High Level Expression Of Two Acidothermophilic α-Amylases

Alpha-amylases (1,4-alpha-D-glucan-glucanohydrolase, EC 3.2.1.1) hydrolyze alpha-1,4-linkages in starch. The action of alpha-amylases on starch leads to a rapid decrease of the starch solution viscosity. Alpha-amylases are widely applied in numerous areas, such as in starch saccharification, fermentation, spin, paper. Thus any research on alpha-amylase is particularly significant in both industorial production and scientific investigation. In this project, in order to explore the way of using P. pastoris as bioreactor to express extremophilic alpha-amylases, two non-coincident acidothermophilic alpha-amylases were expressed in Escherichia coli and Pichia pastoris, respectively, and then characterization of the recombinant alpha-amylases were described.Alpha-amylase AMY from the Gram-positive Alicyclobacillus acidocaldarius is one kind of acidothermophilic enzyme, with the optimal temperature and pH of 75℃ and 3. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3903bp long, comprising one open reading frame encoding a polypeptide of 1301amino acids. The calculated molecular weight of AMY was about 140kD. The gene amy was expressed in E. coli BL21(DE3) and P. pastoris GS115 respectively, and both of the recombinant proteins had bioactivity.rAMY expressed in P. pastoris was further purified and characterized. The apparent molecular weight of rAMY was about 160kD according to SDS-PAGE. When rAMY hydrolyzed soluble starch, the optimal pH value was 3.2, which is the same as that of the native enzyme. However, the optimum of temperature was 65℃, a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes at 70 ℃. Anyway rAMY expressed in P. pastoris is acidothermophilic.Based on analysis of AMY amino acid sequence and homology model of its catalytic domain, the middle segment of the gene amy, which ranged from +1174 bp to +3288 bp, was cloned by PCR. The truncated gene amy' encoded 705 amino acids with the calculated molecular weight of 79kD. amy' was expressed in E. coli BL21(DE3) induced by 1mmol/L IPTG, and the expressed enzyme also retained α-amylase activity. So the truncated peptide AMY' should include essential amino acid sequences which can fold catalytic domain.Acidohermophlic alpha-amylase BD5063 is found in Thermococcus sp.. The encoding gene BD5063 was reconstructed for the optimal expression in P. pastoris. Modification was based on unequal usage of synonymous codon of high-level expressed genes in P. pastoris. The gene BD5063r consisted of 1299bp, encoding 433 amino acids with the calculated molecular mass 49kD. The recombinant a-amylase rBD5063 was secreted by P. pastoris up to a yield of 60mg/L. TLC results for the various hydrolysis products of soluble starch showed that rBD5063 cleaved alpha-1,4-glucosidic linkages, and most products were glucose, maltose, and other oligosaccharides.rBD5063 was purified and characterized. When rBD5063 hydrolyzed soluble starch, the optimalpH was 5.0 and more than 50% of its maximal activity remained after treatment in the buffers ranged from pH 3.5-10.0 for 30 minutes. The optimum temperature was 100-110°C, and the half-life at 90°C, pH 5.0 was 30 minutes. Low content of Ca + is enough to activate and stabilize its activity, and even up to 20mmol/L, Ca2+ only affected its activity slightly. Mg2+, SDS and Trition X-100 partially inhibited its activity, while Cu2+ and EDTA thoroughly inhibited its activity.Most rBD5063 was inclined to aggregate into bioactive polymers form, which would be disaggregated into three bioactive proteins with molecular weight of 64, 57, 44kD, respectively. rBD5063 is not modified by N-linked glycosyiation but by O-linked glycosyiation. The O-linked glycosyiation had little influence on thermostability and the optimal temperature of rBD5063.In conclusion, two acidothermophilic alpha-amylases were high-level expressed in P. pastoris. Those recombinant alpha-amylases expressed in P. pastoris held essential characterization of native alpha-amylases. As a result, P. pastoris has been of great value as the bioreactor to produce extremophilic alpha-amylases.