSU University OF SCIENCE AND TECHNOLOGY Country or region before: China |
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