Cloning, Sequencing, and Expression of the Genes Encoding an Isocyclomaltooligosaccharide Glucanotransferase and an α-Amylase from aBacillus circulansStrain

Abstract

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-alpha-maltosyltransferase, alpha-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to alpha-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

“…BaSBD1 and BaSBD2 as well as sugar binding module from B. halodurans maltohexaose-forming amylase were classified as CBM25 family [18,19,21]. CBM25 member proteins adopt ␤-sandwich folds in the 3-dimentional structures and were found as distinct domain of several amylolitic enzymes [34][35][36].…”

Two potentially novel amylolytic enzyme specificities in the prokaryotic glycoside hydrolase α-amylase family GH57

“…It catalyses the hydrolysis of the internal a-1,4-glucosidic bonds in starch and related poly-and oligosaccharides (Janeček et al, 2013). Based on their amino acid sequence similarities, a-amylases have been classified into three different glycoside hydrolase (GH) families in the Carbohydrate-Active enZyme (CAZy) server (Cantarel et al, 2009): (i) family GH13 -the main a-amylase family, established more than 20 years ago (Henrissat, 1991;Jespersen et al, 1991;Takata et al, 1992), with more than 14 000 sequences and~30 different enzyme specificities from Bacteria, Archaea and Eukarya, forming together with families GH70 and GH77 the a-amylase clan GH-H (MacGregor et al, 2001); (ii) family GH57 -the second and smaller a-amylase family, established in 1996 (Henrissat & Bairoch, 1996), containing a few amylolytic specificities from prokaryotes only, often from extremophiles (Janeček & Blesák, 2011;Blesák & Janeček, 2012); and (iii) family GH119 -created after the study by Watanabe et al (2006) describing the protein product of the igtZ gene as an a-amylase, and recently indicated to be very closely related to family GH57 (Janeček & Kuchtová, 2012). A recently published structure of the amylase from Clostridium perfringens indicated the presence of a-amylase specificity even in family GH126 (Ficko-Blean et al, 2011), but owing to its obvious homology with inverting bglucan-active hydrolases more biochemical characterization is required to confirm the exact enzyme specificity (Janeček et al, 2013).…”

Two structurally related starch-binding domain families CBM25 and CBM26

“…there are examples of other amylolytic families, such as the α-amylase family GH119 (Watanabe et al 2006;Janecek & Kuchtova 2012) and β-amylase family GH14 (Siggens 1987). Interestingly, the representative of related α-glucosidase family GH31 originates from a eukaryotic fungus responsible for potato late blight (Haas et al 2009).…”