Studies On β-galactosidase From Arthrobacter Sp.

β-galactosidase had the two catalytic activity at the same time, one was the hydrolysis and the other was the transfer.It hydrolyzed lactose into glucose and galactose, with the addition of fructose,lactuloses were formed by fructose transferred to galactose. Because of these two fuctions,they determined the main use ofβ-galactosidase in industry: (1) hydrolyzed the lactose in milk,solved the problem lactose intolerane; (2) produced the lactulose by biocatalysis process. One bacterium strain Arthrobacter Z21 producingβ-galactosidase with transglycosylation activity was screened in the past time. The major results of this preliminary study were presented as follow:When the lactose and fructose concentrations were 2.5 % and 1.25 % respectively in transglycosylation reation,the activity ofβ-galactosidase was 2.4 U/mL(lactose).Lactuloses was decteted with the production of 15 g/L and had a high purity. Theβ-galactosidase can hydrolyze 90% lactose in milk, at the 40℃for 5 h, with theβ-galactosidase quantity was 1.22 U/mL(lactose). But only hydrolyzed 65% lactose in milk at the 4℃for 10 h.β-galactosidase from Arthrobacter Z21had the transglycosylation activity and hydrolysis activity in low temperature, the trait was valuable for industry application.Through native-PAGE(native polyacrylamide gel electrophoresis),β-galactosidase from Arthrobacter Z21 had three isozymes, the isozymeⅡwas expressed constitutively, and was not inducd by lactose,the isozymeⅠandⅢwere expressed by lactose induced. The results made a foundation forβ-galactosidase purification and gene cloning and expression .A mutant M2 was abtained by treating Z21with UV.β-galactosidase activity was 0.89 times as that from original strain. The production of lactuloses was increased to 30 g/L that was 1.4 times as that from Arthrobacter Z21.The optimized fermentation medium of Arthrobacter M2 contained(g/L): lactose 10, corn liquor 22, Fe3+ 1.5 mmoL/L,pH7.0. Theβ-galactosidase activity was increased 2.76 times from 0.465 U/mL(ONPG) to 1.875 U/mL(ONPG) after optimization.β-galactosidase from the mutant strain M2 had the good character that temperature stability and pH stability was better than that from original strain. If it was in a certain temperature and pH condition, the activity ofβ-galactosidase did not fluctuate acutely.β-galactosidase from both the original strain and mutation strain could be activated by Mn2+ and Mg2+,and it was strongly inhibited by Cu2+.In order to clone the gene ofβ-galactosidase, the methods of genomic DNA extraction from Arthrobacter sp were researched in this paper. The repeatedly chilled grinding and toluene treatments to the cells were used respectively before CTAB extraction. And the above methods were compared with that of bacterial genomic DNA extraction kit. The modified methods were effective in genomic DNA extraction. the concentration and purity of the DNA was good enough for PCR amplification.