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Bacillus amyloliquefaciens engineering strain for producing 1-deoxynojirimycin at high yield and fermentation method

A technology for deoxynojirimycin and amylolytic spores, applied in the field of microorganisms, can solve problems such as unsatisfactory yield, and achieve the effect of broad application prospects

Inactive Publication Date: 2018-05-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, wild bacteria have been fermented for the production of DNJ. For example, Bacillus subtilis B2 uses bean dregs to ferment DNJ; Bacillus amyloliquefaciens 140N ferments soluble starch as a carbon source, and its DNJ yield reaches 0.8g / L; However, the yield is still unsatisfactory. Obtaining a high-yielding engineered bacterium through biotechnological means has always been the goal of those skilled in the art.

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  • Bacillus amyloliquefaciens engineering strain for producing 1-deoxynojirimycin at high yield and fermentation method
  • Bacillus amyloliquefaciens engineering strain for producing 1-deoxynojirimycin at high yield and fermentation method
  • Bacillus amyloliquefaciens engineering strain for producing 1-deoxynojirimycin at high yield and fermentation method

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Experimental program
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Effect test

Embodiment 1

[0032] Construction of Bacillus amyloliquefaciens HZ-15:

[0033] 1. Construction of gene gabT1, yktC1 and gutB1 recombinant vectors

[0034] According to the genome sequence of Bacillus subtilis 168, the promoter P43SEQ ID NO:1 and the terminator amyL SEQ ID NO:2 were amplified by PCR, and the primers were respectively P43-F SEQ ID NO:3, P43-R SEQ ID NO:4, TamyL-F SEQ ID NO: 5, TamyL-R SEQ ID NO: 6. According to the genome sequence of Bacillus amyloliquefaciens LX-12, PCR amplified gene gabT1SEQ IDNO: 7 and its upstream and downstream homologous arms gabT1-up SEQ IDNO: 8 and gabT1-down SEQ IDNO: 9, the primers were gabT1-F SEQ ID NO:10, gabT1-R SEQ ID NO:11, gabT1-upF SEQ ID NO:12, gabT1-upR SEQ ID NO:13, gabT1-downF SEQ ID NO:14, gabT1-downR SEQ ID NO:15. According to the genome sequence of Bacillus amyloliquefaciens LX-12, the gene yktC1SEQ IDNO:16 and its upstream and downstream homologous arms yktC1-up SEQ IDNO:17 and yktC1-down SEQ IDNO:18 were amplified by PCR, and th...

Embodiment 2

[0042] The solid fermentation method of high yield 1-deoxynojirimycin of Bacillus amyloliquefaciens HZ-15:

[0043] 1-Deoxynojirimycin detection method and quantitative method:

[0044] After the fermentation, distilled water was added to soybeans at a ratio of 1:1 (w / v), treated at 200r / min for 1h to extract DNJ, centrifuged at 10000r / min for 20min, and the supernatant was taken. Derivatize the sample: put 35 μL of fermentation supernatant into a 1.5 mL centrifuge tube, add 169 μL of 0.4mol / L potassium borate buffer (pH 8.5), 5 mmol / L of derivatization reagent FMOC-Cl (dissolved in 50% acetonitrile Medium) 250 μL, mix well, react in a constant temperature water bath at 25°C for 20 min, add 25 μL of 1mol / L glycine to terminate the reaction (neutralize the remaining FMOC-Cl), add 66 μL of 1% (V / V) acetic acid solution, and dilute with water Volume to 707μL, filter with a 0.22μm microporous membrane, collect the filtrate, and measure it by RP-HPLC-UV method. The chromatographic...

Embodiment 3

[0050] The liquid fermentation method of high yield 1-deoxynojirimycin of Bacillus amyloliquefaciens HZ-15:

[0051] Pick a single colony of HZ-15 and transfer it to 5mL LB medium, culture overnight at 37°C with shaking at 180r / min. The culture was inoculated into 50mL LB medium with 2% inoculum amount, 37°C, 180r / min shaking culture for 9h, until OD600 reached 4.0, and then inoculated into 30mL optimized liquid fermentation medium with 2% inoculum amount, 180r / min / min, cultured at 37°C for 48h.

[0052] The optimized liquid fermentation medium formula is: malt extract powder 70g / L, soluble starch 30g / L, K 2 HPO 4 ·3H 2 O0.75g / L, MgSO 4 ·7H 2 O 0.2g / L, MnSO 4 ·H 2 O 0.075g / L; fermentation conditions are: seed age is 9h, inoculum size (v / v) is 2%, fermentation culture temperature is 37 ℃, initial pH of culture medium is 7.5, and liquid volume is 30mL / 250mL Erlenmeyer flask.

[0053] In the optimized liquid fermentation medium, the DNJ yield of the engineered bacteria H...

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Abstract

The invention provides a bacillus amyloliquefaciens engineering strain for producing 1-deoxynojirimycin at high yield and a fermentation method. The preservation number is CCTCC NO.M2017849, and the bacillus amyloliquefaciens engineering strain is named bacillus amyloliquefaciens HZ-15, and the invention further provides a construction method of bacillus amyloliquefaciens HZ-15 and a method for producing 1-deoxynojirimycin (DNJ) at high yield. By the adoption of a molecular biological technique, on the basis of preserved bacillus amyloliquefaciens LX-12 for producing DNJ in a laboratory, the engineering strain HZ-15 for producing the DNJ at high yield is constructed successfully through a homologous recombination method. With soybeans being raw materials, solid fermentation is performed; by adding 1% of malt extract powder and 2% of lactose into a solid fermentation culture medium, the yield of the DNJ is up to 1117.2 mg / kg.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a high-yield 1-deoxynojirimycin Bacillus amyloliquefaciens engineering bacterium and a fermentation method. Background technique: [0002] With the increasing number of diabetic patients, finding a drug for treating diabetes has become a major problem in the medical field. 1-deoxynojirimycin (1-deoxynojirimycin) referred to as DNJ, is a piperidine polyhydroxy alkaloid, its chemical structure is similar to glucose, has α-glucosidase inhibitory activity, and can competitively bind α-glucosidase activity When the glycosidase in the digestive tract or in the process of glycoprotein processing is competitively inhibited by polyhydroxyalkaloids such as DNJ, carbohydrate absorption disorders and abnormal synthesis of glycoprotein oligosaccharide chains will occur, thereby inhibiting the rise in blood sugar , antitumor, antiviral and other biological activities. DNJ ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/12C12R1/07
CPCC12N9/0004C12N9/1096C12N9/16C12P17/12C12Y206/01
Inventor 冀志霞程相锦陈守文卢玉
Owner HUAZHONG AGRI UNIV
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