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Application of heat-resistant beta-glucosidase in preparation of gentiooligosaccharide

A technology of glucosidase and gentiosaccharide oligosaccharide, applied in the application, glycosylase, enzyme and other directions, can solve the problems of little research on β-glucosidase and the application prospect is yet to be developed, etc., and achieves high thermal stability, Efficient expression effect

Active Publication Date: 2020-07-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, most of the current studies on the preparation of gentiooligosaccharides mainly use the β-glucosidase of the GH3 family, and there is little research on the β-glucosidase of the GH1 family. The application prospects in the preparation have yet to be developed

Method used

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  • Application of heat-resistant beta-glucosidase in preparation of gentiooligosaccharide
  • Application of heat-resistant beta-glucosidase in preparation of gentiooligosaccharide
  • Application of heat-resistant beta-glucosidase in preparation of gentiooligosaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: Contain the construction of tsbgl gene expression vector

[0055] According to the amino acid sequence of Thermotoga spKOL6 β-glucosidase Tsbgl (WP_101510358) in the Genbank database, the gene whose nucleotide sequence is shown in SEQ ID NO.1 was chemically synthesized.

[0056] The synthetic gene fragment was digested with pET-24a (restriction sites: Nde I and EcoR I) to obtain a ligation product; the ligation product was transformed into E. coli E. coli. JM109 by heat shock transformation method. The transformation product was obtained; the transformation product was coated on LB solid medium (containing 0.05 mg / mL kanamycin), and cultured upside down in a constant temperature incubator at 37°C for 8-12 hours to obtain a transformant.

[0057] Heat shock conversion method:

[0058] (1) Put E.coli.JM109 competent cells on ice for 5 minutes in advance. After the competent cells are completely melted, add 10 μL of complete plasmid or PCR product to them, b...

Embodiment 2

[0071] Example 2: Bacillus subtilis expression host transformation culture and extraction of crude enzyme solution

[0072] The recombinant plasmid pBSMμL3-tsbgl, which was verified by enzyme digestion and sequenced correctly, was linearized and then electrotransformed into Bacillus subtilisWSH11 (Bacillus subtilisWSH11 is described in the patent publication number CN108102997A). The recombinant plasmid pBSMμL3-tsbgl was electroporated to transform Bacillus subtilisWSH11 competent cells:

[0073] (1) Place the Bacillus subtilisWSH11 competent cells on ice for 5 minutes in advance, and after the competence is completely melted, add 10 μL of recombinant plasmid to them, blow gently and evenly, and place on ice for 15 minutes;

[0074] (2) Turn on the electroporator to preheat for 30 minutes in advance, and set the electric shock voltage to 2400V. Slowly add the competent state after the ice bath to the electric shock cup with a diameter of 2 mm for extraction and pre-cooling. Af...

Embodiment 3

[0080] Embodiment 3: the determination of β-glucosidase TSBG1 application condition

[0081] (1) Optimal temperature of β-glucosidase TSBG1

[0082] Using pNPG as a substrate, add the purified β-glucosidase obtained in Example 3 into the enzyme activity assay reaction system, the pH is 6.0, react at different temperatures, measure the enzyme activity, and calculate its relative enzyme activity , see the result image 3 , the specific data are shown in Table 1, it can be seen that the optimum temperature of β-glucosidase is 90°C, and at 90-100°C, the relative enzyme activity can reach more than 85%.

[0083] The relative enzyme activity of table 1β-glucosidase TSBG1 under different temperatures

[0084]

[0085] (2) Optimum pH of β-glucosidase TSBG1

[0086] Using pNPG as a substrate, the purified β-glucosidase obtained in Example 3 was added to the enzyme activity assay reaction system, and reacted in a constant temperature water bath at 90° C. for 10 minutes at differen...

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Abstract

The invention discloses an application of heat-resistant beta-glucosidase in preparation of gentiooligosaccharide and belongs to the field of genetic engineering and enzyme engineering. A beta-glucosidase TSBGl gene from Thermotoga sp.KOL6 takes Bacillus subtilis WSH11 as an expression host, and high-efficiency expression of the tsbgl gene in bacillus subtilis is achieved. The optimum temperatureof the beta-glucosidase TSBGl is 90 DEG C, the optimum pH is 6.0, and the beta-glucosidase TSBGl has relatively high thermostability at 90 DEG C. The beta-glucosidase is added to a reaction system employing 1200 g / L of glucose as a substrate, enzyme reaction is carried out under the conditions of pH 6.0 and 90 DEG C, and the yield of the gentiooligosaccharide reaches 178.2 g / L. The beta-glucosidase meets the requirements for foods and other industrial applications and can be applied to industrial production of the gentiooligosaccharide.

Description

technical field [0001] The invention relates to the application of a heat-resistant β-glucosidase in the preparation of gentiooligosaccharides, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] β-glucosidase (EC 3.2.1.21) is a type of glycoside hydrolase (Glycoside hydrolysase, GH) that can specifically hydrolyze the β-D-glucosidic bond at the non-reducing end to release glucose and the corresponding ligand. β-glucosidases widely exist in organisms and play an important role in them. According to the differences in their amino acid sequence characteristics, they are mainly distributed in six families: GH1, GH3, GH5, GH9, GH30 and GH116, and most of them are β-glucosidases. Glycosidase belongs to GH1, GH3 family. The β-glucosidases of the GH1 and GH3 families differ in protein folding, physicochemical properties, catalytic properties, and substrate specificity. β-glucosidase can be used in various industries, such as degrad...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/75C12N1/21C12P19/18C12P19/14C12P19/12C12P19/00C12R1/125
CPCC12N9/2445C12N15/75C12P19/18C12P19/14C12P19/12C12P19/00C12Y302/01021
Inventor 吴敬夏伟盛玲玲黄燕
Owner JIANGNAN UNIV
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