Yukio Takii

Alkaline serine protease produced from citric acid by Bacillus alcalophilus subsp. halodurans KP 1239

SummaryMaximum production of alkaline serine protease by Bacillus alcalophilus subsp. halodurans KP 1239 was achieved after 24 h cultivation, at an initial pH of 7.6, on a medium containing 1.0% sodium citrate, 0.3% yeast extract, and 0.3% KH2PO4. The enzyme was purified to crystalline form from culture broth. The enzyme was most active at 60° C and at pH 11.5. The molecular weight, isoelectric point and sedimentation coefficient in water at 20° C were estimated as 29 000, 8.8 and 3.3S, respectively. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-. The enzyme shared its antigenic determinants with B. alcalophilus ATCC 21522 serine protease, but not with the subtilisins Carlsberg and BPN′.

Bacillus stearothermophilus ATCC12016 α-glucosidase specific for α-1,4 bonds of maltosaccharides and α-glucans shows high amino acid sequence similarities to seven α-d-glucohydrolases with different substrate specificity

We have sequenced the gene encoding Bacillus stearothermophilus ATCC12016 α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) specific for non-reducing terminal α-1,4 bonds of maltosaccharides and α-glucans. The amino acid sequence of the enzyme deduced from the nucleotide sequence of the gene (1665 base pairs) consisted of 555 residues with a molecular mass of 65233. The enzyme showed 40%–57% sequence similarities to α-d-glucohydrolases with very different substrate specificity, such as Bacillus cereus ATCC7064 oligo-1,6-glucosidase, Bacillus thermoglucosidasius KP1006 oligo-1,6-glucosidase, Saccharomyces carlsbergensis CB11 α-glucosidase, Bacillus sp. F5 α-glucosidase, Streptococcus mutans (Ingbritt strain) dextran glucosidase, Bacillus sp. SAM1606 α-glucosidase and Escherichia coli ECL116 trehalose-6-phosphate hydrolase. All these enzymes had sequences equivalent to secondary elements revealed in B. cereus oligo-1,6-glucosidase by X-ray crystallography. We have suggested that the B.stearothermophilus enzyme adopts the same polypeptide folding, i.e. an (α/β)8-barrel in the N-terminal active-site domain, as the B.cereus enzyme and other α-glucohydrolases.

Bacillus stearothermophilus KP 1236 neutral protease with strong thermostability comparable to thermolysin

SummaryAn extracellular neutral protease, of Bacillus stearothermophilus KP 1236 (a soil isolate) able to grow at 39°–71°C was purified to homogeneity. The molecular weight, sedimentation coefficient in water at 20°C, and isoelectric point were estimated as 33,000, 3.46 S and 7.5, respectively. The enzyme was most active at 80°C and pH 7.0. The activity was stable for 10 min up to 80°C at pH 7.5 and for 18 h at 60°C over pH 6.0–8.8. The enzyme and thermolysin (microbial metalloproteinase, EC 3.4.24.4) shared their antigenic determinants in part.

Cloning and expression of a thermostable exo-α-1,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli

The gene coding for a thermostable exo-α-1,4-glucosidase (α-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exo-α-1,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62 000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.

A simplified method of the purification of Bacillus cereus ATCC 7064 exo-oligo-1,6-glucosidase by the use of immunosorbent

SummaryA p-nitrophenyl-α-D-glucopyranoside-hydrolyzing exo-oligo-1,6-glucosidase (dextrin 6-α-glucanohydrolase, EC.3.2.1.10) of Bacillus cereus ATCC 7064 was 1,120-fold purified to an electrophoretically- and immunologically-homogeneous form by a simple 4-step method containing as the most efficient step the enzyme elution from immunosorbent with a pH 11 medium including 50% (w/v) glycerol. The final enzyme yield was 47%. The specific activity was 218 μmol p-nitrophenyl-α-D-glucopyranoside hydrolyzed/min/mg protein at 35°C and pH 6.8. The amino-terminal amino acid of the enzyme was determined to be methionine. No antigenic common determinant occurred between this enzyme and each of its homologous counterparts from obligate thermophile Bacillus thermoglucosidius KP 1006 and from facultative thermophile Bacillus coagulans ATCC 7050.