| In recent years, the frequently happened events about Lentinula edodes formaldehyde, has attracted much attention from the public. The research showed that formaldehyde was formed as Lentinula edodes growing, and is the normal metabolites of Lentinula edodes; The formation of Lentinula edodes endogenous involved in two critical enzymes:gamma-glutamyltranspeptidase (GTT) and L-cysteine sulphoxide lyase (C-S lyase). In order to know the function of GGT during Lentinula edodes formaldehyde metabolism, and the relationship between gene expression level and the production of Lentinula edodes formaldehyde, the full length cDNA sequence of Lentinula edodes was cloned by RT-PCR and RACE method, the GGT sequence was predicted by bioinformatics software, GGT was heterogeneously expressed in the strain of E.coli and pichia. Results were summarized as follows:1. The full length of GGT gene was obtained by RACE method. The full length of Lentinula edodes GGT cDNA was 1971 bp, with open reading frame of 1749 bp, the length of 5'untranslated region was 70 bp, and the length of 3'untranslated region was 152 bp. Bioinformatics analysis showed that GGT encoded 582 amino acids, The theoretical molecular weight was 62330.52 Da, the theoretical isoelectronic point was 5.83; The deduced amino acid sequence of Lentinula edodes GGT showed 76% identity with Agaricus bisporus GGT. Peptide analysis showed that Lentinula edodes didn't have signal peptide, it belonged to non-secretory protein, it was hydrophilic protein in the meanwhile. Lentinula edodes GGT was a glycoprotein, there existed the post-translated glycosylation and phosphorylation, and it had 3 potential glycosylate sites and 29 phosphorylate sites. Lentinula edodes GGT were encoded by a single gene and were translated into a unique polypeptide, which then undergone an auto-proteolytic cleavage into a large and small subunit. The potential molecular mass of the big subunit was 40731.29 Da, while the small subunit was 21617.25 Da, the two subunits combined together by hydrogen bonding force, to be the active mature heterodimers.2. We engineered a E.coli expression vector containing the coding sequence of Lentinula edodes GGT:PET28a(+)-GGT, and transferred the vector into E.coli BL21(DE3).The recombinant GGT was induced under the condition of 0.2mM IPTG concentration and the temperature of 18?, after induced 16h, the yields of soluble purified protein per liter of bacterial culture was 13mg. The recombinant GGT was purified by Ni2+-NTA-resin affinity chromatography, and the molecular properties of the recombinant GGT were analyzed by SDS/PAGE, the recombinant Lentinula edodes GGT exhibited three major bands with molecular mass of 68 kD,45 kD and 23 kD, which was the pro-enzyme, GGT large subunit and GGT small subunit, respectively. The catalytic reaction activity was measured. Unfortunately, the recombinant GGT do not have catalytic activity.3. To examine the catalytic properties of Lentinula edodes GGT, We engineered a eukaryotic expression vector containing the coding sequence of Lentinula edodes GGT (pPIC9K-ggtc). The recombinant vector was transferred into histidine defect Pichia GS115 by electronic method. The Pichia was plated to the MD plate, positive Pichia was isolated preliminary. PCR method was used to screen the engineering strains that GGT gene was successfully inserted into the Pichia genome. The plate contrast experiment was used to testify the mut+ strains. The recombinant GGT was induced under the condition of 0.5% methanol concentration and the temperature of 30?, by the analysis of enzyme activity and SDS-PAGE, Pichia strains that can express the active recombinant GGT was screened. It was showed that Lentinula edodes GGT gene was expressed in Pichia with active product, and recombinant GGT was secreted outside the sell by the direction of signal peptide. |
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