Dissertation
Dissertation > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )

Screening of Bacterial Strains with High α-Galactosidase Activity and Its Gene Cloning, Expression, Purification and Characterization

Author ZuoPeiPei
Tutor CuiZhongLi
School Nanjing Agricultural College
Course Microbiology
Keywords Bacillus megaterium α- galactosidase Properties of Gene Cloning Recombinant expression Purification
CLC TQ925
Type Master's thesis
Year 2011
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a-Galactosidase (a-D-galactoside galactohydrolase; EC 3.2.1.22), also knowed as melibiase, is an exoglycosidase. It can remove a-linked terminal non-reducing galactose residues from different substrates. a-Galactosidase is widely distributed in animals, plants and microorganisms. And microorganisms are the main sources of this enzyme for their vast diveristy, wide distribution and strong adaption. This enzyme is widely used in food, feed and pharmaceutical industries, and regarded as one of the most potential enzyme preparations.In this study, a strain with high a-galactosidase production was screend from 236 soil samples. The strain was identified as Bacillus megaterium by 16s rDNA identification and physiological and biochemical identification. The a-galactosidase activity of the crude enzyme extract of Bacillus megaterium MEPE0049 was 1.22 U/mg。Conditions including liquid medium volume, inoculation size, sources of carbon, nitrogen sources, the optimum temperature, optimal pH, and metal ions, were tested to find the optimal enzyme production conditions. The optimal temperature and pH were 35℃,8 respectively.The characters of a-galactosides of strain MEPE0049 were determined. The results showed that its a-galactosidase activity wouldl disappear when temperature was 45℃, it was more sensitive to pH when pH is under 5。Most of the metal ions were no great influence to the enzyme activity, only Co+made enzyme activity fall below 30%.According to the genome sequence of B. megaterium, we designed four primer pairs and amplified the 1323 bps agaA and 2226 bps agaB. Both genes were cloned into expression vector pET-29a and expressed in E.coil BL21. AgaA expressed in E.coli BL21 showed no detectable activity. The activity of crude extract of AgaB in E.coli BL21 was 9.86 U/mg. AgaB was purified by Ni-NTA affinity purification to homogenity. Its specific enzyme activity was 380.66 U/mg. The characteristics of the purified enzyme showed no significant difference with the crude enzyme. Results indicated that a-galactoside detected in B. megaterium MEPE0049 was mainly from AgaB.

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