Bacillus amyloliquefaciens and application thereof in preparation of 1-deoxynojirimycin

A technology for deoxynojirimycin and amylolytic spores, applied in the directions of bacteria, microorganism-based methods, biochemical equipment and methods, etc., and can solve the problems of low yield of 1-deoxynojirimycin and long fermentation time, etc.

Active Publication Date: 2020-12-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the reported microorganisms that can produce 1-deoxynojirimycin generally

Method used

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  • Bacillus amyloliquefaciens and application thereof in preparation of 1-deoxynojirimycin
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  • Bacillus amyloliquefaciens and application thereof in preparation of 1-deoxynojirimycin

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Experimental program
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Embodiment 1

[0023] Embodiment 1: the screening method establishment of producing 1-DNJ strain

[0024]By measuring the inhibition rate of 1-DNJ produced in the fermentation process of different strains on the glycosidase gene, the high-yielding 1-DNJ strains were screened. Sulfolobus solfataricus The β-galactosidase LacS was used as an indicator enzyme, and the feasibility of the screening method was tested with 1-DNJ standard.

[0025] (1) Induced expression of β-galactosidase LacS

[0026] from Sulfolobus solfataricus β-galactosidase gene lac (Genbank accession no.AF133096.1) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and connected to the pET-28a(+) vector. Transform the constructed vector E.coli BL21 (DE3) Competent cells, coated with LB solid plates containing 100μg / mL Amp antibiotics, cultivated overnight in a 37°C incubator to obtain expressible lac host cells. Pick a single colony from the plate to LB liquid medium containing 100 μg / mL Amp antibiotic, cu...

Embodiment 2

[0036] Example 2: Screening of 1-DNJ-producing strains

[0037] (1) Primary screening of 1-DNJ-producing strains

[0038] To screen Bacillus from fermented bean curd samples, the specific method is: filter the samples with three layers of gauze, treat the filtrate at 80°C for 20 min, and then use sterile normal saline according to 10 -3 to 10 -7 Gradient dilution was carried out, and 0.1 mL of the diluted solution was spread on an LB solid plate, and incubated in a 37°C incubator for 48 h, and the single colony growing on the plate was streaked and purified, and a total of about 230 single colonies were obtained.

[0039] Pick a single colony and inoculate it into a 30 mL test tube containing 2 mL LB glucose medium, and incubate at 37 °C, 200 rpm for 72 h. Take 1 mL of fermentation broth into a 1.5 mL centrifuge tube, centrifuge at 14,000 rpm for 10 min at 4°C to obtain the fermentation supernatant. Add 60 μL of Buffer Z, 50 μL of 12 mM oNPG, 20 μL of fermentation supernata...

Embodiment 3

[0048] Embodiment 3: bacillus amyloliquefaciens of the present invention ( Bacillus amyloliquefaciens ) Optimization of medium composition for 1-DNJ production

[0049] Put 30ml of fermentation medium into a 250mL Erlenmeyer flask, sterilize with high-temperature steam at 121°C for 30 minutes, and inoculate the seed liquid cultivated to the late logarithmic phase into the Erlenmeyer flask aseptically, with an inoculum volume of 1%, at 37°C and 100 rpm After culturing for 5 days, the appropriately diluted fermentation supernatant was taken to determine the inhibition rate of LacS, and all experiments were designed in triplicate. The results of the previous optimization step are used in subsequent experiments.

[0050] (1) Effects of different carbon sources on the production of 1-DNJ by strains

[0051] Lactose in the fermentation medium was replaced with other carbon sources such as glucose, fructose, xylose, galactose, mannose, sorbose, mannitol, sucrose, maltose, xylitol,...

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Abstract

The invention discloses bacillus amyloliquefaciens capable of producing 1-deoxynojirimycin at high yield. The strain preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 19098. The invention also discloses a method for producing the 1-deoxynojirimycin by fermenting the bacillus amyloliquefaciens, and the yield of the 1-deoxynojirimycinreaches 0.9 g/L. The 1-deoxynojirimycin has alpha-glucosidase inhibitory activity and can be applied to blood glucose control of diabetics, so that the strain and the method for preparing the 1-deoxynojirimycin through fermentation of the strain have wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of industrial biology, and in particular relates to a strain of Bacillus amyloliquefaciens capable of producing 1-deoxynojirimycin, and a method and application for fermenting and producing 1-deoxynojirimycin using the strain. Background technique [0002] Diabetes is a chronic metabolic disease characterized by high blood sugar. According to statistics from the International Diabetes Federation, as of 2017, the number of diabetic patients in the world has reached 425 million, while the number of diabetic patients in China has reached 121 million. There is a potential diabetic. If blood sugar is not well controlled for a long time in diabetic patients, it can lead to organ tissue damage and become the "source of all diseases", accompanied by various organs, especially eye, heart, blood vessel, kidney, nerve damage or organ insufficiency or failure. The patient becomes disabled or dies. Based on the pathoge...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/12C12R1/07
CPCC12N1/20C12P17/12C12N1/205C12R2001/07
Inventor 陈朋朱玥明门燕刘德川刘汉民孙媛霞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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