• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.925-935
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• 2014

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the ${\alpha}/{\beta}$-class of protein consisted of a central six-stranded ${\beta}$-sheet flanked by eight ${\alpha}$-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were $15^{\circ}C$ and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at $50^{\circ}C$ or $60^{\circ}C$ for 6 h, and 89% of its enzyme activity when preincubated at $70^{\circ}C$ for 1 h. The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by $Na^+$ and $Mg^{2+}$ ions but was strongly inhibited by $Cu^+$ and $Hg^{2+}$ ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a $K_m$ of 0.278 mM and a $k_{cat}$ of $1.9s^{-1}$. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

• YAKHAK HOEJI
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• v.2 no.1_2
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• pp.41-47
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• 1953

"Kimchi" is a most popular pickled Vegetables in Korea. In this paper, eight strains of gram positive rocls and two strains of gram positive cocci are isolated from "Kimchi". For the each isolated strains, the acid production test against carbohydrate and production test of amylase, protease, esterase, and general biological tests are investigated. The most active one for the amylase production is No.2 strain the most active one for the Protease production is No.5,6, and 9 strains. And that for Esterase production is No.9 strain (see the original paper on the results in this journal page)

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.155-162
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• 2020

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50℃, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana β-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.11
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• pp.1555-1565
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• 2009

Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastix frontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five $\beta$-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis.

• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.13-17
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• 1993

When mandarin orange peel was used for a substrate of citric aCid fermentation by Aspergillus niger, principal enzyme activities were investigated. Not only the activity of polygalacturonase and pectin esterase being capable of digesting pectin and crude fiber of mandarin orange peel. but also that of carboxymethyl cellulase, xylanase and amylase was high. In carbohydrate metabolism, the activity of enzymes related in HMP pathway was higher than that in EMP pathway at the orange peel medium designed hereby rather than synthetic medium. Productivity of citric acid was significantly increased when the activity of citrate synthetase was high and 5imultaneously those of aconitase and NADP-dependent dehydrogenase were low.

• Microbiology and Biotechnology Letters
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• v.33 no.1
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• pp.9-15
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• 2005

Chitin deacetylase (CDA; EC 3.5.1.41) catalyzes the hydrolysis of N-acetamide bonds of chitin, converting it to chitosan. Chitosan has several applications in areas such as biomedicine, food ingredients, cosmetics, pharmaceuticals, and agriculture. In this paper, occurrence, assay and purification protocols, enzymatic characteristics, substrate specificity, and mode of action of microbial CDAs have been described. Several lines of evidence have substantiated the biological roles involved in cell wall formation and plant-pathogen interactions for fungal CDAs. The gene structure of CDAs has been compared with other family 4 carbohydrate esterases which deacetylate a wide variety of acetylated poly/oligo-saccharides. The use of CDAs for the conversion of chitin to chitosan, in contrast to the presently used chemical procedure, offers the possibility of a controlled, non-degradable process, resulting in the production of well-defined chitosan oligomers and polymers. Insect pathogen that can secrete high levels of chitin-metab­olizing enzymes including CDA can be a possible alternative for new pest management tools.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.802-810
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• 2007

Fibrobacter succinogenes, a Gram-negative, anaerobic ruminal bacterium is a major fibre digesting species in the rumen. It intensively degrades plant cell walls by an erosion type of mechanism, burrowing its way through the complex matrix of cellulose and hemicellulose with the release of digestible and undigested cell wall fragments. The enzymes involved in this process include a combination of glucanases, xylanases, arabinofuranosidase(s) and esterases. The genome of the bacterium has been sequenced and this has revealed in excess of 100 putative glycosyl hydrolase, pectate lyase and carbohydrate esterase genes, which is greater than the numbers reported present in other major cellulolytic organisms for which genomes have been sequenced. Modelling of the amino acid sequences of two glycanases, CedA and EGB, by reference to crystallized homologs has enabled prediction of the major features of their tertiary structures. Two dimensional gel electrophoresis in conjunction with mass spectroscopy has permitted the documentation of proteins over expressed in F. succinogenes grown on cellulose, and analysis of the cell surfaces of mutant strains unable to bind to cellulose has enabled the identification of candidate proteins with roles in adhesion to the plant cell wall substrate, the precursor to cellulose biodegradation.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.28 no.3
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• pp.323-327
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• 1983

The intercellular material was extracted from rice leaf tissue. The quantitative tests of protein and soluble carbohydrate, and activity of peroxidase, showed differences between tissue extract and intercellular material. Also electrophoretic patterns of peroxidase and esterase isozymes were not similar between them. It indicated that the intercellular material which was extracted, was not the mixture of cellular materials.

• Journal of fish pathology
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• v.20 no.3
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• pp.249-255
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• 2007

We investigated the phenotypic characteristics by using API20E, APIZYM and determined minimum inhibitory concentrations (MICs) of 7 antibiotics in motile aeromonads isolated from freshwater fishes in Korea and Japan, and 4 American Type Culture Collection (ATCC) strains. All isolates (n=7) were identified as motile Aeromonas species according to API20E test. Lysine decarboxylase activity and acid production from 4 different carbohydrates including mannitol, rhamnose, amygdalin and arabinose were observed in various strains. In enzymatic activities by APIZYM, all isolates showed negative reactions in valine and cystine arylamidases, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase and α-fucosidase. Although the intensities of each enzymatic activity were diverse in alkaline phosphatase, esterase-lipase, leucine arylamidase, β-galactosidase and N-acetyl-β-glucosaminidase, all isolates showed positive reactions. All isolates were resistant to ampicillin sodium (MIC>100㎍/ml), but sensitive to chloramphenicol (MIC≤1.6㎍/ml). However, recently isolated strains (AC9804, AC0202 and GMA0361) were commonly resistant to tetracycline (MIC=50㎍/ml). Furthermore, AC9804 was resistant to oxolinic acid (MIC=12.5㎍/ml). GMA0361 was resistant to kanamycin sulfate (MIC>100㎍/ml) and streptomycin sulfate (MIC>100 ㎍/ml).

• Food Science of Animal Resources
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• v.28 no.5
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• pp.587-595
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• 2008

In order to develop a new starter for fermented milk, 1037 bacterial strains were isolated from raw milk. The strain that showed excellent acid producing and angiotensin converting enzyme (ACE) inhibitory activity (88.6%) was selected and identified as a Lactobacillus zeae based on the result of API carbohydrate fermentation pattern and 16S rDNA sequence. Lactobacillus zeae RMK354 was investigated further to study its physiological characteristics. It showed strong ACE inhibitory activity compared with commercial LAB starters tested. The optimum growth temperature of L. zeae RMK354 was $40^{\circ}C$ and it took 10 hr to reach pH 4.3 under this condition. L. zeae RMK354 showed more sensitive to penicillin-G, bacitracin, novobiocin, in a comparison of 14 different antibiotics, and showed most resistance to polymyxin B and vancomycin. It showed higher esterase and leucine arylamidase activities compared with 16 other enzymes. It was comparatively tolerant to bile juice and able to survive at pH 2 for 3 hr. It showed inhibitory activity against Salmonella Typhimurium with the rate of 60%. Based on these and previous results, L. zeae RMK354 could be an excellent starter culture for fermented milk with high level of ACE inhibitory activity.