Gene Cloning And Functional Characterization Of A L-Glutamic Acid Decarboxylase Gad3 From Contaminated Lake Metagenome

Gamma amino acid (GABA) is a neurotransmitter in mammals, and it has the function of inhibiting the nerve stimulation. It is widely used in the fields such as drugs, food additives and so on. Glutamic acid decarboxylase in catalytic glutamic acid decarboxylate, which can generate GABA, is an important means for the synthesis of GABA by biological method. Compared with the chemical synthesis, the use of microbial technology to produce gamma amino acid has incomparable superiority. Uncultured microorganisms contain a large number of new genetic resources, but ordinary separation purification and pure culture technology is difficult to perform. Therefore, in nature of the glutamic acid decarboxylase gene mining, this paper explores through the establishment of a metagenomic library, which is rarely reported in both domestic and international glutamic acid decarboxylase research.In this paper, we use acidic polluted samples to construct a metagenomic library, which uses pGEM-3Zf (+) as the carrier, Escherichia coli DH5a as the host, with a total of 1.2 million clones in its capacity. That equals about 50.4 MB Information. We randomly sequenced some part of the library, and searched the GenBank database for consistency comparison, a large number of new gene emerged, which demonstrats the effectiveness of this method.Through an activity screening strategy, two positive clones with obvious color reaction stood out from the library, namely pGEMWl and pGEMW2.We searched against the GenBank database with the ORFs (open reading frame) contained in the two positive clones, functional identification was performed. Finally, a glutamate decarboxylase gene gad3 was found out. The total length of the gene was about 1.4 KB, encodes a polypeptide chain which composes 469 amino acids, with a molecular weight of about 53 kDa, and the isoelectric point is 5.61. It shares a 99% consistency with the glutamic acid decarboxylase gene in Lactobacillus plantarum, at the amino acid level,And the latter one came from genome annotation, none action was performed to identify the function.The gad3 gene was amplified by PCR, and the product was ligased to the expression vector pET-30a(+). After transferring the recombinant plasmid into the expression system E.coli BL21 (DE3) pLysS, recombinant protein was finally obtained. The recombinant protein was purified by Ni-NTA purification, and the enzymatic properties were measured. It shows maxmum activity at the temperature of 37 ℃, and the optimum pH value is 4.7. Sr2+, CO2+, Fe2+, Zn2+, Mg2+ and Zn2+ activates the enzymatic reaction, while Al3+ and Fe3+ inhibites the enzymatic reaction. The inhibition of EDTA on the activity indicates its dependence on metal ions.The Km of the pure enzyme was 0.619 mg/mL, kcat/ Km=19.483 mL/(mg·s). Compared with glutamic acid decarboxylases from other sources, this enzyme presents a middle level in the enzyme property aspect.This study established a set of the metagenomic library cloning method in mining of glutamic acid decarboxylase gene, which shows an example to the subsequent researches.