• Journal of Microbiology and Biotechnology
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• v.6 no.3
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• pp.173-178
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• 1996

Synergic effects among endo-xylanase, $\beta$-xylosidase, and acetyl xylan esterase of Bacillus stearothermophilus in the hydrolysis of xylan were studied by using birchwood, oat spelt, and acetylated xylan as substrates. Synergism between endo-xylanase and $\beta$-xylosidase was observed on all three substrates tested, indicating that $\beta$-xylosidase enhanced the production of xylose by relieving the end-product inhibition upon endo-xylanase conferred by xylooligomers. Endo-xylanase and $\beta$-xylosidase also showed synergism with acetyl xylan esterase in the hydrolysis of birchwood and acetylated xylan, while no synergic effect was detected in oat spelt xylan hydrolysis. Thus, the hydrolysis of xylan containing acetic acid side chains required the action of acetyl xylan esterase, which eliminated the steric hindrance of the side chains, leading to the better hydrolysis by endo-xylanase and $\beta$-xylosidase , and the acetyl xylan esterase activity was also enhanced by endo-xylanase and $\beta$-xylosidase for the latter enzymes provided acetyl xylan esterase with shorter xylan oligomers, the better substrate for the enzyme.

• Microbiology and Biotechnology Letters
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• v.22 no.5
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• pp.507-514
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• 1994

Acetyl xylan esterase was produced by E. coli HB101 harboring a recombinant plasmid pKMG6 which contained the estI gene of Bacillus stearothermophilus. The maximum production was observed when the E. coli strain was grown at 37$\circC for 12 hours in the medium containing 0.5% acetyl xylan, 1.0% tryptons, 1.0% sodium chloride, and 0.5% yeast extract. The esterase produced was purified to homogeneity using a combination of ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration. The native enzyme had an apparent molecular mass of 60 kd and was composed of two identical subunits of 29 kd. The N-terminal amino acid sequence of the polypeptide was Ala-X-Leu-Gln- Ile-Gln-Phe-X-X-Gln. The acetyl esterase displayed a pH optimum of 6.5 and a temperature opti- mum of 45$\circC. The heavy metal ions such as Ag$^{++}$, Hg$^{++}$ and Cu$^{++}$ inhibited nearly completely the activity of the esterase, and no specific metal ion was found to be required for the enzyme activity. The enzyme readily cleaved MAS, $\beta$-D-glucose pentaacetate, $\alpha$-naphthyl acetate, $\rho$-nitrophenyl acetate as well as acetyl xylan, but had no activity on $\rho$-nitrophenyl propionate, $\beta$-nitrophenyl butyrate or $\beta$-nitrophenyl valerate. The Km and Vmax values for MAS were 2.87 mM and 11.55 $\mu$mole/min, respectively. Synergistic behavior was demonstrated with a combination of xylanase and esterase from B. stearothermophilus in hydrolyzing acetyl xylan.

• Journal of the Korean Graphic Arts Communication Society
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• v.26 no.1
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• pp.17-28
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• 2008

The wood-pulp is treated with the three enzymes; Endo-xylanase, exo-xylanase and acetyl-esterase. The maximum value of relative activity appeared 0.95 in acetyl-esterase at $40^{\circ}C$, 0.9 in exo-xylanase at $40^{\circ}C$, and 0.8 in endo-xylanase at $50^{\circ}C$, respectively. And it has measured 0.8 in endo-xylanase, 0.95 in acetyl-esterase at pH 6 and 0.9 in exo-xylanase at pH 5, while the maximum value of relative activity does not rely on reaction time for three enzymes treatment, and the value was about 0.9, respectively. We have watched that decreased Kappa number and increased brightness. And it turned out that the three enzyme produced a lot of reducing sugar with wood-pulp treatment.

• Microbiology and Biotechnology Letters
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• v.21 no.6
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• pp.542-548
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• 1993

Bacillus stearothermophilus was shown to express multiple xylanolytic enzymes including acetyl xylan esterase. Genomic DNA of the strain partially digested with HindIII was ligated into the HindIII site of pBR322, and expressed in E. coli HB101 cells in order to clone the gene for acetyl xylan esterase. One transformant among 4000 screened formed a clear zone around its colony on the LB agar supplemented with 1.0% tributyrin. The functional clone harbored the recombinant plasmid pKMG5 with an insert of 5.1kb.

• Korean Journal of Microbiology
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• v.39 no.2
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• pp.83-88
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• 2003

We cloned a gene encoding acetyl xylan esterase(axeA) of Streptomyces coelicolor A3(2) and studied its expression pattern in Escherichia coli. The full sequence of axeA was amplified by PCR. Sequence analysis of the PCR product revealed an open reading frame of 1,008 nucleotides encoding a protein consisted of 335 amino acid residues, with a calculated molecular mass of about 38 kDa. The base sequence showed 98% homology to the same gene of Streptomyces lividans. Two different kinds of acetyl xylan esterases were produced in Escherichia coli(pLacI) by IPTG induction; their molecular weights were 38 kDa and 34 kDa, respectively. Of these, 38 kDa protein seemed to be a total protein holding N-terminal signal peptide region, whereas 34 kDa protein seemed to be a matured protein without signal peptide which was produced by peptide bond cleavage between two amino acid residues of alanine 41 and alanine 42.

• 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.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.145-148
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• 2006

A new functional lipolytic enzyme (AT4) has recently been found from Agrobacterium tumefaciens C58 Cereon using a genome-wide approach. The enzyme has some sequence similarity to E. coli acetyl hydrolase, Emericella nidulans lipase, Moraxella sp. lipase, Acinetobacter lwoffii esterase, and Streptomyces hygroscopicus acetyl hydrolase. However, the sequence similarities are very low (less than $25\%$), suggesting that it is a new lipase/esterase enzyme. ill the present study, intact cell of the A. tumefaciens strain was shown to have lipolytic activity on a tributyrin-LB plate. The AT4 gene was then expressed at a high level in E. coli BL21 (DE3) cells and the enzyme was purified simply by Ni-NTA column chromatography. The purified enzyme showed hydrolytic activity toward p-nitrophenyl caproate, but not toward olive oil, suggesting that the AT4 enzyme was a typical esterase rather than lipase. AT4 esterase had a maximum hydrolytic activity at $45^{\circ}C$ and pH 8.0, when p-nitrophenyl caproate was used as a substrate. It was relatively stable up to $40^{\circ}C$ and at pH 5.0-9.0. Calcium ion and EDT A did not affect the activity and thermal stability of the enzyme. As for substrate specificity, AT4 enzyme could rapidly hydrolyze acetyl and butyl groups from p-nitrophenyl esters and 1-naphthyl esters. In addition, it also released acetyl residues from acetylated glucose and xylose substrates. Therefore, this new esterase enzyme might be used as a biocatalyst in acetylation and deacetylation reactions performed in the fine chemical industry.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.305-311
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• 2002

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZ$\alpha$C-AXE (4.6 kb), was constructed, in which the AXE gene was under the control of the AOXI promoter and connected downstream of mating factor u-1 signal sequence. The plasmid linearized by Sacl was integrated into the 5'AOXI region of the chromosomal DNA of P. pastoris. In the flask batch culture of P. pastoris transformant on methanol medium, the cell concentration and total AXEase activity reached at 6.0 g-dry cell weight/1 and 77 unit/ml after 36 h cultivation, respectively. In the fed-batch culture employing the optimized methanol and histidine feeding strategy, the cell concentration and total AXEase activity were significantly increased to about 97 g-dry cell weight/1 and 930 unit/ml. Most of AXEase activity (>90%) was found in the extracellular medium and the majority of extracellular protein (>80%) was AXEase enzyme (33.5 kDa). This result means that about 9.8 g/1 of AXEase protein was produced in the extracellular medium.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.232-236
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• 2004

We investigated hydrogen peroxide resistance of Escherichia coli possessing acetyl xylan esterase(AxeA) of Streptomyces coelicolor A3(2). The induction of AxeA production by isopropyl-$\beta$-thiogalactoside was confirmed by SDS-polyacrylamide gel electrophoresis. The differences in growth between induced and non-induced E. coli were determined by the changes in optical density of cultures after hydrogen peroxide treatment The lethal effect of hydrogen peroxide was observed for non-induced cultures at all concentrations tested in this study (lmM, 2.5mM and 5mM). However, cultures induced for AxeA production resisted the lethal effect, except at 5mM where cells were killed irrespective of the AxeA production. The axeA induction increased survival against 1.5mM hydrogen peroxide from 59% to 74%. In addition, AxeA producing E. coli showed increased survival at $45^{\circ}C$, near maximum growth temperature. Therefore, it was concluded that AxeA conferred a cross-resistance upon the bacterium against both oxidative- and heat stress.

• 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.