Large-island saline-alkali bacillus capable of producing cyclodextrin glucosyltransferase and application of large-island saline-alkali bacillus

A bacillus and glucose-based technology, applied in the field of enzyme engineering, can solve the problems of few species and high price, and achieve the effects of high conversion rate, low material cost and low energy consumption

Pending Publication Date: 2022-04-19
HANGZHOU WAHAHA TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are fewer types of cyclodextrin glucosyltransferases widely used in the market, and the price is higher.

Method used

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  • Large-island saline-alkali bacillus capable of producing cyclodextrin glucosyltransferase and application of large-island saline-alkali bacillus
  • Large-island saline-alkali bacillus capable of producing cyclodextrin glucosyltransferase and application of large-island saline-alkali bacillus
  • Large-island saline-alkali bacillus capable of producing cyclodextrin glucosyltransferase and application of large-island saline-alkali bacillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: strain screening

[0036] From the soil of the stevia planting base of Zhucheng Haotian Pharmaceutical Co., Ltd. in Shandong Province, 10 soil samples were taken according to the "five-point sampling method", and the strains were isolated from them. Weigh 5g of the sample and add it to a small Erlenmeyer flask filled with 45ml of sterile normal saline, shake it on a shaker at 150rpm at 28°C for 1 hour, draw 0.5ml of soil bacteria suspension and place it in 4.5ml of sterile normal saline, mix well, and dilute gradually 10 -1 、10 -2 、10 -3 、10 -4 and 10 -5 5 gradients. Draw 100 μL from the bacterial suspension of each dilution gradient and spread it on the separation and screening plate, and incubate at 28°C and 37°C incubators for 2 days respectively. Select the strain that produces a yellow transparent circle on the plate as the strain that produces CGTase. Pick a single colony and streak the plate 4 times. Afterwards, after the bacteria obtained ...

Embodiment 2

[0039] Embodiment 2: fermentation produces enzyme

[0040] (1) Fermentation culture: Inoculate the bacterial strain screened in Example 1 into the fermentation medium, culture at 28° C. for 2-3 days, and collect the supernatant by centrifugation to obtain the crude enzyme liquid. Fermentation medium (g / L): soluble starch 10, peptone 5, yeast powder 5, dipotassium hydrogen phosphate 1, magnesium sulfate heptahydrate 0.2, sodium carbonate 5, among which sodium carbonate was first formulated to 30% (w / v) The mother liquor was sterilized separately and then added to the culture medium, and autoclaved at 121°C for 20 minutes. Natural pH.

[0041] (2) Product identification: prepare 2% (w / v) steviol glycoside (Zhucheng Haotian Pharmaceutical Co., Ltd. SG90) + 2% (w / v) soluble starch solution, add crude enzyme solution (culture medium 10000rpm, centrifuge 20min , take the supernatant), and react at 28-50°C for 24-48h. The resulting reaction solution was filtered through a 0.22 μm ...

Embodiment 3

[0044] Embodiment 3: the concentration of crude enzyme liquid

[0045] Concentrate the enzyme solution obtained in Example 2 with an ultrafiltration centrifuge tube Millipore 15ml / 10kd (concentrate 100 times), and exchange the buffer (> 1000 times) to remove the medium components, so that the enzyme is placed in ultrapure water to avoid Impurities are introduced in subsequent reactions.

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Abstract

The invention relates to the field of enzyme engineering, and discloses a large-island saline-alkali bacillus for producing cyclodextrin glucosyltransferase and application of the large-island saline-alkali bacillus. According to the invention, a large island saline-alkali bacillus (WahahaMZJ-1) for expressing cyclodextrin glucosyltransferase is screened, and the preservation number of the large island saline-alkali bacillus is CGMCC (China General Microbiological Culture Collection Center) No.23164. The invention also discloses a preparation method of the large island saline-alkali bacillus. Compared with the existing commercial similar enzymes, the glucosyl stevioside is generated by carrying out enzyme reaction on the obtained enzyme, and the contents of RebD, RebD isomeride, RebM and RebM isomeride in the converted product are high, so that the taste is better; meanwhile, the method has the advantages of simple reaction method, low energy consumption, low material cost, high conversion efficiency and the like, and lays a foundation for industrial large-scale production.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to a cyclodextrin glucosyltransferase-producing bacillus saline-alkali and its application. Background technique [0002] In recent years, the natural sweetener steviol glycoside has attracted more and more attention from consumers and food manufacturers because of its high sweetness (200-350 times that of sucrose) and low calorific value (only 1 / 300 of sucrose). . However, the commercially available steviol glycosides have strong bitterness and astringency in the mouthfeel, and the taste is slower than that of sucrose, which seriously affects its market share. Steviol glycoside is a mixture of diterpene glycosides extracted from Stevia rebaudiana and contains different components. They all have the same aglycone or ligand-steviol, which is a typical kaurane-type tetracyclic diterpene structure steviol The C13 hydroxyl group and the C19 carboxyl group on the ligand have two activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/10C12P19/18C12P19/56C12R1/01
CPCC12N1/20C12N9/1074C12P19/18C12P19/56C12Y204/01019
Inventor 张睿钦曹成浩毕家华赵志红沈珊珊李言郡余腾斐
Owner HANGZHOU WAHAHA TECH
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