AN -AMYLASE OF A CORAL-REEF BACTERIUM B. AQUIMARIS MKSC 6.2: A NOVEL RAW-STARCH-DEGRADING ENZYME

• Main Author: Puspasari, Fernita
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/54657
• Institution: Institut Teknologi Bandung

Amylolytic enzymes are involved in carbohydrate metabolism of various organisms. One of the widely used amylolytic enzymes is ????-amylase (EC 3.2.1.1). Plant ????-amylases are used in seed germination, whereas in animals they contribute to the digestive system. Various bacteria secrete ????-amylases to hydrolyze starch from its environment to produce maltooligosaccharides that can be used for cells. Bacterial ????-amylases have been widely studied, especially those from the genus Bacillus. ????-Amylase belongs to family glycoside hydrolase 13 (GH13). Three-dimensional structures of GH13 enzymes at least consist of three domains, namely domain A, B, and C. Domain A has the folding of (????/????)8-barrel, domain B is a loop between ????3-strand and ????3-helix of domain A, whereas domain C lies on C-terminal end of the polypeptide. ????-Amylase are widely used in starch processing industries. Conventional starch processing requires gelatinization step at high temperature to open up the starch granule stucture prior to the addition of thermostable ????- amylase. Starch hydrolysis can be performed without gelatinization step if the thermostable ????-amylase is replaced by a raw-starch-degrading ????-amylase. Currently, information of raw-starch-degrading ????-amylase derived from marine bacteria is very limited. One of coral-reef bacteria that is associated with soft-coral Sinularia sp. from Merak Kecil Island, Banten, namely MKSC 6.2 isolate, produces raw-starchdegrading enzymes. The aims of this study were to identify MKSC 6.2 isolate, to determine biochemical properties of its amylolytic enzymes, to discover an open reading frame (ORF) encoding ????-amylase, to express ????-amylase gene in Escherichia coli, and to determine the ability of recombinant ????-amylase to degrade raw starch. Molecular identification of MKSC 6.2 isolate by using 16S rRNA gene sequence homology analysis method suggested that the isolate had the closest relationship with Bacillus aquimaris MSU1110 with homology of 99.9%. Identification based on its physiological and morphological properties indicated that MKSC 6.2 isolate belongs to the group of Bacillus aquimaris. Amylolytic enzymes are involved in carbohydrate metabolism of various organisms. One of the widely used amylolytic enzymes is ????-amylase (EC 3.2.1.1). Plant ????-amylases are used in seed germination, whereas in animals they contribute to the digestive system. Various bacteria secrete ????-amylases to hydrolyze starch from its environment to produce maltooligosaccharides that can be used for cells. Bacterial ????-amylases have been widely studied, especially those from the genus Bacillus. ????-Amylase belongs to family glycoside hydrolase 13 (GH13). Three-dimensional structures of GH13 enzymes at least consist of three domains, namely domain A, B, and C. Domain A has the folding of (????/????)8-barrel, domain B is a loop between ????3-strand and ????3-helix of domain A, whereas domain C lies on C-terminal end of the polypeptide. ????-Amylase are widely used in starch processing industries. Conventional starch processing requires gelatinization step at high temperature to open up the starch granule stucture prior to the addition of thermostable ????- amylase. Starch hydrolysis can be performed without gelatinization step if the thermostable ????-amylase is replaced by a raw-starch-degrading ????-amylase. Currently, information of raw-starch-degrading ????-amylase derived from marine bacteria is very limited. One of coral-reef bacteria that is associated with soft-coral Sinularia sp. from Merak Kecil Island, Banten, namely MKSC 6.2 isolate, produces raw-starchdegrading enzymes. The aims of this study were to identify MKSC 6.2 isolate, to determine biochemical properties of its amylolytic enzymes, to discover an open reading frame (ORF) encoding ????-amylase, to express ????-amylase gene in Escherichia coli, and to determine the ability of recombinant ????-amylase to degrade raw starch. Molecular identification of MKSC 6.2 isolate by using 16S rRNA gene sequence homology analysis method suggested that the isolate had the closest relationship with Bacillus aquimaris MSU1110 with homology of 99.9%. Identification based on its physiological and morphological properties indicated that MKSC 6.2 isolate belongs to the group of Bacillus aquimaris. Three-dimensional structure model of BaqA showed that it has no starch binding domain commonly found in a raw-starch-degrading enzyme. In BaqA, the role of raw-starch binding is proposed to be played by the two consecutive tryptophan residues (Trp201 and Trp202) and triad (Asp246, Arg247, and Asp248) located in domain A; and a ‘sugar-tongs’ residue (Tyr400) in domain C. Its domain C structure which consists of five antiparallel ????-strands is predicted to have an important role for raw-starch binding.