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Arthrobacetr Sp. Mutant Selection and Fermentative Optimization for β-galactosidase Production and β-galactosidase Conserved Gene Sequence Analysis

Author: DongXiaoZuo
Tutor: TangLei
School: Jiangnan University
Course: Biochemistry and Molecular Biology
Keywords: β-galactosidase composite mutagenesis response surface method conserved region ofβ-galactosidase genes cloning
CLC: TQ925
Type: Master's thesis
Year: 2010
Downloads: 91
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Abstract


β-galactosidase (EC 3.2.1.23) is a kind of enzymes which can catalyze hydrolysis and transglycosylation. It can hydrolyze lactose into glucose and galactose. Under the turn of transglycosylation, lactulose was formed by fructose transferred to galactose.β-galactosidase enzymology character itself determines that it can be widely applied in the degradation of lactose in the dairy products (such as lactose to solve resistance syndrome, etc.) and functional oligosaccharide production (such as galactooligo saccharide, GOS etc.). In the previous study, Arthrobacter bacillus sp. was screening by our laboratory, which is aβ-galactosidase producer. The following study was performed to further understand the stain and itsβ-galactosidase:In order to improve hydrolysis ability of the strain, the NTG-LiCl composite mutagenesis and ultraviolet radiation-LiCl composite mutagenesis were carry out. A strain Arthrobacter sp. U14 was selected. The hydrolysis ability was improved to 2.65 U/mL, 470% higher than that of the Arthrobacter sp..In the selection ofβ-galactosidase improved strains process, a screening merhod was establish using lysozyme and SDS treatment for the Arthrobacter bacillus sp..4 mg/mL of lysozyme was addad to the bactera suspension for about 1.5 h at 37℃, and then SDS was added and kept at 37℃for 20 min.The response surface method was used to investigate the influence of Arthrobacter sp. U14β-galactosidase production.Optimized composition of fermentation medium:natural pH,5.4% lactose,5.1%yeast extracts,0.93 mM Fe3+.The predicted maximum productivity was 3.7 U/mL. After the actual yield ofβ-galactosidase was 3.85 U/mL,45.3% higher than before.The primers were designed according to the published genes of Arthrobactorβ-galactosidase.PCR was performed. The PCR products were analysis, and 97.37% similarity was for found between the parent strain and mutant in the conserved region ofβ-galactosidase genes.

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