Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-␤-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95°C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k cat /K m values are 155.2, 76.0, and 9.9 mM ؊1 s ؊1 for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-␤-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrateand end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85°C. It has specific activity of 11.8 U/mg and a K m of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K m for cellobiose (compared to GghA's K m of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.To utilize polysaccharides such as glucans to meet carbon and energy requirements, heterotrophic organisms depend on a catabolic pathway involving the interaction of multiple hydrolytic enzymes, transporter complexes, and regulatory systems coordinating gene expression of pathway-specific proteins (41). During hydrolysis of the homopolymer cellulose, for example, the consortium of catalytic enzymes consists of endoglucanases (1,4-␤-D-glucan 4-glucohydrolase [EC 3.2.1.4]), which randomly hydrolyze internal ␤-1,4 glycosidic bonds; cellobiohydrolases (␤-D-glucan cellobiohydrolase [EC 3.2.1.91]), also referred to as exoglucanases, which remove cellobiose from either the nonreducing or reducing ends of cellooligomers; and ␤-glucosidases (␤-D-glucoside glucohydrolase [EC 3.2.1.21]), which convert cellobiose to glucose. These classes of enzymes have been documented in a number of fungal and bacterial systems (4, 5, 39), while other glucan-catabolyzing enzymes are less well understood.Thermotoga neapolitana, a marine hyperthermophile isolated from geothermally heated biotopes, belongs to the order Thermotogales. T. neapolitana shares with other Thermotogales, specifically Thermotoga maritima, both the capacity to catabolize a wide variety of ␣-and ␤-linked glucans and a fermentative metabolism. While Thermotogales elaborate hydrolases such as amylases, cellulases, glucosidases, galac...

Two thermostable endocellulases, CelA and CelB, were purified fromThermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high V max value and low apparentKm value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside theV max values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding thecelA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA andcelB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.

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Two overlapping cDNA clones have been isolated from bovine adipose tissue by the reverse-transcription-polymerase chain reaction (RT-PCR) method. The combined sequence of the two clones was 1039 bp in length and encoded 345 amino acids. The deduced amino acid sequence of the clones showed 96.5% similarity to that of sheep SCD and more than 88% similarities to other mammalian SCD1s, indicating that the clones are the cDNAs for the bovine SCD1. The transcript size of the bovine SCD1 was about 4.9 kb, the message was detected in the bovine adipose tissues but not in the liver. Female cattle expressed threefold higher levels of SCD1 mRNA than male animals.

BackgroundConcerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine.Results and conclusionWe designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136–162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21–35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.