Yasuyuki Kawabata

Isolation of Bacillus and Paenibacillus Bacterial Strains That Produce Large Molecules of Cyclic Isomaltooligosaccharides

Cyclic isomaltooligosaccharides (CIs) usually consist of 7 to 12 glucose units, although only CI-10 has strong inclusion complex-forming ability. Four Bacillus strains and two Paenibacillus strains were isolated as novel CI-producing bacteria. Among these, five strains produced small amounts of CI-7 to CI-9, but mainly produced CI-10 to CI-12. Larger CIs, up to CI-17, were also identified.

Deletion analysis of regions at the C-terminal part of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040.

Cycloisomaltooligosaccharide glucanotransferase (CITase) belongs to glycoside hydrolase family 66. According to the sequence alignment of enzymes in the same family, we divided the structure of CITase into five regions from the N terminus to the C terminus: an N-terminal conserved region (Ser1-Gly403), an insertion region (R1; Tyr404-Tyr492), two conserved regions (R2; Glu493-Ser596 and R3; Gly597-Met700), and a C-terminal variable region (R4; Lys701-Ser934). CITase catalyzes the synthesis of cycloisomaltooligosaccharides (CIs) with 7-17 glucose units (CI-7 to CI-17) from dextran. In order to clarify the functions of these C-terminal regions (R1-R4), we constructed 15 deletion mutant enzymes. M123Δ (R4-deleted), MΔ234 (R1-deleted), and MΔ23Δ (R1/R4-deleted) catalyzed CI synthesis, but other mutants were inactive. M123Δ, MΔ234, and MΔ23Δ increased their K(m) values against dextran 40. The wild-type enzyme and M123Δ produced CI-8 predominantly, but MΔ234 and MΔ23Δ lost CI-8 production specificity. The k(cat) values of MΔ234 and MΔ23Δ decreased, and these mutants showed narrowed temperature and pH stability ranges. Our deletion analysis suggests that (i) R2 and R3 are crucial for CITase to generate an active form; (ii) both R1 and R4 contribute to substrate binding; and (iii) R1 also contributes to preference of CI-8 production and enzyme stability.

Production of Arabinose from Corn Hull and Beet Pulp by High Pressure Steaming.

食品加工副産物であるコーンハルおよびビートパルプからの高温蒸煮によるアラビノースの効率的な生産法を検討した.その結果,コーンハルを処理圧力2～15Hkg/cm2で蒸煮すると,コーンハルから最大7.9%のアラビノースが遊離した.これはコーンハル中のアラビノースの63%に相当した.コーンハルの高圧蒸煮の際に燐酸(終濃度0.1M)を添加するとにより,アラビノースの生成量が増加し,その生成量はコーンハル中のアラビノースの82%に相当した.一方,ビートパルプを2～15kg/cm2で蒸煮すると,ビートパルプから最大6.6%のアラビノースが遊離した.これはビートパルプ中のアラビノースの30%に相当した.ビートパルプの高圧蒸煮においても燐酸(終濃度0.1M)を添加することにより,アラビノースの生成量が増加し,ビートパルプから最大16%のアラビノースが生成した.その生成量はビートパルプ中のアラビノースの72%に相当した.