Rhizopus Oryzae CICIM F0071 ?-amylase: Gene Cloning, Identification And Heterologous Expression

Fungal?-amylase is one of the most important enzymes used in industry, which hydrolyzes starch by cleaving internal?-1, 4-glucosidic linkages and the main hydrolyzed product is maltose. Fungal?-amylase has a vast application in the modern industries of starch syrup, baking, brewing and fuel alcohol.The study of fungal?-amylase in our country has fallen behind and almost all of the researches have no relate to gene cloning and expression. The traditional solid-state fermentation has not converted to liquid-state fermentation and also the production level is much lower than the international advanced level. The commercial fungal?-amylase, which is a mixture of many?-amylases, glucoamylase and glucose glycosidase, can not be effectively applied in the production of some special products. Therefore, with the aim to study and develop new type fungal?-amylase with special characteristics, the?-amylase gene of Rhizopus oryzae (CICIM F0071 and CICIM F0072) was cloned and functional identified. One of the?-amylase genes was obtained heterologous expression in several eukaryotic expression systems. The recombinant R. oryzae?-amylase was purified and characterized. The main results are as follows:(1) Two putative fungal?-amylase genes from R. oryzae strains were cloned, sequenced and functional expressed. The size of two?-amylase coding sequences is both 1389 bp and with no intron existed. The two genes have a disimilarity of 3.02% and the disimilarity between the gene products was 1.95%. The gene products belong to the amylase family 13 and have four classical highly conserved regions that observed in most?-amylases. Both RoAmy1 and RoAmy2 shared the highest amino acid similarity (42.8%) with other?-amylases. Recombinant plasmids for expression of the cloned?-amylase genes were constructed based on the plasmid vector pET-28a (+) and the highest expression levels obtained in E. coli for RoAmy1 and RoAmy2 were 1.3 U/mL and 0.5 U/mL, respectively.(2) The recombinant?-amylases were purified and subsequently characterized. The recombinant?-amylases were purified through affinity chromatography, the molecular weight of the purified enzymes determined by SDS-PAGE was approximately 48 kDa. The two enzymes exhibited similar properties. The optimal pH and temperature were 4.0 to 6.0 and 60°C, respectively. The enzymes were stable at pH ranges of 4.5 to 6.5 and temperatures below 50°C. Purified RoAmy1 has a K_m and V_max of 0.27 mg/mL and 0.068 mg/min, respectively with a specific activity was 1123.4 U/mg on soluble starch, and purified RoAmy2 has a K_m and V_max of 0.27 mg/mL and 0.064 mg/min, respectively with a specific activity was 1118.6 U/mg on soluble starch. Amylase activity was strongly inhibited by Cu²⁺ and Fe²⁺ (5 mmol/L), whereas Ca²⁺ (5 mmol/L) showed no significant effect. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K_m=0.22 mmol/L) to maltotriose. A high ratio of glucose : maltose of 1 : 4 was obtained when maltotriose was used at an initial concentration of 40 mmol/L. (3) A genetic transformation protocol for Aspergillus niger was constructed and R. oryzae?-amylase was successfully expressed in A. niger by using verious constructed expression vectors. Based on the selection maker of Hygromycin B, studies on the effect of germination time, field strength and plasmid concentration on the transformation efficiency revealed that the appropriate transformation conditions for A. niger MGG029-?aamA were: 4 d growth time of spores was suitable, 2 h germination time was preferred and 5 kV/cm of field strength was optimal. By using Hygromycin B as the selection marker, the A. niger expression vectors pPTH-RoAmy, pPglaA-RoAmy and pPglaAs-RoAmy for expressing R. oryzae?-amylase under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpdA) and the glucoamylase (glaA) promoters of A. niger were constructed, and the highest?-amylase expression levels obtained were around 14-15 U/mL. By comparing the intracellular and extracellular amylase activities, we concluded that the R. oryzae?-amylase could successfully expressed in A. niger under the direction of signal sequences of glucoamylase and the R. oryzae?-amylase, respectively, and the expression levels were almost the same. When R. oryzae?-amylase was expressed in A. niger strain F0521, the relative amylase expression levels were around 45 U/mL.(4) The expression of R. oryzae?-amylase was studied in several yeast expression systems and achieved a high expression level in Pichia pastoris. The expression of RoAmy in S. cerevisiae and Kluyveromyces lactis was investigated. Under shake flask fermentation, the higest?-amylase production levels obtained by the recombinant S. cerevisiae W303-1A and K. lactis was 1.3 U/mL and 22.4 U/mL, respectively. P. pastoris strains with the capability to express recombinant?-amylase under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) and the methanol-inducible alcohol oxidase 1 (AOX1) promoters were obtained. The levels of inducibly expressed?-amylase were higher than those expressed constitutively, especially for the Mut+-phenotype strains, in which the maximal inducible?-amylase expression levels (41 U/mL) were approximately 8 times higher than those expressed in the MutS-phenotype strains and 24 times higher than those expressed under the control of the GAP promoter. In both inducible and constitutive expression modes, the S. cerevisiae?-prepro sequence and the native signal sequence of R. oryzae?-amylase were used separately for directing the secretion of?-amylase into the culture medium of P. pastoris. Low levels of intracellular amylase activities detected at the end of shake flask fermentation indicated that both of the signal sequences could effectively direct the secretion of the enzyme under all studied conditions; moreover, the secretion levels of the enzyme directed with its own signal peptide were 7–10% higher than the levels directed by the?-prepro sequence. Scale up fermentation conditions based on basal salts medium in a 15 L fermenter were adjusted to 30°C, pH 6.0, inoculum size 10% (v/v), the flow rate of methanol was controlled by DO-Start mode and the highest amylase expression level obtained by the combining use of AOX1 and GAP promoters was 462.8 U/mL (0.42 mg/mL), which was about 9 times higher than that achieved in shake flask cultures.