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A high catalytic efficiency beta-glucosidase mutant and its coding gene and application

A glucosidase and catalytic efficiency technology, applied in the field of high catalytic efficiency β-glucosidase mutants and their preparation, can solve the problems of not being completely consistent, different types and degrees, etc.

Active Publication Date: 2020-01-21
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, there are great differences in the post-translational modification of proteins in different hosts, even if they are all eukaryotes, they will not be completely consistent, mainly manifested in differences in types and degrees

Method used

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  • A high catalytic efficiency beta-glucosidase mutant and its coding gene and application
  • A high catalytic efficiency beta-glucosidase mutant and its coding gene and application
  • A high catalytic efficiency beta-glucosidase mutant and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Cloning of High Catalytic Efficiency β-Glucosidase Mutant Encoding Gene A-M36E

[0040] The present invention takes the acid β-glucosidase (its amino acid sequence as SEQ ID NO.5) 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] SEQ ID NO.5 is shown below:

[0042] YGFGGSGWDAAYGRAKAALNKLNQTEKVGIVTGVKWMGGPCVGNTYKPSSIDYPSLCLQDSPLGVRFANPVTAFPAGINAGATWDRSLINARGAAMGAEAKGLGVNVQLGPVAGPLGKNPNSGRIWEGFSNDPYLSGVAMEETIAGMQGSGVQACAKHYIGNEQEHNRETISSNIDDRTLHELYVWPFMNAVKANVASVMCSYNEVNGSWSCENDALLNGLLKTELGFPGYIMSDWNAQHTTVNSANSGLDMTMPGSDFNNPPGSIYWGPNLEAAVANGSVPQSRLDDMVTRILASWYLVGQDEGYPPVAFSSWNGGKANVDVTGDHKSVVRAVARDSIVLLKNDNNALPLRKPKSLAIIGQDATVNPAGPNACSDRGCDTGTLAMGWGSGTAQFPYIVGPLDAIQSQAAADGTNITTSTTDDTTAAASAAASAGTAIVFINSDSGEGYITVEGNAGDRNNLDPWHNGNELVQAVAAVNKNVIVVVHSVGPVILEAILAQPNVKAIVWPGLPGQESGNALVDVLYGSTSPSGKLPYTIAKQFS...

Embodiment 2

[0046] Example 2 Preparation of high catalytic efficiency β-glucosidase mutants with improved pH stability.

[0047] The expression vector pPIC9r was subjected to double enzyme digestion (EcoR I+Not I), and at the same time, the gene A-M36E 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 (removing the signal peptide fragment) of the high catalytic efficiency β-glucosidase mutant was connected with the expression vector pPIC9r to obtain the recombinant plasmid pPIC9r- Bgl3A-M1 and pPIC9r-Bgl3A-M2 were transformed into Pichia pastoris GS115 to obtain recombinant yeast strains GS115 / Bgl3A-M1 and GS115 / Bgl3A-M2.

[0048] 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 supernatan...

Embodiment 3

[0049] The activity analysis of the high catalytic efficiency β-glucosidase mutant that embodiment 3 pH stability improves

[0050] 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.

[0051] Reaction steps: mix 125μl 2mM pNPG substrate with 125μl buffer, add 250μl appropriately diluted enzyme solution, react at 70°C for 10min, add 1.5mL 1M Na 2 CO 3 Terminate the reaction and measure the OD using a spectrophotometer 405 value.

[0052] 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 field of genetic engineering, in particular to beta-glucosaccharase mutants high in catalysis efficiency and a preparation method and application thereof. A beta-glucosaccharase sequence is subjected to multipoint mutation through a molecular biological technology, so that part of O-galactosylated modification sites are removed; the two mutants M1 and M2 with pH stability improved are constructed; the amino acid sequences of the two mutants are as indicated in SEQ ID NO.1 and SEQ ID NO.2. The pH stability of enzyme is stably widened to the range that pH ranges from 3.0 to 10.0 from the range of proenzyme that pH range from 3.0 to 5.0, and good stability is always maintained. By means of the mutants, pH stability of beta-glucosaccharase Bgl3A can be greatly improved, and the foundation is laid for applying the mutants to the industrial production industries such as food and bioethanol.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a beta-glucosidase mutant with high catalytic efficiency and its preparation method and application. Background technique [0002] β-glucosidase (EC3.2.1.21), is a β-glucosidase that can specifically catalyze the non-reducing ends of oligosaccharides (usually 2-6 monosaccharide residues), alkyl glucosides and aryl glucosides The hydrolysis of D-glucoside bonds, thereby releasing glucose monosaccharide molecules and corresponding ligands, is a general term for glycoside hydrolase (GH), also known as β-D-glucoside glucohydrolase. Because it was first isolated from bitter almonds and has significant activity on gentiobiose and cellobiose, it is also known as gentiobiase, cellobiase and amygdalase. β-glucosidase is an important part of the cellulose degrading enzyme system, which can efficiently act on the degradation products of upstream cellulase to further degrade cellulose mol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12P7/06C12R1/84
CPCC12N9/2445C12P7/06C12Y302/01021Y02E50/10
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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