Production of recombinant bacterial alpha amylase in Pichia pastoris

α-amylase which is one of the most important and commonly used forms of industrial amylases, is a biocatalyst preferred in many fields such as food, pharmaceutical, textile, detergent, biofuel production and treatment of contaminated water. α-amylases (α-1,4-D-glucan glucanohydrolase, EC 3.2.1.1), catalyze the hydrolysis of starch by acting on the inner parts of α-(1,4)-glucosidic bonds in the starch molecule. As a result, α-amylases create of monomers and polymers of different lengths with α-conformation such as glucose, maltose, maltotriose and α-limit dextrins. α-amylases are commonly found in many plants, animals, bacteria and fungi. However, depending on the need in applications, recombinant strains are preferred for the production of high efficiency enzymes and to obtained of α-amylases having desirable properties in industrial conditions. Pichia pastoris (Komagataella phaffii) is a methylotrophilic yeast which is used in commonly recombinant protein production. Despite P. pastoris has several promotors, ADH2 (alcohol dehydrogenase) promoter which is responsible for ethanol metabolism is remarkable because of high expression level. In this study, Bacillus subtilis PY22 α-amylase gene (AmyE); the sequence encoding the full-length and mature form was ligated into the expression vector containing the synthetic ADH2 (ADH2SNT5) promoter and was cloned to P. pastoris GS115 strain. The obtained clones were selected P. pastoris GS115-pADH2SNT5α-α-Amylase-Full Lenght (FL) and P. pastoris GS115-pADH2SNT5α-α-Amylase-Mature (MF) production clones which were determined single copy by Real-Time Polymerase Chain Reactions (RT-PCR) analysis. This clones (FL and MF) were produced recombinant protein by inducing with ethanol during 96 hours in shake flasks. The highest α-amylase activities of FL clone and MF clone are 74.2±0.94 U/ml and 215±3.18 U/ml, respectively. Due to approximately 3-fold difference between the production of FL and MF clones, the MF clone was chosen for large scale production. The MF clone was induced with ethanol according to the constant µ (µ: 0.028 sa-1) strategy, at 28°C, pH 6.0 and normoxic conditions (dissolved oxygen %20), in 5 L volume bioreactor and α-amylase production was carried out during 90 hours. As a result of the large scale production of MF clone, α-amylase enzyme was increased in time and the highest α-amylase activity (90th hour) was calculated 2219±52.18 U/ml (1403 U/mg). When the production of bioreactor and shake flask were compared, it was observed that α-amylase enzyme increased approximately 10 times. The characterization of the recombinant α-amylase enzyme was carried out and according to SDS-PAGE analysis, the molecular weight of the protein was detected approximately 80 kDa. The optimum working conditions of the enzyme were determined temperature of 60°C and pH 7.0. Consequently, incubation of the α-amylase at different temperatures for a hour, it was found that 80% and 65% activity was maintained at 50°C and 60°C, respectively. The enzyme activity was stable above 80% at pH 6.0-7.5 during a hour. The α-amylase activity was increased by Ca2+ and Mg2+ ions at 10 mM concentration but it was decreased by Fe2+, Zn2+ and Cu2+ ions at the same concentration. As a result of this study, it was observed that recombinant bacterial α-amylase enzyme was successfully expressed in large scale production under the control of ADH2 promoter, which is strong and can used as alternative in P. pastoris expression system.