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A kind of beta-glucosidase improved mutant e168q and its coding gene and application
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A technology of glucosidase and mutant, applied in the field of genetic engineering and genetic engineering, can solve the problem of high cost
Active Publication Date: 2019-03-12
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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However, due to the special structure of lignocellulose, the resulting anti-biodegradation barrier keeps the cost of the conversion process high, which constitutes the biggest bottleneck in the production of bioenergy from cellulose materials.
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Embodiment 1
[0034] Cloning of embodiment 1 high catalytic efficiency β-glucosidase mutant coding gene A-E168Q
[0035] The present invention takes the acid β-glucosidase (its amino acid sequence as SEQ ID NO.3) derived from the thermophilic fungus Talaromyces leycettanus JCM12802 as the parent, and uses molecular biology techniques to carry out the acid β-glucosidase sequence Expression after region replacement.
[0041] Example 2 Preparation of β-glucosidase mutants with high catalytic efficiency.
[0042] The expression vector pPIC9r was subjected to double enzyme digestion (EcoR I+Not I), and at the same time, the gene A-E168Q encoding the high catalytic efficiency β-glucosidase mutant was double enzyme digested (EcoR I+Not I), and the cut code was mature The gene fragment of the high catalytic efficiency β-glucosidase mutant was connected with the expression vector pPIC9r to obtain the recombinant plasmid pPIC9r-A-E168Q containing the high catalytic efficiency β-glucosidase mutant gene A-E168Q and transformed into Pichia pastoris GS115, The recombinant yeast strain GS115 / A-E168Q was obtained.
[0043] Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, place it at 30°C, and culture it on a shaker at 220rpm for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernatant, and use 100mL of 0...
Embodiment 3
[0045] Example 3 Activity Analysis of Recombinant High Catalytic Efficiency β-Glucosidase Mutant and Wild Type
[0046] Determination of β-glucosidase activity: the amount of the product p-nitrophenol (pNP) generated by enzymatic hydrolysis of the substrate pNPG was measured at 405 nm.
[0047] Reaction steps: Mix 125μl 2mM pNPG substrate with 125μl buffer, add 250μl appropriately diluted enzyme solution, react at 75°C for 10min, add 1.5mL 1M Na 2 CO 3 Terminate the reaction and measure the OD using a spectrophotometer 405 value.
[0048] Definition of enzyme activity unit: 1 β-glucosidase activity unit (U) is defined as the amount of enzyme required to decompose the substrate pNPG to generate 1 μmol p-nitrophenol (pNP) per minute under given reaction conditions.
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Abstract
The invention relates to the fields of gene engineering and genetic engineering, and in particular relates to beta-glucosaccharase improved mutant E168Q as well as a coding gene and applications of the beta-glucosaccharase improved mutant E168Q. According to the beta-glucosaccharase improved mutant E168Q, high-temperature acid beta-glucosaccharase BGL3A derived from Talaromyces leycettanus JCM12802 is taken as a female parent, site-specific mutagenesis is carried out on the sequence of the beta-glucosaccharase by adopting a molecular biological technology, and expression is carried out. Under the modification condition, the affinity of the mutant for cellobiose is improved by 2.0 times compared with that of the wild type (before mutation), the catalytic efficiency is improved by 1.5 times, and the optimum reaction pH and temperature are invariable. With the adoption of the strategy, the catalytic efficiency of beta-glucosaccharase can be greatly improved, and an application foundation is provided for the beta-glucosaccharase in the industrial production fields including food, bioethanol and the like. The strategy has great guiding significances for the improvement of the catalytic efficiency of beta-glucosaccharase and other enzymes.
Description
technical field [0001] The present invention relates to the field of genetic engineering and genetic engineering, in particular, the present invention relates to an improved mutant E168Q of β-glucosidase, its coding gene and application. Background technique [0002] Cellulose is the most abundant renewable resource in nature. Its degradation not only constitutes an important part of the carbon cycle in nature, but also provides a potential application space for the production of biofuels. However, due to the special structure of lignocellulose, the resulting anti-biodegradation barrier makes the conversion process cost high, which constitutes the biggest bottleneck in the production of bioenergy from cellulose materials. There are three enzymes that synergistically degrade cellulose in nature: endo-β-1, 4-glucanase (EC 3.2.1.4), exoglucanase (exoglucanase, EC 3.2.1.91) and β-glucosidase (β-glucosidase, EC 3.2.1.21). Wherein the endoglucanase mainly acts on the non-crystal...
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