Cloning, expression and application of a β-glucosidase gene

A glucosidase and gene technology, applied in the field of genetically engineered bacteria that secrete β-glucosidase, can solve the problems of unsuitable lactose decomposition, β-glucosidase cannot take into account both high temperature activity and low temperature activity, etc., and achieves safety The effect of strengthening, increasing security, and strong stability

Active Publication Date: 2011-12-07
山西恩泽生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] For this reason, the technical problem to be solved by the present invention is that the β-glucosidase in the prior art cannot take into account high temperature activity and low temperature activity at the same time, so it is not suitable for the problem of lactose decomposition in China's dairy industry. β-glucosidase in product industry and genetically engineered bacteria capable of obtaining the β-glucosidase

Method used

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  • Cloning, expression and application of a β-glucosidase gene
  • Cloning, expression and application of a β-glucosidase gene
  • Cloning, expression and application of a β-glucosidase gene

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1: Construction of recombinant plasmid

[0054] Extract the total DNA template: take pyrococcus furiosus 20 mg of bacterial cells were added to DNA extraction solution (including 20 ml Tris-HCl pH8, 25 mM EDTA, 0.1 M NaCl, 0.5% SDS, 10 μg / ml proteinase K) to digest for 12 hours. Extract twice with phenol / chloroform (24:1 by volume) and once with chloroform. Separate the supernatant and add twice the volume of ethanol (100% concentration) to precipitate the DNA. The DNA was collected by centrifugation, dissolved in TE buffer, and PCR reaction was performed after the concentration was determined.

[0055] Construct the recombinant plasmid β-glucosidase / pCR2.1: According to the β-glucosidase gene sequence (Genebank pF0073), design two PCR primers:

[0056] They are: G001 5′-atgaagttcccaaaaaacttcatgtttgg-3′;

[0057] G002 5'-ctactttcttgtaacaaatttgaggtctgcg-3';

[0058] The gene was amplified by PCR, and then directly cloned into the PCR2.1-Top10 plasmid, a...

Embodiment 2

[0059] Example 2: Site-directed mutagenesis of recombinant plasmids

[0060] Refer to the online primer design software QuikChange?? Primer Design Program of Stratagene Company, and use the recombinant plasmid β-glucosidase / pCR2.1 as a template to design two mutation primers, respectively:

[0061] 371-F: 5′-ctaccaatgataatt gca gagaacggtatggccgatgcagca-3′;

[0062] 371-R: 5′-ggccataccgttctc tgc aattatcattggtagctcgatgg-3′;

[0063] The 371st amino acid of the recombinant plasmid β-glucosidase / pCR2.1 was subjected to site-directed mutation to prepare the recombinant mutant plasmid pGAPHa / β-glucosidase. Among them, TAC at positions 1111-1113 of the target gene was mutated into GCA, so that threonine at position 371 was mutated into leucine. The gene site-directed mutagenesis reaction system is shown in Table 1, and the PCR reaction parameters are shown in Table 2. .

[0064] After PCR, after hydrolysis with DpnI enzyme at 37°C for 1 hour, the mutant plasmid was transformed...

Embodiment 3

[0070] Example 3: Preparation of target DNA and electrotransformation of Pichia pastoris

[0071] Extract the plasmid pGAPHa / β-glucosidase, use AvrII / BglII for enzyme digestion and linearization, and then purify by agarose electrophoresis to obtain linear target DNA; prepare electrocompetent cells of Pichia pastoris GS115; mix 50ul of electrocompetent cells with 5ul (3 micrograms) of linearized DNA was mixed, transferred to a 0.4cm spacing electroporation cuvette, and electroporated at a voltage of 800V, and transformed into Pichia pastoris host strain GS115, which was constructed to express β-glucosidase yeast engineering bacteria.

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a beta-glucosaccharase which can decompose lactose and has high activity under both high-temperature and low-temperature conditions, and a gene engineering bacterium capable of efficiently expressing secretion-type beta-glucosaccharase. The invention discloses a beta-glucosaccharase having an amino acid sequence disclosed as SEQ ID NO:2, and constructs a gene engineering bacterium capable of efficiently secreting the beta-glucosaccharase. The gene engineering bacterium is collected in Common Microorganism Center of Committee for Culture Collection of Microorganisms, and the collection number is CGMCC No.4891. The beta-glucosaccharase produced by the method disclosed by the invention has high activity of galactosidase, and can be applied to dairy industry; and meanwhile, the beta-glucosaccharase has stable activity within wide pH value range and temperature range.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a genetically engineered β-glucosidase capable of decomposing lactose with strong activity under high temperature and low temperature conditions and capable of highly expressing secreted β-glucosidase. Background technique [0002] The system name of β-glucosidase (β-Glucosidase, EC3.2.1.21) is β-D-glucoside hydrolase, which belongs to hydrolase. It can catalyze the hydrolysis of the terminal non-reducing β-D-glycosidic bond, releasing the ligand and glucosome at the same time. In 1837, Liebig and Wohler first discovered β-glucosidase in bitter almonds. Over the years, many scholars have isolated and purified β-glucosidase from bitter almonds, grapes, sword beans, corn, black cherries, rice, and soybeans. . There is also β-glucosidase in the epithelial cells of the intestinal mucosa of mammals. If the enzyme is deficient, the absorption of corresponding carbohydrates wil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12N15/81C12P19/14C12R1/84
Inventor 李侍武
Owner 山西恩泽生物技术有限公司
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