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Novel efficient beta-glucosidase CsBGL as well as encoding gene and application thereof

A technology of glucosidase and coding gene, applied in the field of β-glucosidase CsBGL and its coding gene and application, and biology, can solve the problems of low efficiency, low enzyme yield, poor specificity, etc., and achieve operational production The method is simple, the catalytic specificity is strong, and the effect of good industrialization prospect

Active Publication Date: 2020-11-06
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims at the problems such as low enzyme yield, low efficiency and poor specificity faced by rubusoside in the production, and the present invention provides a novel high-efficiency beta-glucose derived from Chryseobacterium sp. 1433 Glucosidase CsBGL and its coding gene and the application of this enzyme in the preparation of rubusoside

Method used

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  • Novel efficient beta-glucosidase CsBGL as well as encoding gene and application thereof
  • Novel efficient beta-glucosidase CsBGL as well as encoding gene and application thereof
  • Novel efficient beta-glucosidase CsBGL as well as encoding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1: Preparation of the crude enzyme liquid of β-glucosidase CsBGL

[0046] Chryseobacterium sp. 1433 was inoculated in LB antibiotic-free medium, cultured at 30°C and 200rpm for 24h, and then centrifuged at 3000-10000rpm for 2-10min to collect bacterial precipitates. With 50mM Na at pH 6.0-8.0 2 HPO 4 / NaH 2 PO 4 Resuspend the cells in the buffer, then centrifuge at 3000-10000rpm for 2-10min to collect the bacterial pellet again, and then use 10-20mL of 50mM Na at pH 6.0-8.0 2 HPO 4 / NaH 2 PO 4 The cells were resuspended in buffer, disrupted by ultrasonic waves, centrifuged at 12,000 rpm for 20 min, and the supernatant was collected as crude enzyme solution of β-glucosidase CsBGL.

Embodiment 2

[0047] Embodiment 2: Native-PAGE electrophoresis under acidic conditions of β-glucosidase CsBGL crude enzyme solution

[0048] The β-glucosidase CsBGL crude enzyme solution obtained in Example 1 was subjected to Native-PAGE electrophoresis. The specific parameters of the electrophoresis were: the positive and negative electrodes were inverted, traced with methyl green (0.002%), and a constant current of 10mA was used at 4°C. Electrophoresis 2 ~ 3h.

[0049] The formula of Native-PAGE gel under acidic conditions is as follows:

[0050] 1) Separating gel: 0.06M KOH, 0.376M HAc, pH 4.3 (7.7% gel concentration, acrylamide:methylene acrylamide is 37.5:1);

[0051] 2) Stacking gel: 0.06M KOH, 0.063M HAc, pH 6.8 (3.125% gel concentration, acrylamide: methylene acrylamide is 3:1);

[0052] 3) Electrophoresis buffer: 0.14M L-alanine, 0.35M glacial acetic acid, pH 4.5.

[0053] Use a clean scalpel to cut off the corresponding swimming lane in the PAGE gel, and use pH7.4, 50mM Na 2 H...

Embodiment 3

[0054] Embodiment 3: Cloning of gene encoding β-glucosidase CsBGL, construction of recombinant vector and heterologous expression

[0055] 1. Extraction of Genomic DNA from Chryseobacterium sp. 1433

[0056] Chryseobacterium sp. 1433 was inoculated in LB medium, cultured at 30° C. and 200 rpm for 24 hours, and then centrifuged at 3000 to 10000 rpm for 2 to 10 minutes to collect bacterial precipitates. Using the Tiangen Genomic DNA Extraction Kit, follow the steps in the manual to extract DNA from the collected bacterial precipitates to obtain Chryseobacterium sp. 1433 genomic DNA.

[0057] 2. Identification of the amino acid sequence of β-glucosidase CsBGL and the nucleotide sequence of the coding gene

[0058] The genomic DNA of Chryseobacterium sp. 1433 extracted in step 1 was sequenced.

[0059] The Native-PAGE electrophoresis activity staining band that embodiment 2 obtains is cut into 1mm 3 Size, in-gel digestion with trypsin, and comparison with the Chryseobacterium s...

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Abstract

The invention relates to a novel efficient beta-glucosidase CsBGL as well as an encoding gene and application thereof. The amino acid sequence of the beta-glucosidase CsBGL is shown as SEQ ID NO. 2. The nucleotide sequence of the encoding gene of the beta-glucosidase CsBGL is shown as SEQ ID NO. 1. The beta-glucosidase CsBGL is derived from Chryseobacterium sp. 1433, the Chryseobacterium sp. 1433was preserved in the China General Microbiological Culture Collection Center on July 6, 2020, the preservation number is CGMCC No. 20188, and the address is Institute of Microbiology, Chinese Academyof Sciences, No. 3, No. 1 Yard, Beichenxi Road, Chaoyang District, Beijing City. The novel efficient beta-glucosidase CsBGL provided by the invention can hydrolyze sophorose of stevioside into glucosyl to generate rubusoside, the rubusoside cannot be further hydrolyzed, so that the conversion rate of the rubusoside reaches 100%, and the yield of the rubusoside reaches 100%.

Description

technical field [0001] The invention relates to a novel high-efficiency beta-glucosidase CsBGL and its coding gene and application, belonging to the technical field of biotechnology. Background technique [0002] rubusoside is the extract of sweet tea from Rubus genus Rosaceae which grows in Guangxi Zhuang Autonomous Region, China. It is a natural and high-efficiency sweetener. Its sweetness is 300 times that of sucrose, and its taste is close to that of sucrose. It has the effects of lowering blood sugar, lowering blood fat and preventing dental caries (Kim et al., 2019). It is a good sugar substitute natural sweetener for people with diabetes, hyperglycemia and obesity. It is widely used in natural calorie-free sweetness such as food and health products. drug market. In addition, rubusoside is also a good natural co-solvent, which can help insoluble drugs dissolve to improve drug efficacy, such as anticancer drugs paclitaxel, resveratrol and curcumin (Chen et al., 2020; Z...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/56C12R1/19
CPCC12N9/2445C12N15/70C12P19/56C12Y302/01021
Inventor 肖敏阎振鑫徐莉
Owner SHANDONG UNIV
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