Comparison Of Two Metagenoms From Two Hot Springs In Eryuan And Cloning And Expression Of Aβ-galactosidase Gene

β-galactosidase(EC3.2.1.23) was known as a lactase, it is a member of glycoside hydrolases. β-galactosidase hydrolyses the β-1,4-D-galactosidic linkage of lactose, as well as those of related chromogens substrate that contain β-1,4-D-galactosidic linkage such as ONPG β-galactosidase is widely used in the food industry and molecular biology, which could alleviate the lactose intolerance of human effectively. β-galactosidase also can be used as an additive in animal feed production to improve digestibility of feed and reduce the diarrhea caused by lactose intolerance at the same time.Metagenomics technology is a powerful tool for biodiversity research of natural environmental sample, it can avoid the microbial pure culture in Laboratory and study on uncultured microorganisms directly. Two samples from hot springs with the temperature of55℃and65℃respectively in Eryuan in Dali, Yunnan province were collected and their metagenomic DNA were extracted, sequenced and the data were analyzed. After assembly,864942and275787scaffolds were obtained from5℃and65℃hot spring respectively, and the scaffolds average length were205bp and343bp of55℃and65℃hot spring, respectively. Genemark.hmm soft used for Gene prediction showed that the numbers of ORF were571158and228039of55℃and65℃hot spring, respectively. Generally, the ORF derived from65℃hot spring was greater than that derived from55℃hot spring in size. Taxonomic analysis showed that the most of the55℃hot spring microorgnisms belonged to30phyla of thernophiles, covering Proteobacteria, uryarchaeota,acteroidetes, etc. Most of the65℃hot spring microorgnisms belonged to43phyla of ancestral thermophiles, covering Euryarchaeota, ermotogae, imicutes, etc. Gene functional annotation showed that the quantity of genes that matched to the secondary pathways of KEGG in65℃hot spring was larger than that in55℃,this proved that the metabolic functions of microorganisms in65℃hot spring were stronger than that in55℃hot spring, so microoganisms in65℃hot spring can adapt to higher temperatures.The diversity analysis showed that only8sequences related to β-galactosidase gene in55℃hot spring were found, all the8sequences derived from6species. However, there were55sequences related to β-galactosidase gene in65℃hot spring. These sequences derived frorm19species, and a few sequences still could not be identified.One complete β-galactosidase gene was obtained from the65℃hot spring metagenomic database. Sequence analysis result showed that the sequence potentially encoded a β-galactosidase, since the encoded amino aicd shared71%sequence homology with that of Thermotoga lettingae TMO glycoside hydrolase. After heterologous expression in E.coli Rosetta and affinity purification, the purified β-galactosidase was further subjected to analysis of its enzymatic characteristics. The results showed that the optimum catalytic temperature and pH value of the expressed β-galactosidase were55℃and7.5, respectively. The β-galactosidase could keep about65%of its enzyme activity after treated at58℃for30min. The β-galactosidase could hydrolyze lactose in soybean into galactose and glucose, indicating a good application prospect in feed production.