Identification Of A Strain Of Bacteria Producing α-galactosidase And Cloning The Enzyme Gene

α-galactosidase (α-galactosidase,α-D-galactoside galactohydrolase; EC 3.2.1.22) catalyze theα-banded nonreducing D-galactose in different substrates such as melibiose. Theα-galactosidase exists widely in microorganism, animals and plants. It has a wide application in feed industry and sugar producing and medical field, etc. Experiments showed thatα-galactosidase add in soybean meal feed can effectively degraded galactoside and reduce intestinal diseases in animals. Because the costs ofα-galactosidase that it can not meet the requirements of presenting feed industry. It is necessary to development some novelα-galactosidase especially origin in bacterium for a better engineering possibility.Theα-galactosidase can be produced in many organisms. It is reasonable considering that theα-galactosidase produced in prokaryotes of single-peptid and relatively easier to manipulate. We tried to identify someα-galactosidase producing bacteria from wild. A strain ofα-galactosidase producing bacteria was screened out from soil rich in soybean wastes in Western Hunan. The bacterial morphology observation, molecular biological identification and enzymatic characteristics analysis had been carried out. It is identified that the strain of screened bacterium is Gram positive and the 16S rDNA sequence shares high homologous (98%) with Lactoccus.The p-Nitrophenol methods are applied to detect theα-galactosidase activity of the bacterium after shaking incubation. The results showed that the bacteria produce an excretiveα-galactosidase with the maximalα-galactosidase activity of shaking cultured supernatant 6.21 U/mL. The enzyme is in the highest activity when the value of pH is 5.5 and in the tempertured of 45℃. The enzyme gene expression is regulated in the typical glycolytic operon model that induced by the substrate and inhibited by glucose. The best induction effect obtained when the D-raffinose concentration reached 30μmol/mL. And it is inhibited when glucose concentration reached 30μmol/mL.The enzyme gene was cloned by random fragement cloning. The genomic DNA was extracted and then digested with Sau3AI by partial digestion. The random fragment of 3 kb were inserted into pUC19 that linearize by BamHI. The recombinates were transformaned into Escherichia coli InvaF and screen of positive clone byα-galactosidase activity detection. Theα-galactosidase gene (agaH) of we identified Lactococcus was cloned and sequenced. Bioinformatics analysis reveales that the cloned 3039 bp DNA contains a 1230 bp opening reading frame. It can encode a putative protein of 409 amino acid that with a conserved domain of glycoside hydrolase 2 super family (10-220 aa). We can confer the cloned fragment is the gene forα-galactosidase. But the gene shared no obvious nucleotide sequences homology with other reported a-galactosidase gene. So we regard it a novelα-galactosidase gene from bactiria.