Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

• Published in: Biochimica et biophysica acta Vol. 1824; no. 7; pp. 919 - 924
• Main Authors: Suzuki, Ryuichiro, Terasawa, Kazue, Kimura, Keitarou, Fujimoto, Zui, Momma, Mitsuru, Kobayashi, Mikihiko, Kimura, Atsuo, Funane, Kazumi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2012
• Subjects: Amino Acid Sequence; Aspartic Acid - chemistry; Aspartic Acid - genetics; Bacillus - chemistry; Bacillus - enzymology; Bacillus circulans; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Catalytic residue; Chromatography, High Pressure Liquid; Cyclodextran; Cyclodextrins - biosynthesis; Cycloisomaltooligosaccharide; Cycloisomaltooligosaccharide glucanotransferase; Dextran; Dextrans - metabolism; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Glutamic Acid - chemistry; Glutamic Acid - genetics; glycoside hydrolase; Hot Temperature; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Molecular Sequence Data; Paenibacillus; Paenibacillus - chemistry; Paenibacillus - enzymology; proteomics; Reaction mechanisms; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity; Thermal stability; CITase; SmuDXA; DP; Cycloisomaltooligosaccharide; CBM; CITase-598K; IG3; IG2; IG5; IG4; IG7; IG6; HPLC; IG; CITase-T3040; GH; CI; PsDex; Cyclodextran; EDTA; EGTA; SDS-PAGE; Catalytic residue; PCR; Cycloisomaltooligosaccharide glucanotransferase
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2012.04.001

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the kcat/KM value of CITase-598K was approximately two times higher (32.2s−1mM−1) than that of CITase-T3040 (17.8s−1mM−1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes. [Display omitted] ► CITse-598K is a novel cycloisomaltooligosaccharide glucanotransferase. ► CITase-598K exhibited higher activity and thermal stability. ► The major product of CITase-598K was cycloisomaltoheptaose. ► The minimum size of substrate for CITase is isomaltotetraose. ► Asp144, Asp269, and Glu341 of CITase-598K are catalytically important.