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Method for preparing gentiosaccharide oligosaccharides by using β-1,6-glucanase and its application

A technology of gentiooligosaccharides and glucanase, which is applied in the field of preparation of gentiooligosaccharides, and can solve the problem of undetected gentiotriose oligosaccharide components and low yield of gentiooligosaccharides , high cost of enzymes, etc.

Active Publication Date: 2022-07-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The research related to the enzymatic production of gentiooligosaccharides mainly focuses on the use of β-glucosidase to form products through reverse hydrolysis and condensation with glucose as the substrate. Mainly there are three problems, one is that the yield of gentiooligosaccharides is low; It is expensive to use enzymes

Method used

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  • Method for preparing gentiosaccharide oligosaccharides by using β-1,6-glucanase and its application
  • Method for preparing gentiosaccharide oligosaccharides by using β-1,6-glucanase and its application
  • Method for preparing gentiosaccharide oligosaccharides by using β-1,6-glucanase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of β-1,6 glucanase and β-glucosidase Pichia genetically engineered bacteria

[0052] (1) Construction of β-1,6 glucanase Pichia genetically engineered bacteria

[0053] The gene encoding β-1,6-glucanase TcBgn (nucleotide sequence shown in SEQ ID NO. 3) was chemically synthesized and linked with the Pichia pastoris expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-TcBgn and transferred into Escherichia coli JM109 In the middle, after the digestion verification, the recombinant plasmid pPIC9K-TcBgn was electro-transformed into Pichia pastoris KM71, and then the transformation solution was spread on the MD plate, and a single clone was grown on the MD plate, and then 96 transformants were picked and transferred to The transformants with higher enzyme activity were screened on a new MD plate and a 10 mL tube was used to obtain the β-1,6 glucanase Pichia pastoris genetically engineered bacteria.

[0054] Primers used for PCR amplifica...

Embodiment 2

[0059] Example 2: Production of β-glucosidase and β-1,6-glucanase by 3.6L tank fermentation

[0060] (1) The β-1,6 glucanase Pichia genetically engineered bacteria and the β-glucosidase Pichia genetically engineered bacteria prepared in Example 1 were inoculated into YPD medium, respectively, at 30° C., 200 rpm Cultivated for 24h, cultivated to the OD of the seed liquid 600 is 1.3-1.5.

[0061] (2) Batch fermentation stage: the seed liquid is inoculated into the fermenter with 8%-12% inoculation amount, and the temperature is controlled at 28-30°C, the initial rotation speed is 180-220rpm, the initial ventilation rate is 7L / min, and the dissolved oxygen is 28-32 %, pH 4.5-5.5;

[0062] (3) Feed-feed fermentation stage: when the dissolved oxygen rises to 80-100%, feed-feed culture is carried out by adding glycerol at a constant speed, and the temperature is controlled at 28-30°C, dissolved oxygen at 28-32%, and pH at 4.5-5.5;

[0063] (4) Induction culture stage: when the ba...

Embodiment 3

[0076] Example 3: Enzymatic properties of β-glucosidase

[0077] The obtained β-glucosidase enzyme solution is subjected to enzymatic characterization with the above-mentioned enzyme activity assay method, and the enzyme activity is measured at different temperatures with pNPG as a substrate. The results show that the optimum temperature of β-glucosidase is Then, different pH gradients were set to measure the enzymatic activity of β-glucosidase under the optimum temperature conditions, and the optimum pH of the enzyme was obtained as 4.5 ( Figure 4 ).

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Abstract

The invention relates to a method for preparing oligogentisose by using β-1,6-glucanase and its application, and belongs to the technical field of genetic engineering and fermentation engineering. The present invention first provides a β-1,6-glucanase TcBgn with transglycosylation activity on gentiobiose, which can convert glucose into oligomeric gentian with a low amount of added enzyme sugar, and the conversion rate is high, which can significantly reduce production costs. In the present invention, β-1,6-glucanase and β-glucosidase double enzymes are also compounded to prepare oligosaccharides, and the double-enzyme compounding system is used to convert glucose into gentiobiose and gentiotriose. , the conversion rate is high, and the proportion of gentiotriose in the product is significantly increased. Therefore, the method for preparing oligosaccharide gentisose by β-1,6-glucanase single-enzyme or double-enzyme compounding has good industrial application value.

Description

technical field [0001] The invention relates to a method for preparing oligogentisose by using beta-1,6-glucanase and its application, and belongs to the technical field of genetic engineering and fermentation engineering. Background technique [0002] Gentiose oligosaccharides are functional oligosaccharides formed by the linking of glucose with β-1,6 glycosidic bonds, including gentiobiose and a small amount of tris and tetrasaccharides. Gentiose oligosaccharide is not digested and absorbed by the human intestine, but it is beneficial to the reproduction and growth of bifidobacteria and lactic acid bacteria, and is suitable for people with diabetes and other people. At the same time, gentiosaccharide oligosaccharide has high heat resistance and is suitable for foods that require high temperature treatment; the gentiotriose in its composition has a unique, soft and refreshing bitter taste, and has good health care effects. Currently, gentiosaccharides are widely used in ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N9/42C12N15/81C12N1/19C12P19/14C12P19/12C12P19/00C12R1/84
CPCC12N9/2402C12N9/2445C12N15/815C12P19/14C12P19/12C12P19/00C12Y302/01021C12Y302/01075
Inventor 吴敬夏伟徐星豪
Owner JIANGNAN UNIV
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