Kyo Shimada

Nucleotide Sequence of the Clostridium stercorarium xylA Gene Encoding a Bifunctional Protein with β-D-Xylosidase and α-L-Arabinofuranosidase Activities, and Properties of the Translated Product

The nucleotides of the β-xylosidase (xylA) gene from Clostridium stercorarium were sequenced. A single open reading frame of 473 codons specifying the subunit (MW 53,340) of xylosidase was identified. The N-terminal amino acid sequence and molecular weight estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme were quite in agreement with those deduced from the nucleotide sequence. Analysis of the enzyme by gel filtration on an HPLC column gave a molecular weight of 220,000, suggesting that the native enzyme is a tetramer composed of 4 identical subunits. The pH optimum was 7.0 and quite stable over the pH range of 5 to 10 at 4°C. The optimum temperature was 65°C. Vm was estimated to be 5.9nmol/min/μg for p-nitrophenyl-β-D-xylopyranoside and 16.7nmol/min/μg for p-nitrophenyl-α-L-arabinofuranoside, while Km was estimated to be 2.5 mM for p-nitrophenyl-β-D-xylopyranoside and 17.6 mM for p-nitrophenyl-α-L-arabinofuranoside.

Cloning and sequencing of a novel endo-1,4-β-glucanase gene from Ruminococcus albus

Abstract The gene encoding an endo-1,4-β-glucanase from Ruminococcus albus F-40 was cloned in Escherichia coli JM109 using pBR322. The nucleotide sequence of the 1,798 bp PstI-PvuII fragment which includes a cellulase gene was determined. There was a single open reading frame (ORF) consisting of 936 bp encoding a peptide of 312 amino acid residues with a molecular weight of 35,766. The N-terminal amino acid sequence determined for the enzyme expressed in E. coli was identical to that deduced from the beginning of the ORF. A putative ribosome-binding site and a promoter were located upstream of the ORF. Activity was expressed from this fragment when it was subcloned in both orientations in pUC118 and pUC119, indicating that its own promoter functioned in E. coli. The amino acid sequence of the endoglucanase deduced from the nucleotide sequence showed 44% homology with CelA from Butyrivibrio fibrisolvens A46, suggesting that this enzyme was a member of family A2. The enzyme purified from E. coli exhibited the highest activity against carboxymethyl cellulose (CMC) at 40°C and pH around 7.0.

Effects of EDTA on Alkaline-Protease of Aspergillus oryzae

麹菌のアルカリ性プロテアーゼに対するEDTAの影響を試験した (1)アルカリ性側のpHにおけるほど失活を蒙り易い. (2) 温度が高いほど容易に,かつ強い失活を受ける. (3) Na+その他の1価のカチオンの存在は酵素を失活から保護する効果がある. (4) EDTAによって失活を受けた酵素は, Co2+その他の金属イオンによって活性を回復しない.