• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.23-29
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• 1997

The nucleotide sequence of the estI gene encoding acetylxylan esterase I of Bacillus stearothermophilus was determined and analyzed. The estI gene was found to consist of a 810 base pair open reading frame coding for a polypeptide of 270 amino acids with a deduced molecular weight of 30 kDa. This was in well agreement with the molecular weight (29 kDa) estimated by SDS-PAGE of the purified esterase. The coding sequence was preceded by a putative ribo some binding site 10 bp upsteam of the ATG codon. Further 53 bp upstream, the transcription initiation signals were identified. The putative $_{-}$10 sequence (TCCAAT) and $_{-}$35 seqence (TTGAAT) corresponded closely to the respective consensus sequences for the Bacillus subtiis major RNA polymerase. The G+C content of the coding region of the estI was 51% whereas that of the third position of codone was 60.2%. The N-terminal amino acid sequence of the EstI deduced from the nucleotide sequence perfectly matched the corresponding region of the purified esterase described previously. Comparison with the amino acid sequence of other esterases and lipases reported so far allowed us to identify a sequence, GLSMG at positions 123 to 127 of the EstI which was reported to be the highly conserved active site sequence for those enzymes. The nucleotide sequence of the estI revealed 55.7% homology to that of the xylC coding for the acetylxylan esterase of Caldocellum saccharolyticum.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.454-460
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• 1995

Acetylxylan esterase II was produced by Escherichia coli HB101 harboring the recombinant plasmid pKMG7 which contained the estII gene of Bacillus stearothermophilus. Optimal medium for the production of the acetylxylan esterase by E. coli HB101/pKMG7 was determined to contain 0.5% galactose, 1% yeast extract and 1% NaCl. The enzyme produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The temperature and pH optimum of the esterase were 45$\circ$C and pH 6, respectively. The essential amino acids for the esterase activity were found to be methionine, serine, and cysteine. Molecular weight of the esterase was determined to be 28 kDa by SDS-polyacrylamide gel electrophoresis, and 120 kDa by gel filtration. This suggests that the functional enzyme is a homomeric tetramer. The esterase had an isoelectric point of pH 3.4. The N-terminal amino acid sequence of the enzyme was Ala-Leu-Phe-Glu-Ser-Arg-Phe-Phe-Ser-Glu-Val-Leu-Gly-Leu.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.599-606
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• 1994

Bacillus stearothermomophilus, a strong xylan degrader, was confirmed to express multiple esterase activities in addition to the major xylanolytic enzymes. One of the genes encoding the esterases was isolated from the genomic library of B. stearothermophilus constructed with EcoRl restriction endonuclease and pBR322 plasmid. Three recombinant plasmids showing the tributyrin degrading activity were selected from approximately 7, 000 E. coli HB101 transformants, and were found to have the same insert of a 3.2 kb DNA fragment. Restriction mapping and hybridization studies revealed that the gene(estII) on the hybrid plasmid (pKMG7) had originated from the B. stearothermophilus chromosome, and was distinct from the estl, another esterase gene of B. stearothermophilus isolated in the previous work. The E. coli cells harboring pKMG7 produced an acetylxylan esterase that exibited similar substrate specificity to the esterase encoded by the estI gene.