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A novel high-efficiency β-glucosidase csbgl and its coding gene and application

A technology of glucosidase and stevioside, which is applied in biology, beta-glucosidase CsBGL and its encoding gene and application field, can solve the problems of low efficiency, low enzyme yield, poor specificity, etc., and achieves operational production. The method is simple, the catalytic specificity is strong, and the effect of good industrialization prospects

Active Publication Date: 2022-06-07
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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  • A novel high-efficiency β-glucosidase csbgl and its coding gene and application
  • A novel high-efficiency β-glucosidase csbgl and its coding gene and application
  • A novel high-efficiency β-glucosidase csbgl and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Preparation of crude enzyme solution of β-glucosidase CsBGL

[0046] Chryseobacterium sp. 1433 was inoculated into LB antibiotic-free medium, cultured at 30° C. and 200 rpm for 24 h, and then centrifuged at 3,000-10,000 rpm for 2-10 min to collect bacterial pellets. With 50mM Na at pH 6.0-8.0 2 HPO 4 / NaH 2 PO 4 The cells were resuspended in the buffer, and then centrifuged at 3000-10000rpm for 2-10min to collect the cell pellet again, and then use 10-20mL pH 6.0-8.0 50mM Na 2 HPO 4 / NaH 2 PO 4 The cells were resuspended in the buffer, disrupted by ultrasonic waves, centrifuged at 12,000 rpm for 20 min, and the supernatant was collected as the crude β-glucosidase CsBGL enzyme solution.

Embodiment 2

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

[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 10 mA at 4°C was used. Electrophoresis 2 ~ 3h.

[0049] Native-PAGE gels for acidic conditions are formulated as follows:

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

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

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

[0053] Cut out the corresponding lanes in the PAGE gel with a clean scalpel, use pH 7.4, 50 mM Na 2 HPO 4 / NaH 2 PO 4 The buffer was wa...

Embodiment 3

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

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

[0056] Chryseobacterium sp. 1433 was inoculated into LB medium, cultured at 30°C and 200rpm for 24h, and then centrifuged at 3000-10000rpm for 2-10min to collect the bacterial pellet. Using the Tiangen genomic DNA extraction kit, DNA was extracted from the collected bacterial cell pellets according to the instructions to obtain the genomic DNA of Chryseobacterium sp. 1433.

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

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

[0059] The Native-PAGE electrophoresis activity staining band obtained in Example 2 was cut into 1mm 3 The nucleotide sequence of β-glucosidase CsBGL was determined, as shown in SEQ ID NO. .1, a total of 2271bp; the ...

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Abstract

The invention relates to a novel high-efficiency β-glucosidase CsBGL and its coding gene and application. A β-glucosidase CsBGL, the amino acid sequence of which is shown in SEQ ID NO.2. The nucleotide sequence of the coding gene of the β-glucosidase CsBGL is shown in SEQ ID NO.1. The β-glucosidase CsBGL is derived from Chryseobacterium sp. 1433, and the Chryseobacterium sp. 1433 was preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms on July 6, 2020. Collection number CGMCC No.20188, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The invention provides a novel and efficient β-glucosidase CsBGL, which can hydrolyze the sophoryl group of stevioside into glucose group, generate rubusoside, and will not further hydrolyze rubusoside, so that the conversion rate of stevioside reaches 100%, sweet tea The yield of glycosides reaches 100%.

Description

technical field [0001] The invention relates to a novel and efficient beta-glucosidase CsBGL and its encoding gene and application, and belongs to the technical field of biotechnology. Background technique [0002] Rubusoside is an extract of sweet tea from the Rosaceae Rubus plant grown in Guangxi Zhuang Autonomous Region of China. It is a natural high-efficiency sweetener, 300 times sweeter than sucrose, and tastes close to sucrose. It is safe, non-toxic, and has good properties. It has the functions of lowering blood sugar, blood lipid and anti-caries (Kim et al., 2019). It is a good natural sugar substitute for people with diabetes, hyperglycemia and obesity. It is widely used in food, health products and other natural calorie-free sweeteners agent market. In addition, rubusoside is also a good natural co-solvent, which can help the insoluble drugs dissolve to improve their efficacy, such as anticancer drugs paclitaxel, resveratrol and curcumin, etc. (Chen et al., 2020;...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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