Gene Cloning And Prokaryotic Expression Of Amylomaltase From Streptomyces Ssp. And Research On Production Of Amylomaltase

Cyclodextrin is the cyclizing product of starch degraded by enzyme with the structure of a hydrophobic inner cavity that looks like a column. Amylomaltase, existing in a number of microbe and plants, is one enzyme that can catalyze this cycliztion. But its low content in nature resources cannot meet the need of lib research or prodution of large cyclodextrins. This study was aimed to construct a recombinant strain that can produce amylomaltase. Gene Mal Q was cloned from Streptomyces ssp. ST66 and then transformed into E.coli. A recombinant engineering strains E.coli. BL21/pET-GST-Mal Q was constructed. Expression, purification and fermentation of amylomaltase were studied in detail. And also successfully catalyzing amylose cyclization to yield large cyclodextrins. The main results are described as follows.(1) The gene Mal Q was amplified from Streptomyces ssp. ST66 by PCR, then integrated into vector pET-GST and sequenced. The sequence was analyzed and found having a open reading frame with the length of 2133 bp, it translated a protein with 711 amino acids and molecular weight(MW) of 76.56 kDa. Mal Q sequence was analyzed by BLAST and it displayed high similarity(97%) to gene Mal Q of Streptomyces coelicolor reported in the GenBank. The recommend plasmid pET-GST-Mal Q was transformated into E.coli BL21 to construct the recombinant engineering strains E.coli. BL21/pET-GST-Mal Q. After induced by IPTG, the interest protein was produced and seperated by ultrasonication. SDS-PAGE indicated the successful expression. The mixture of crude enzyme treating maltose demonstrated 4-α-glucanotransferase activity and this 4-a-glucanotransferase was an endoenzyme by TLC.(2) Purified by Ni-NTA affinity chromatography and cut by HRV 14 3C to remove 6His-tag, interest protein got purified. SDS-PAGE showed a clear 76 kDa band. This crude enzyme was used to treat amylose. The resulting mixture solution was treated by glucoamylase. Result displayed an accumulation of starch which was resistant to an exo-amylase (glucoamylase). TLC showed that there were existance of cycloamylose.These all above indicated that the crude enzyme could catalyzed cyclization of amylose to produce cycloamylose.(3) The factors that affected the expression of exogenous gene in E.coli. were studied to get maximum enzyme activity from E.coli. BL21/pET-GST-Mal Q. The optimal inducing time was 5 hr, inducing temperature was 35℃, initial OD600 before inducing was 0.8, concentration of inducer IPTG was 0.9 mmol/L and inducing speed was 150 rpm, respectively. Under the optimal inducing conditions, we got the maximum enzyme activity which is 2.84 U. Compared with 0.31 U of wild fungus Streptomyces ssp. ST66, the result was increased nine-fold or so.