Cloning, Expression And Characterization Of Amylase And Agarase Genes From Pseudoalteromonas Species

Glycosidases catalyze the hydrolysis of the glycosidic linkage,α-Amylases are classified as family 13 of the glycosidases;they are among the most important commolercial enzymes,have wide applications in starch processing,brewing,alcohol production, textile,detergent and other industries.Agarases,catalyzing the hydrolysis of agar,are characterized asα-agarases,β-agarases;β-agarases produce neoagarooligosaccharides which have potential applications for the food,cosmetic,pharmaceutical and medical industries.In this study,two glycosidase genes were cloned from Pseudoalteromonas species;they were expressed and characterized.A gene(amyA) encoding an extracellularα-amylase from marine bacterium, Pseudoalteromonas sp.MY-1,was cloned and expressed in Escherichia coli.It comprised an open reading frame of 2,007 base pairs and encodes a protein of 668 amino acids with a predicted molecular weight of 74 daltons and a pI of 5.15.The entire amino acid sequence of amyA gene showed 86%similarity to theα-amylase preproprotein from Pseudoalteromonas haloplanktis.It consists of a signal peptide,α-amylase catalytic domain and an Amy_C domain.The recombinant amylase was purified to homogeneity and biochemically characterized.The enzyme revealed maximum activity at pH 7.0 and 40℃.The enzyme hydrolyzed soluble starch and some maltooligosaccharides to several oligosaccharides,and maltose was the commolon product from different substrates.The results also suggest that Ca²⁺ plays important roles inα-amylase.A gene(agaA6) encoding an extracellular agarase from Pseudoalteromonas sp.JT-6,was cloned,sequenced and expressed in Escherichia coli.It comprised an open reading frame of 1,338 base pairs and encodes a protein of 445 amino acids with a predicted molecular weight of 50 daltons.The entire amino acid sequence of this agarase gene showed 99% identity with the agaA gene from Janthinobacterium sp.SY12.It consists of a signal peptide,a glycoside hydrolase family 16β-agarase domain and a carbohydrate-binding domain.The recombinant agarase was overexpressed and purified to homogeneity. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant enzyme were around 40℃and 9.0.The enzyme was an endo-typeβ-agarase and hydrolyzed agarose to several oligosaccharides.