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Producing microorganism for trans-glycosylation beta-galactosidase

A technology of galactosidase and transglycosylation, applied in the field of Enterobacteriaceae

Inactive Publication Date: 2006-09-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After searching, there is currently no report in the world that Enterobacter cloacae β-galactosidase catalyzes the production of galactooligosaccharides using lactose as a substrate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: strain screening

[0026] 1. Isolation and purification of growth strains that can use lactose as the only carbon source

[0027] Add 10g of soil sample to 100mL of culture solution with lactose as the only carbon source for enrichment culture, and dilute the culture solution with sterile water to 10 -1 、10 -3 、10 -5 、10 -7 For each gradient, 200 μL was evenly spread on the lactose selection plate. Bacterial screening plates were incubated at 37°C for 24 hours, and fungal screening plates were incubated at 28°C for 48-60 hours. 10 -5 There are many types of colonies grown on gradient plates, and they are easy to purify. Colonies with different shapes were picked, separated and purified by streaking on the plate, and repeated 3 times. Perform microscopic examination on the isolated strains to determine the purity: the bacteria use Staphylococcus aureus (Staphylococcusaureus) and Escherichia coli (Escherichia coli) as controls, and carry out Gram stai...

Embodiment 2

[0045] Embodiment 2: strain identification

[0046] 1. Morphological and physiological and biochemical characteristics

[0047] The bacterial strain Enterobacter cloacae (Enterobacter cloacae) B5CGMCC No.1401 that the present invention isolates and screens out, morphological feature is: Gram-negative bacillus; Shaped to short rod-shaped, ellipsoid, and finally reduced to spherical, the size of the stain is 0.5-1.5×0.7-9.0 microns; arranged in single, paired or short chains; moves with perinatal flagella; does not form spores; physiological and biochemical characteristics Yes: can use citrate or acetate as the only carbon source; ferment glucose at 37°C to produce acid and gas; glycerol fermentation does not produce gas; does not hydrolyze escin; does not produce indole; positive for V.P. determination; determination of methyl red Negative; reduced nitrate; no decarboxylation of lysine, but with ornithine decarboxylase, arginine dihydrolase, urease; see Table 1 for other chara...

Embodiment 3

[0081] Embodiment 3: Utilize Enterobacter cloacae (Enterobacter cloacae) B5CGMCC No.1401 to produce galactooligosaccharides

[0082] 1. Strain culture

[0083] The bacterial strain Enterobacter cloacae (Enterobacter cloacae) B5CGMCC No. 1401 isolated and screened by the present invention was selected, activated by a conventional method, and then transferred into the enzyme-producing fermentation medium for culture.

[0084] The formula of the above enzyme-producing fermentation medium is: lactose 10g / L, peptone 5g / L, yeast powder 10g / L, CaCl 2 0.11g / L, MnSO 4 0.001g / L, MgSO 4 ·7H 2 O 0.3g / L, KH 2 PO 4 0.05g / L, FeSO 4 ·7H 2 O 0.03g / L, pH7.0-7.5 (bacteria) or pH6.0-6.5 (fungi).

[0085] After a series of experiments to explore and optimize the enzyme-producing fermentation medium, the formulation of the enzyme-producing fermentation medium for Enterobacter cloacae B5 CGMCC No.1401 was simplified as follows: lactose 10g / L, peptone 5g / L, yeast powder 10g / L, Sodium chlorid...

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Abstract

The invention discloses a glycosyl-transferbeta-galactosidase production bacterium, which is characterized by the following: the name of this bacterium is Enterobacter cloacae B5, which is Gram-negative bacterium; the conservation code is CGMCC No.1401; the bacterium shape changes along culture time extension on the LB culture medium from long rod to short rod, ellipsoid shape and ball; the colouring size is 0.5-1.5*0.7-9.0 mm with single, couple or short-chain arrangement; the bacterium can move by peripheral flagellar without gemma and can apply citrate or acetate as only one carbon source, which products acid and gas by fermenting glucose at 37 deg.c and glycerin without gas, hydrolyzing aesculin and indole; V.P. detects positive and methyl red detects negative; the nitrate is reduced and Lys is not decarboxylation with ornithine decarboxylase, arginine dihydrolase and urease; the nucleotide sequence of this bacterium 16S rDNA is described as SEQ ID No.1; the beta-galactosidase can take lactin as substrate, which catalyzes to produce oligomer galactose.

Description

technical field [0001] The invention relates to an enterobacterium, in particular to an enterobacter cloacae capable of producing transglycosyl beta-galactosidase. Background technique [0002] Galactooligosaccharide is one of the natural components in breast milk, and its most important and fundamental physiological function comes from its effect on the proliferation of bifidobacteria. Because galactooligosaccharide has good heat stability and acid resistance, its health care effect has aroused great concern of people. At present, the production of galacto-oligosaccharides is mainly synthesized by microbial enzymatic methods, that is, β-galactosidase or lactase uses lactose as a substrate to transglycosylate synthesis. The development and research of galactooligosaccharides in my country is still in the initial stage, and the independent microbial enzymatic industrial production is still blank, and the lactase used in the dairy industry is all dependent on imports. Theref...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/38C12P19/00C12P1/04
Inventor 肖敏卢丽丽
Owner SHANDONG UNIV
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