Characterization and molecular cloning of secreted α-amylase with dominant activity from mon thong durian (Durio zibethinus murr. cv. mon thong)

• Published in: Revista Brasileira de Fruticultura 2021-01, Vol.43 (3)
• Main Authors: Posoongnoen, Saijai, Ubonbal, Raksmont, Klaynongsruang, Sompong, Daduang, Jureerut, Roytrakul, Sittiruk, Daduang, Sakda
• Format: Article
• Language: English
• Subjects: characterization; Durio zibethinus Murr. cv. Mon Thong; Escherichia coli; HORTICULTURE; secreted α-amylase
• ISSN: 0100-2945; 1806-9967; 1806-9967
• DOI: 10.1590/0100-29452021231

Abstract The secreted α-amylase with dominant activity was purified from the crude extract of Mon Thong durian by steps of ammonium sulphate precipitation and the affinity column chromatography. The purified α-amylase (DzAmy1) had a molecular mass of approximately 44 kDa. Its optimum pH and temperature for activity were 7.0 and 50°C, respectively. The enzyme was stable from pH 6 to 10 and from 30 to 60°C. Many metal ions did not affect amylase activity. The gene cloning of DzAmy1 was carried out and it was confirmed that DzAmy1 gene consisted of 1,254 bp open reading frame, which encoded 23 amino acids of the signal peptide and 395 amino acids of mature protein with a calculated molecular mass of 43.7 kDa. The isoelectric point of the enzyme was 5.78. DzAmy1 was shown to belong to sub-family one of the plant α-amylases based on phylogenetic tree analysis. Structural characterization by homology modelling suggested that it consisted of 3 domains with a catalytic triad in domain A. Recombinant DzAmy1 (rDzAmy1) was successfully expressed in Escherichia coli and had hydrolysis activity for starch and ethylidene-pNP-G7, which was clearly confirmed the authenticity of DzAmy1 as a functional α-amylase. Resumo A α-amilase secretada, com atividade dominante, foi purificada a partir do extrato bruto de Mon Thong durian através de passos de precipitação utilizando sulfato de amônia e cromatografia de afinidade. A α-amilase purificada (DzAmy1) tem uma massa molecular de aproximadamente 44 kDa. O pH e temperatura ótimos para a atividade foram 7.0 e 50°C, respectivamente. A enzima ficou estável no pH entre 6 a 10 e temperatura entre 30 a 60°C. Vários íons metais não afetaram a atividade amilásica. A clonagem do gene DzAmy1 foi feita e confirmou que esse gene consiste em 1.254 bp de região codificante, na qual se codificam 23 aminoácidos de peptídeo sinal e 395 aminoácidos da proteína madura com massa molecular calculada de 43.7 kDa. O ponto isoelétrico da enzima é de 5.78. A DzAmy1 pertence a subfamília das α-amilases de planta, de acordo com dados de filogenia. A caracterização estrutural através de modelagem por homologia sugeriu que a DzAmyl consiste em 3 domínios, com sua tríade catalítica presente no domínio A. A DzAmy1 recombinante (rDzAmy1) foi expressa em Escherichia coli com sucesso e teve sua atividade de hidrólise de amido e etilideno-pNP-G7, as quais claramente confirmaram a autenticidade da DzAmy1 como uma α-amilase funcional.