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The nucleotide sequence of the Clostridium stercorarium F-9 xynC gene, encoding a xylanase XynC, consists of 3,093 bp and encodes a 1,031-amino acids with a molecular weight of 115,322. XynC is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family IX cellulose-binding domain, and two S-layer homologous domains. Immunological analysis indicated the presence of XynC in the culture supernatant of C. stercorarium F-9 and in the cells, most likely on the cell surface. XynC purified from a recombinant E. coli was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethylcellulose. XynC hydrolyzed xylan and xylooligosaccharides larger than xylotriose to produce xylose and xylobiose. This enzyme was optimally active at 85 degrees C and was stable up to 75 degrees C at pH 5.0 and over the pH range of 4 to 7 at 25 degrees C.

Chitinase B (ChiB) was purified from the culture supernatant of Xanthomonas sp. strain AK by Phenyl-Toyopearl 650M and DEAE-Toyopearl 650M column chromatographies. The purified enzyme preparation gave a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight of ChiB was estimated to be 48,000. The enzyme was optimally active at pH 6.0 and 60°C. N-Terminal amino acid sequence analysis suggested that ChiB is a member of glycosyl hydrolase family 18 and that it is genetically different from ChiA recently reported (Sakka et al., J. Ferment. Bioeng., 86, 527–533, 1998). Immunological analysis suggested that ChiB was the major chitinase species in the culture supernatant of Xanthomonas sp. strain AK and that production of the enzyme was induced by the presence of chitin.

L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE. The absorption spectrum of the hydroxylapatite column-purified GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDH-ase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo. N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined. The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene. The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa. The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms. The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently. We conclude that GLDHase, L-gulono-gamma-lactone oxidase and L-galactono-gamma-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors. Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.

Restriction endonuclease Ral F40I was purified from cell-free extracts of the rumen cellulolytic bacterium Ruminococcus albus F-40 by heparin-Sepharose chromatography. The preparation was active only on DNA substrates that were not Dam-methylated. Ral F40I recognizes the 4-bp palindrome, 5′-/GATC-3′, and cleaves DNA at the 5′ side of G in the sequence, producing 5′ tetranucleotide protruding ends. Ral F40I is a class II restriction endonuclease and an isoschizomer of Mbo I and Dpn II.

The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. The pH and temperature optima of the enzyme were 6.0 and 45 degrees C, respectively. The Km and Vmax values for 4-MU-(GlcNAc)2 were estimated to be 6.3 microM and 46 micromol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin.

The Clostridium thermocellum endoglucanase CelJ contains two different catalytic domains in a polypeptide, i.e., a subfamily E1 catalytic domain and a family J catalytic domain [J. Bacteriol., 178, 5732-5740 (1996)]. The family J catalytic domain (CDJ-CelJ) was produced by a recombinant Escherichia coli and purified. The purified CDJ-CelJ gave a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight of this enzyme (60,000) was consistent with the value (60,333) calculated from the DNA sequence. CDJ-CelJ hydrolyzed various cellulosic substrates, xylan, and lichenan but not p-nitrophenyl (PNP)-cellobioside, PNP-glucoside, or PNP-xyloside at all. CDJ-CelJ was active on Avicel, a microcrystalline cellulose, and the specific activity of CDJ-CelJ on Avicel (0.0078 U/mg protein) was comparable to that of CelS, which is recognized as the most important catalytic subunit of the C. thermocellum, cellulosome, suggesting that CelJ is also an important catalytic subunit in the cellulosome of this bacterium, in addition to CelS.

The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The structural gene consists of an open reading frame of 1533 bp encoding a protein of 511 amino acids with an M(r) of 56,519. XynA consists of a catalytic domain belonging to family G at the NH2-terminus and two direct repeats of about 90 amino acids with a short spacing at the COOH-terminus. The repeated sequences, CBDI and CBDII, were not homologous with amino acid sequences of the CBDs classified into families I to V. Nevertheless, XynA showed an affinity for insoluble cellulose such as Avicel. Binding of XynA to Avicel was strongly dependent on the concentration of the incubation buffer and was inhibited by Triton X-100. XynA bound to Avicel (2.4 nmol/g-cellulose) and acid-swollen cellulose (180 nmol/g-cellulose), suggesting that this enzyme has higher affinity for amorphous cellulose than for crystalline cellulose. Functions of CBDI and CBDII were investigated by constructing the mutant enzymes and evaluating the cellulose-binding ability of each of them. XynA4 lacking CBDI and XynA5 lacking CBDII bound to Avicel to a lesser extent than the parental enzyme XynA; but XynA6, devoid of both CBDs, did not bind at all, indicating that CBDI and CBDII each functioned independently as CBD in XynA and their binding capacity was additive. Although the Ruminococcus albus endoglucanase EgIV that was joined to CBDs of XynA acquired cellulose-binding ability, the substrate specificity of EgIV was not altered in the presence or absence of CBDs.

焼酎白麹抽出液からDEAE Bio-Gel Aイオン交換クロマトグラフィー, Sephacryl S-300ゲルクロマトグラフィー(pH3.6),ω-アミノドデシルアガロースクロマトグラフィーおよびSephacryl S-200ゲルクロマトグラフィーを組み合わせた系統的分離精製法により,A. kawachiiのα-アミラーゼIおよびII,グルコアミラーゼII,α-グルコシダーゼ,酸性プロテアーゼおよび酸性カルボキシペプチダーゼを単離精製した. 各精製酵素の分子量は, Sephacryl S-300ゲルクロマトグラフィーおよびSDS-PAGEにより,グルコアミラーゼIIが101,000,α-グルコシダーゼが200,000,酸性プロテアーゼが43,000,そして酸性カルボキシペプチダーゼが215,000と推定された. 最適反応pHは,グルコアミラーゼIIが4.0～5.0,α-グルコシダーゼが4.5～5.5,酸性プロテアーゼが3.0,酸性カルボキシペプチダーゼが3.5であった.グルコアミラーゼIIはpH3.0～8,0,α-グルコシダーゼはpH3.0～7.0,酸性プロテアーゼはpH3.0～6.0,酸性カルボキシペプチダーゼはpH3.0～5.5でそれぞれ安定であった. 最適反応温度は,グルコアミラーゼIIおよびα-グルコシダーゼが70°C,酸性プロテアーゼが60°C,酸性カルボキシペプチダーゼが50°Cであった.グルコアミラーゼIIおよびα-グルコシダーゼは60°C,酸性プロテアーゼおよび酸性カルボキシペプチダーゼは50°Cまで安定であった. また,α-グルコシダーゼはCu2+およびHg2+イオンにより80%以上阻害された.酸性プロテアーゼはCu2+イオンにより1.8倍に活性化され, pepstatinにより95%以上阻害された.酸性カルボキシペプチダーゼはFe3+イオンにより80%以上, monoiodoacetateにより50%程度阻害された. さらに,グルコアミラーゼIIはアミロペクチン,グリコーゲン,可溶性デンプン,デキストリン,アミロースの順に,高分子基質をよく加水分解した.α-グルコシダーゼはマルトースを最もよく水解し,可溶性デンプンの水解速度は非常に遅かった.酸性カルボキシペプチダーゼはカルボキシル末端から2番目のアミノ酸残基が,L-Tyr, L-Phe, L-Leu, L-Aa, L-Gluの順に水解しやすい傾向があった.

The crystal structures of a carbohydrate-binding module (CBM) family 28 domain of endoglucanase Cel5A from Clostridium josui have been determined in ligand-free and complex forms with cellobiose, cellotetraose, and cellopentaose as the first complex structures of this family. In the cleft of a β-sandwich fold, the ligands are recognized by stacking interactions and hydrogen bonds. Conformations of the bound cellooligosaccharides are similar to those in crystals and solution but clearly different from the cellulose structure. Interestingly, the glucan chain bound on CBM28 is in the opposite direction of that bound to CBM17, although these families share significant structural similarity.