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Humicola-sourced high-temperature acid beta-glucosidase HiBgl3C as well as gene and application thereof

A glucosidase, high temperature technology, applied in the field of genetic engineering, can solve the problems such as the inability to meet the application requirements of the medium-alkaline hydrolysis process

Active Publication Date: 2015-11-04
黑龙江卫诺恩生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most of the β-glucosidase pI values ​​from existing microorganisms are in the acidic range, and the optimum pH value is generally between 3.5 and 5.5, which can be applied to many acidic hydrolysis processes, but cannot meet the requirements of some parts such as detergents, papermaking and Application requirements of medium-alkaline hydrolysis process in textile industry, etc.

Method used

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  • Humicola-sourced high-temperature acid beta-glucosidase HiBgl3C as well as gene and application thereof
  • Humicola-sourced high-temperature acid beta-glucosidase HiBgl3C as well as gene and application thereof
  • Humicola-sourced high-temperature acid beta-glucosidase HiBgl3C as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Cloning of Humicola insolens Y1 CGMCC 4573 β-glucosidase coding gene bgl3C

[0047] According to the H.insolens genome sequencing sequence, design gene-specific primers:

[0048] PF:GGG ACTAGT ATGGGTATCTTCGGTCTCGC

[0049] PR: GGG GCGGCCGC TCAAACATCAATGCTGCCCGTC

[0050]The RNA of Humicola insolens Y1 CGMCC 4573 was extracted and cDNA was obtained by reverse transcription. PCR amplification was performed using Humicola insolens Y1 CGMCC 4573cDNA as a template. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 2 min and 30 s, and after 35 cycles, incubation at 72°C for 10 min. A fragment of about 2200bp was obtained, which was recovered and connected with the pEASY-T3 vector and sent to Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0051] The sequencing result is β-glucosidase gene Hibgl3C gene 2151bp (signal peptide removed), encoding 717 ...

Embodiment 2

[0052] The preparation of embodiment 2 recombinant xylanase

[0053] The expression vector pPIC9 was subjected to double digestion (Spe I+Not I), and the gene bgl3C encoding β-glucosidase HiBgl3C was double-digested (Spe I+Not I) at the same time to cut out the gene encoding mature β-glucosidase The fragment (not including the signal peptide sequence) was connected with the expression vector pPIC9 to obtain the recombinant plasmid pPIC-Hibgl3C containing the Humicola β-glucosidase gene Hibgl3C and transform it into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / Hibgl3C.

[0054] A recombinant Pichia strain containing the complete gene was constructed in the same way.

[0055] The GS115 strain containing the recombinant plasmid was inoculated in 400mL of BMGY culture medium, shaken at 250rpm at 30°C for 48h, and then collected by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 48 hours of induction, the...

Embodiment 3

[0056] Example 3 Activity analysis of recombinant β-glucosidase

[0057] 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.

[0058] Reaction steps: mix 125μl 2mM pNPG substrate with 125μl buffer, add 250μl appropriately diluted enzyme solution, react at 60°C for 10min, add 1.5mL 1M Na 2 CO 3 Terminate the reaction and measure the OD using a spectrophotometer 405 value.

[0059] 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, and specifically relates to a novel specific humicola-sourced high-temperature acid beta-glucosidase HiBgl3C as well as a gene and an application thereof. The invention provides a novel high-temperature acid beta-glucosidase HiBgl3C, which has an amino acid sequence shown as SEQ ID No.1 or 2. The invention also provides a gene for coding the high-temperature neutral beta-glucosidase HiBgl3A, a nucleotide sequence of which is shown as SEQ ID NO.4 or 5, as well as a recombinant vector comprising the gene and a recombinant strain and an application thereof. An optimum temperature of the high-temperature acid beta-glucosidase HiBgl3C sourced from the specific humicola is 60 DEG C, and an optimum pH value is 5.5; moreover, the pH stability is good, and the high-temperature acid beta-glucosidase HiBgl3C can resist the alkaline environment; and the high-temperature acid beta-glucosidase HiBgl3C has good resistance to a majority of chemical reagents, metal ions and glucoses and is remarkable in application prospect in various industries such as washing agent, biological ethanol and the like in a complicated environment.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high-temperature acidic β-glucosidase HiBgl3C derived from Humicola and its gene and application. Background technique [0002] Cellulose is a polymer composed of multiple glucose residues connected by β-1,4-glucosidic bonds, and its basic repeating unit is cellobiose. The utilization and transformation of cellulose is of great significance to solve the world's energy crisis, food shortage, environmental pollution and other problems. Cellulose can be degraded into glucose through the action of cellulase, which can be used as an important industrial raw material to produce chemical products such as alcohol and acetone. Cellulase is a general term for three types of enzymes that can degrade cellulose into glucose, namely endo-β-1,4-glucanase (endo-β-1,4-glucanase, EC 3.2.1.4), exo Glucanase (exoglucanase, also known as cellobiohydrolase, EC 3.2.1.91) an...

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 张王照付华廷付海廷孙光王淑媛杨静
Owner 黑龙江卫诺恩生物技术有限公司
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