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Improved beta-glucosidase gene and preparation of recombinase thereof

A technology of glucosidase and gene, applied in the field of recombinant enzyme preparation

Active Publication Date: 2012-04-04
江苏好多收农业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no research reports on the directional transformation of the Aspergillus niger β-glucosidase gene and the high-efficiency expression of the recombinant enzyme

Method used

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  • Improved beta-glucosidase gene and preparation of recombinase thereof
  • Improved beta-glucosidase gene and preparation of recombinase thereof
  • Improved beta-glucosidase gene and preparation of recombinase thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Aspergillus niger beta - Cloning of glucosidase gene and construction and expression of recombinant plasmid pPICZα-bglI

[0030] 1.1 RNA extraction (Refined Molecular Biology Experiment Guide (Fourth Edition) Beijing: Science Press, 2005):

[0031] (1) Consumables treatment: The plastic consumables used are soaked in 0.1% DEPC solution at 37°C. After overnight, by 1.05 kg / cm 3 , Sterilize at 121°C for 30 min to remove residual DEPC. Dry the mortar and glassware at 200°C for more than 8 hours to inactivate RNase.

[0032] (2) Extraction method: Invitrogen Micro-to-midi Total RNA purification system is used, the specific method is as follows: wash the fresh mycelium with 1×PBS for 3 to 4 times, drain it, wrap it in tin foil, and put it in liquid nitrogen freezing.

[0033] (a) Precool the mortar with liquid nitrogen. Take out the frozen Aspergillus niger mycelium, weigh 200 mg, put it into a mortar, grind it with a pestle, and add liquid nitrogen ...

Embodiment 2

[0063] Embodiment 2: Directional transformation of Aspergillus niger β-glucosidase gene and construction of recombinant plasmid pPICZα-bglII

[0064] 2.1 Aspergillus niger beta - Directed modification of glucosidase gene

[0065] The Pichia pastoris expression system is a eukaryotic expression system that has developed rapidly in recent years. It is very suitable for the expression of eukaryotic proteins. It is manifested in its strong eukaryotic protein modification function, which can correctly process and fold proteins after translation. and glycosylation modifications. The difference in codon usage frequency among different hosts is the main factor affecting the expression efficiency of genes in heterologous hosts. Like other microorganisms, Pichia pastoris expresses foreign genes with codon preference. β-glucosidase gene from Aspergillus niger bgl There are a lot of rare codons in I that are less utilized by Pichia pastoris, so that bgl I cannot get more effic...

Embodiment 3

[0073] Example 3: Transformation of Pichia pastoris with recombinant plasmid pPICZα-bglII and preparation of recombinant enzyme

[0074] 3.1 Restriction linearization of expression vector and recovery of plasmid

[0075] Correctly identified positive transformants were cultured in large quantities, and the recombinant plasmid pPICZα-bglII was extracted by the method of extracting plasmids. use Pme I Carry out single enzyme digestion on the recombinant plasmid pPICZα-bglII. Electrophoresis detection, after the digestion is complete, purify the digested product, and use 10 μL ddH 2 O Resuspend the concentrated DNA.

[0076]

[0077] 3.2 Electroporation transformation of Pichia pastoris

[0078] After the Pichia pastoris strain GS115 was activated, it was inoculated into 500 mL YPD medium and cultured at 30°C until OD 600 Centrifuge at 1.3 to 1.5 at 4°C for 5 min at 1500 rpm, collect the cells, and wash the cells with 500 mL and 250 mL of pre-cooled sterilized water...

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Abstract

The invention relates to an improved beta-glucosidase gene and preparation of a recombinase thereof. The improved beta-glucosidase gene bgl II has an anucleotide sequence as shown in SEQ ID NO.1. Meanwhile, the invention provides a method for expressing the beta-glucosidase by the recombinant bacterium with a basic salt culture medium. Through artificial evolution and high density fermentation ofthe gene, the expression level of the improved aspergillus niger beta-glucosidase gene bgl II in Pichia pastoris is enhanced by 36 times compared with the original gene bgl I. The improved beta-glucosidase gene and preparation of a recombinase thereof in the invention can be used for molecular modification of beta-glucosidase gene and high density fermentation of its recombinant Pichia pastoris gene engineering bacterium, and can substantially improve the expression level of beta-glucosidase.

Description

[0001] technical field [0002] The invention relates to a coding β-glucosidase gene, its recombinant plasmid and its construction method, its cloning, mutation and its high-efficiency expression, and relates to the fields of molecular biology, bioinformatics, enzymology, genetic engineering and the like. It specifically relates to bioinformatics analysis and directional transformation of the β-glucosidase gene of the original Aspergillus niger, as well as the preparation of the recombinant enzyme. [0003] technical background [0004] β-glucosidase (β-glucosidase) is also known as β-D-glucoside hydrolase, also known as cellobiase, gentiobiase and amygdalinase. It is an enzyme that can catalyze the hydrolysis of glycosidic bonds between aryl or hydrocarbon groups and sugar groups to generate glucose. β-glucosidase can be divided into three categories according to its substrate specificity: the first category is the enzyme that can hydrolyze hydrocarbon-β-glucoside or aryl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/63C12N15/81C12N15/66C12N1/19C12N9/42C12R1/685
Inventor 赵林果周潭澈李迅
Owner 江苏好多收农业科技有限公司
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