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Beta-glucosaccharase improved mutant E168Q as well as coding gene and applications thereof

A technology of glucosidase and mutants, which is applied in the field of genetic engineering and genetic engineering, and can solve problems such as high cost

Active Publication Date: 2016-04-27
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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.

Method used

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  • Beta-glucosaccharase improved mutant E168Q as well as coding gene and applications thereof
  • Beta-glucosaccharase improved mutant E168Q as well as coding gene and applications thereof
  • Beta-glucosaccharase improved mutant E168Q as well as coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cloning of embodiment 1 high catalytic efficiency β-glucosidase mutant coding gene A-E168Q

[0035] The present invention uses the acid β-glucosidase (its amino acid sequence as SEQ ID NO.3) derived from the thermophilic fungus Talaromycesleycettanus JCM12802 as the parent, and uses molecular biology techniques to replace the sequence of the acid β-glucosidase and then express it .

[0036] SEQ ID NO.3 is as follows:

[0037] YGFGGSGWDAAYGRAKAALNKLNQTEKVGIVTGVKWMGGPCVGNTYKPSSIDYPSLCLQDSPLGVRFANPVTAFPAGINAGATWDRSLINARGAAMGAEAKGLGVNVQLGPVAGPLGKNPNSGRIWEGFSNDPYLSGVAMEETIAGMQGSGVQACAKHYIGNEQEHNRETISSNIDDRTLHELYVWPFMNAVKANVASVMCSYNEVNGSWSCENDALLNGLLKTELGFPGYIMSDWNAQHTTVNSANSGLDMTMPGSDFNNPPGSIYWGPNLEAAVANGSVPQSRLDDMVTRILASWYLVGQDEGYPPVAFSSWNGGKANVDVTGDHKSVVRAVARDSIVLLKNDNNALPLRKPKSLAIIGQDATVNPAGPNACSDRGCDTGTLAMGWGSGTAQFPYIVGPLDAIQSQAAADGTNITTSTTDDTTAAASAAASAGTAIVFINSDSGEGYITVEGNAGDRNNLDPWHNGNELVQAVAAVNKNVIVVVHSVGPVILEAILAQPNVKAIVWPGLPGQESGNALVDVLYGSTSPSGKLPYTIAKQFSDYGTTWTTSLV...

Embodiment 2

[0041] Example 2 Preparation of β-glucosidase mutants with high catalytic efficiency.

[0042] The expression vector pPIC9r was double digested (EcoRI+NotI), and the gene A-E168Q encoding the high catalytic efficiency β-glucosidase mutant was double digested (EcoRI+NotI). The gene fragment of the -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 to obtain the recombinant yeast strain GS115 / A-E168Q.

[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 0.5% methanol for precipitation. The BMMY medium was resuspended, and placed again at 30°C, 220rpm to induce ...

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 125 μl 2mM pNPG substrate with 125 μl buffer, add 250 μl appropriately diluted enzyme solution, react at 75°C for 10 min, add 1.5 mL 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 (EC3.2.1.4), exoglucanase (EC3.2.1.91 ) and β-glucosidase (β-glucosidase, EC3.2.1.21). Wherein the endoglucanase mainly acts on the non-crystalline region of ce...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2445C12Y302/01021
Inventor 姚斌柏映国夏伟石鹏君罗会颖黄火清王亚茹苏小运王苑
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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