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Clonagem e expressão heteróloga do gene codificante da α-Amilase de Bacillus licheniformis DSM13 em Pichia pastoris
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Instituto Nacional de Pesquisa da Amazônia - INPA
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Genetic manipulation of microorganisms allows the production of enzymes with high biotechnological value with application in various industrial processes. The enzyme α-amylase from Bacillus licheniformis is widely used in industries to produce alcohol, sugar and beer, and its activity involves hydrolysis of starch cleaving the glycosidic bonds α-(1,4). The methylotrophic yeast Pichia pastoris has been used successfully for heterologous protein expression, becoming an efficient expression system. The objective of this study was to clone and to express the gene encoding α-amylase from Bacillus licheniformis DSM13 in the methylotrophic yeast Pichia pastoris and also to analyze the recombinant protein secreted. The gene was inserted into the genome of P. Pastoris using the pPIC9 vector and after the induction process the protein was produced and secreted by the yeast. The maximum enzyme activity was observed in the supernatant of the clone B8 (345,4 U/mL). Variations in molecular weight of the enzymes among the clones were detected by SDS-PAGE gel analysis. The enzyme showed optimum activity at 70 ºC, pH optimum of 7.0 and its activity remained stable for a period of two months when maintained at 4 °C. The Km and Vmáx apparent values calculated for the crude extract clone A7 were 10.74 mg/mL and 416.66 U/mL, respectively.
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