Beta-glucosidase, coding gene, vector, engineering bacteria and application thereof

A technology of glucosidase and genetically engineered bacteria, applied in the direction of genetic engineering, application, plant gene improvement, etc., can solve the problem of reducing activity and so on

Active Publication Date: 2012-01-18
南通九思医疗器械有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Studies have found that many substances with high biological activity in nature have reduced their activity because most of them exist in the form of glycosides

Method used

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  • Beta-glucosidase, coding gene, vector, engineering bacteria and application thereof
  • Beta-glucosidase, coding gene, vector, engineering bacteria and application thereof
  • Beta-glucosidase, coding gene, vector, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0185] Example 1: Aescin screening method for β-glucosidase Y1 gene

[0186] Prepare LB solid medium. The final concentration of LB solid medium is composed of: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L, adjust pH to 7.0 with 5mol / L NaOH solution, solvent is water, At 15psi (1.05kg / cm 2 ) steam sterilization under high pressure for 20min. Cool the medium, add ferric ammonium citrate (sterilized by filter) and 0.1% aescin (sterilized by filter) with a final concentration of 2.5% before the medium is not solidified, and inoculate the microorganisms to be screened after the medium is cooled In this culture medium, cultivate at 37°C for 24 hours, and those with black spots around the microbial colonies are positive ( figure 1 ).

Embodiment 2

[0187] Example 2: Screening and Cloning of Y1 Gene Full-length cDNA

[0188] Construction of a soil genome library. According to the method of Example 1, positive clones were screened and a sub-library was constructed, and the nucleotide sequence shown in SEQ ID NO.1 was obtained by gene sequence determination (measured by Shanghai Sangon Bioengineering Technology Service Co., Ltd.). According to the nucleotide sequence shown in SEQ ID NO.1, design primers that amplify the complete coding reading frame, and introduce restriction endonuclease sites on the upstream and downstream primers respectively (this can be determined by the vector selected) , obtain the β-glucosidase gene of the nucleotide sequence shown in SEQ ID NO.1 by in vitro amplification technology, this gene is cloned into the pET 28a intermediate vector (Hangzhou Normal University Biomedicine and Health Center preservation), in guarantee The expression vector identified under the premise of correct reading frame...

Embodiment 3

[0189] Example 3: Expression of Y1 protein

[0190] The E. Coil BL21 / pET 28a / Y1 obtained in Example 2 was cultured overnight on a shaker in LB liquid medium containing 100 mL of 50 μg / mL kanamycin. Pour 10ml of the overnight cultured bacterial solution into 1L 50μg / mL kanamycin LB liquid medium and cultivate until the bacterial solution OD 600 When it reaches 0.6-0.8, add IPTG to a final concentration of 1.0mmol / L, and after induction at 28°C for 5 hours, collect the bacteria by centrifugation, resuspend the bacteria with 25ml of phosphate buffer pH7.4, add lysozyme, and the final concentration of lysozyme is 1mg / ml, and repeated freezing and thawing to disrupt the cells. The supernatant was collected by centrifugation, and the supernatant containing the Y1 protein was filtered through a 0.22 μm cellulose acetate filter membrane and then purified by nickel column affinity chromatography to obtain Y1 pure enzyme.

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Abstract

The present invention discloses beta-glucosidase, coding gene, a vector, engineering bacteria and an application of the beta-glucosidase in preparing resveratrol through hydrolysis of polydatin. The beta-glucosidase gene shares 70-100% sequence identity with the polynucleotide sequence represented by the SEQ ID NO:2. The beta-glucosidase coded by the beta-glucosidase gene shares 95-100% sequence identity with the amino acid sequence represented by the SEQ ID NO.1. With the beta-glucosidase provided by the present invention, the resveratrol can be prepared through hydrolyzing the polydatin, the preparation process is environmentally-friendly, the yield is high and the application prospect is broad.

Description

(1) Technical field [0001] The invention relates to a β-glucosidase, a coding gene, a carrier, an engineering bacterium and applications thereof. (2) Background technology [0002] β-glucosidase can catalyze the hydrolysis of various β-glucosidases, has a broad spectrum of substrates, and a variety of biological functions: [0003] (1) β-glucosidase for the hydrolysis of cellulose [0004] Cellulose is the most abundant carbon resource in nature, and the decomposition of cellulose into glucose requires the synergy of three enzymes. β-glucosidase can hydrolyze cellobiose into glucose, but the content of β-glucosidase in the cellulase component is low and the activity is low, so it becomes the bottleneck of cellulose enzymatic hydrolysis. Therefore, screening or genetic engineering to obtain highly active β-glucosidase is of great significance for the effective degradation of cellulose. [0005] (2) β-glucosidase is used in the synthesis of alkyl glycosides and aryl glycosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N1/15C12N1/19C12N1/21C12P7/22
Inventor 黄黎锋李海峰蓝袁洋
Owner 南通九思医疗器械有限公司
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