关键词: 未培养微生物, b-葡萄糖苷酶, 碱性蛋白酶, 文库, 宏基因组

Abstract: The metagenomic DNAs were extracted and purified from alkalescence environmental samples directly. On the basis of the metagenomic DNA, the alkaline soil 16S rDNA library composed of 5 562 positive clones was constructed. The phylogenic tree indicated that the bacteria from the alkaline soils were bio-diversity. The metagenomic DNA library named AL01 was constructed by inserting restriction fragments of the purified DNAs into plasmids pGEM-3Zf(+) vector. This library contained 23 650 positive clones and the average foreign DNA fragments were about 3.2 kb. The length of the library covered 75.68 Mb. The efficiency of the metagenomic library was approximately 6 000 clones from 1g dry soil samples. After screening AL01 DNA library with the screening tactics of enzymes, we confirmed that a positive clone, designated pGXAA2011, contained an alkaline protease gene AP01. Enzymatic analysis proved that its reaction optimum pH was 9.5 and the optimum temperature was 40℃. Furthermore, a clone, designated pGXAG142 was screened from metagenomic DNA library, which expresses β-glucosidase. DNA sequence indicated that the potential ORF of pGXAG142, which was named unglu01, there was no DNA or amino acids identity with the known β-glucosidase genes in the Genbank. The integrated ORF was cloned into pETBlue-2 vector and was then transformed into Tuner(DE3)pLacI. The recombinant expression clone could express β-glucosidase on the screening plate clearly and the analysis of SDS-PAGE indicated that the target protein was about 29 kDa.