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Beta-glucosaccharase mutant M36E with high catalytic efficiency as well as coding gene and applications thereof

A glucosidase and catalytic efficiency technology, which is applied to the high catalytic efficiency β-glucosidase mutant M36E and its encoding gene and application fields, can solve the problem of large workload of artificial mutagenesis, low frequency of beneficial mutations, and difficult to obtain purposes. strains, etc.

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

Problems solved by technology

Mutagenesis is divided into natural mutation and artificial mutagenesis. The probability of success of natural mutation is very small, and the workload of artificial mutagenesis is relatively large and the frequency of beneficial mutation is still low. The direction and nature of mutation are difficult to control
The blindness of the screening is large, and it is not easy to obtain the target strain

Method used

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  • Beta-glucosaccharase mutant M36E with high catalytic efficiency as well as coding gene and applications thereof
  • Beta-glucosaccharase mutant M36E with high catalytic efficiency as well as coding gene and applications thereof
  • Beta-glucosaccharase mutant M36E with high catalytic efficiency 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-M36E

[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] YGFGGSGWDAAYGRAKAALNKLNQTEKVGIVTGVKWMGGPCVGNTYKPSSIDYPSLCLQDSPLGVRFANPVTAFPAGINAGATWDRSLINARGAAMGAEAKGLGVNVQLGPVAGPLGKNPNSGRIWEGFSNDPYLSGVAMEETIAGMQGSGVQACAKHYIGNEQEHNRETISSNIDDRTLHELYVWPFMNAVKANVASVMCSYNEVNGSWSCENDALLNGLLKTELGFPGYIMSDWNAQHTTVNSANSGLDMTMPGSDFNNPPGSIYWGPNLEAAVANGSVPQSRLDDMVTRILASWYLVGQDEGYPPVAFSSWNGGKANVDVTGDHKSVVRAVARDSIVLLKNDNNALPLRKPKSLAIIGQDATVNPAGPNACSDRGCDTGTLAMGWGSGTAQFPYIVGPLDAIQSQAAADGTNITTSTTDDTTAAASAAASAGTAIVFINSDSGEGYITVEGNAGDRNNLDPWHNGNELVQAVAAVNKNVIVVVHSVGPVILEAILAQPNVKAIVWPGLPGQESGNALVDVLYGSTSPSGKLPYTIAKQFSDYGTTWTTSLVD...

Embodiment 2

[0042] Example 2 Preparation of β-glucosidase mutants with high catalytic efficiency.

[0043] The expression vector pPIC9r was double digested (EcoRI+NotI), and the gene A-M36E 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 a recombinant plasmid pPIC9r-A-M36E containing the high catalytic efficiency β-glucosidase mutant gene A-M36E and transformed into Pichia pastoris GS115 to obtain a recombinant yeast strain GS115 / A-M36E.

[0044] 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 culture....

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 minutes, add 1.5mL 1M Na2CO 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 mutant M36E with high catalytic efficiency as well as a coding gene and applications of the beta-glucosaccharase mutant M36E. For the mutant M36E of beta-glucosaccharase with heat resistance, 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 the amino acid sequence is shown in SEQ ID NO.1. Under the modification condition, the affinity of the mutant for cellobiose is improved by 2.1 times compared with that of the wild type (before mutation), the catalytic efficiency is improved by 2.3 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 efficiencies of beta-glucosaccharase and other enzymes.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology, in particular, the invention relates to a high catalytic efficiency beta-glucosidase mutant M36E and its coding gene and application. Background technique [0002] β-glucosidase (EC3.2.1.21), also known as β-D-glucoside glucohydrolase, can hydrolyze the non-reducing β-D-glucosidic bond bound to the terminal and release β-D- Glucose and the corresponding ligands. In 1837, Liebig and Wohler first discovered bitter almonds, and later research found that β-glucosidase exists in many plants, insects, yeast, Aspergillus, Trichoderma and bacteria in nature. β-glucosidase hydrolyzes cellobiose to generate two molecules of glucose, which is the key enzyme for the synergy of the cellulase complex enzyme system, and has a strong promotion effect on the overall hydrolysis activity of the cellulase system. The activity of β-glucosidase in the system can improve the function of the whole...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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