Molecular Cloning and Sequencing of Raw Starch Degrading Gene from Laceyella sacchari LP175 and Its Functional Expression in Escherichia coli

Abstract

Raw starch degrading enzyme (RSDE) produced by Laceyella sacchari LP175 was purified 14.7 fold to a 40.5 % yield. The first 15 N-terminal amino acids were sequenced and showed 100% homology with α-amylase from Laceyella sp. DS3 and Thermoactinomyces vulgaris. The RSDE gene was functionally annotated with the Laceyella sacchari strain GS1-1 available genome, which showed the presence of a putative gene of 1362 bp encoding 453 amino acids. The RSDE gene was amplified from Laceyella sacchari LP175 genomic DNA and cloned for expression in Escherichia coli, which showed the highest activity on raw cassava starch at pH 6.5 and a temperature of 50 °C. Homology structure analysis revealed the presence of three domains that are conserved among the structures of GH13 α-amylases, where the active and binding sites both play an important role in starch hydrolysis. The recombinant LsA175 could hydrolyze raw cassava starch at below gelatinization temperature, and showed higher efficiency for hydrolysis than commercial α-amylase (Termamyl®) at 50 °C. This shows the possibility for application of recombinant LsA175 at an industrial level, particularly in terms of energy consumption savings.