Expression of α-Galactosidases from Thermoascus crustaceus JCM12803 in Pichia pastoris

doi:10.13560/j.cnki.biotech.bull.1985.2016-1083

Abstract

Abstract:

This work is to clone the α-galactosidase gene from Thermoascus crustaceus JCM12803 and systematically study its enzymatic properties for obtaining high-quality α-galactosidase in industry. The gene sequence of α-galactosidase was cloned from T. crustaceus JCM12803 by RT-PCR and its enzymatic properties were systematically analyzed. As results revealed,the full-length of tcgal27A belonging to glycoside hydrolase 27 was 1 918 bp,contained 4 introns,the cDNA of tcgal27 was 1 419 bp,and encoded 472 amino acids. SignalP analysis indicated 24 residues in the N-terminal of tcgal27 might be signal peptides. Recombinant TCGal27A successfully expressed in Pichia pastoris had a high specific activity（336.5 U/mg）,and the broad substrate specificity,i.e.,presenting different degrees of degradation to melibiose（14.4 U/mg）,raffinose（9.1 U/mg）,gum tragon（3.6 U/mg）,konjaku flour（1.6 U/mg）,guar gum（1.3 U/mg）,stachyose（0.7 U/mg）. Using pNPG as the substrate,the Km and Vmax of TCGal27A were determined to be 1.6 mol/mL and 536.8 μmoL/（min·mg）_,respectively. Like most fungal a-galactosidases,TCGal27A had an optimal acidic pH 4.5. Purified recombinant TCGal27A was thermophilic,exhibiting the maximum activity at 65℃,and the enzyme remained 86% of initial activity at 50℃ for 1 h. All above results imply that heat-tolerant protein TCGal27A with excellent enzymatic properties can be additive for feedstuff,and will be solid potential applicable value in digesting and improving the energy utilization ratio of soybean meal protein.

Key words: Thermoascus crustaceus JCM12803, α-galactosidase, thermophilic, heterologous expression

ZHANG Duo-duo, ZHENG Fei, LUO Hui-ying, LI Zhong-yuan, LUO Xue-gang. Expression of α-Galactosidases from Thermoascus crustaceus JCM12803 in Pichia pastoris[J]. Biotechnology Bulletin, 2017, 33(6): 207-213.

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