• Journal of Life Science
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• v.10 no.1
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• pp.20-23
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• 2000

Mono-ADP-ribosylation, catalyzed by ADP-ribosyltransferases, is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to an acceptor protein. Previously, we have identified and cloned a glycosylphosphatidylinositol-linked ADP-ribosyltransferase (Yac-1) from mouse lymphoma cells. Yac-1 enzyme contains three regions (region I,II,III) similar to those found in several bacterial toxins and vertebrate ADP-ribosyltransferases. Site-directed mutagenesis was performed to verify the role of Glu 233 in region III. Mutants E233Q, E233D and E233A were inactive for ADP-ribosyltransferase activity. Thus Glu 233 in Yac-1 is essential for enzyme activity, suggesting that Glu 233 in Glu-rich motif near the carboxy terminus plays a catalytic role in ADP-ribosyltransferase activity.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.480-484
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• 2003

A stereoselective-hydrolysing enzyme was cloned from Pseudomonas fluorescens KCTC1767 which had high enantiomeric activity toward (S)-ketoprofen ethyl ester. Analyses of typical properties resulted in low thermostability and substrate specificity. A round error-prone PCR and StEP(STaggered Extension Process) was adopted to evolute this character. As a result, the best clone 6-52 was selected which was represented to increased thermostability(40 fold) compared to wild type enzyme in $50^{\circ}C$. Additionally, specific activity toward (S)-ketoprofen ethyl ester and p-nitrophenyl derivatives improved 3 fold and 1.5 fold, respectively. DNA sequence analyses was showed some exchanged amino acid residue that was L120p, 1208v, T249A, D287H and T357A. Which the 120th's leucine substituted for proline was presumed structurally important residue concerning with catalytic activity.

• Food Science and Industry
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• v.53 no.1
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• pp.43-55
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• 2020

Most reports demonstrated the substrate specificity-based kinetic properties of chitin or chitosan degrading enzymes. However, there is virtually less information on the high quality and quantity production of chitin or chitosan hydrolysates having a larger than (GlcN)₇ from the hydrolysis of high molecular weight chitosan using specific enzymes and their biological activity. Therefore, the production of such molecules and the discovery of such enzyme sources are very important. Fortunately, the author has established a mass production method of chitosan hydrolysates (GlcN)_n, n=2-13 that have been characterized as a potent antioxidant substance, as well as antifungal and antibacterial activities against Penicillium species and highly selective pathogenic bacteria. In addition, preclinical studies using (GlcN)_n, n=5-25 demonstrated that these molecules played a very important role in maintaining biometric balance. Collectively, it is implicated that the application of these mixed substances to foods with significant biological activity is very encouraging.

• Journal of Microbiology
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• v.43 no.3
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• pp.237-243
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• 2005

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, $60^{\circ}C$. The endopeptidase activity was stimulated by $Ca^{++},\;Co^{++},\;Mn^{++},\;Mg^{++},\;and\;Ni^{++}$, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM $Ca^{++}$, and was partially restored by $Co^{++}\;and\;Mn^{++}$, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of $Ca^{++}$ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the $K_m$ value of endopeptidase is 0.315 mM and $V_{max}$ is 0.222 ) is $0.222\;{\mu}mol$ of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.70-76
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• 2003

Three experiments were conducted to determine the effects of a sorghum-specific enzyme system, derived from an Aspergillus niger and Bacillus subtilis fermentation extract (carbohydrase activity of 1,650 $\alpha$-amylase units and cellulase activity of 30 fibrinolytic units/mL), on growth performance of finishing pigs. In Exp. 1,192 pigs (average initial BW of 46.1 kg) were fed sorghum-based diets without or with 360 mL of enzyme system per ton of sorghum in a 78 d growth assay. For d 0 to 39, gain/feed was improved (p<0.03) with enzyme supplementation, but ADG was not affected (p>0.15). For d 39 to 78 and overall (d 0 to 78), ADG, gain/feed, and digestibilities of DM and N were not affected (p>0.13) by enzyme supplementation. Backfat thickness, fat-free lean index, and scores for stomach keratinization and ulcers also were not affected (p>0.15) by the dietary treatments. In Exp. 2,168 pigs (average initial BW of 58.4 kg) were fed diets without or with 150, 300, or 450 mL/ton of the same enzyme system used in Exp. 1. Adding as much as 450 mL enzyme system / ton of sorghum did not affect (p>0.15) ADG or gain/feed for d 0 to 29 of the growth assay. However, during d 29 to 63, ADG increased by 11% (linear effect, p<0.02) and gain/feed increased by 10% (linear effect, p<0.06) as enzyme concentration was increased from none to 450 mL/ton of sorghum. For the overall period (d 0 to 63), ADG tended to increase (p<0.08) with enzyme supplementation, but gain/feed and digestibilities of DM and N were not affected (p>0.14). Carcass characteristics (dressing percentage, backfat thickness, and fat free lean index) also were not affected (p>0.20) by addition of the enzyme system. In Exp. 3,176 pigs (average initial BW of 46.7 kg) were fed diets without or with 450, 900, or 1,350 mL/ton of the same enzyme system used in Exp. 1 and 2 in a 71 d growth assay. Adding up to 1,350 mL/ton of enzyme had no effects (p>0.15) on ADG, gain/feed, digestibilities of DM and N, and carcass characteristics (dressing percentage, backfat thickness, and fat-free lean index). In conclusion, finishing pigs fed diets with a sorghum-specific enzyme system showed some positive trends for improved growth performance, but those effects were not large and (or) consistent.

• Fisheries and Aquatic Sciences
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• v.6 no.1
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• pp.7-12
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• 2003

Chitinase and chitobiase produced by Aeromonas sp. J-5003 were purified and characterized. The chitinase was purified to 19.4 folds by gel chromatography and ion-exchange chromatography with the overall yield of $2.2\%$ and the specific activity of 93.1 unit/mg. The purified enzyme showed a single band on SDS-PAGE with MW 54kDa. The optimum pH and temperature of the purified chitinase were 7.0 and $37^{\circ}C$, respectively, and this enzyme stable in the range of pH 6.0 to 10.0 below $37^{\circ}C$. $Mg^{2+},\;Ca^{2+}\;and\;Na^+$ slightly stimulated the chitinase activity. However, $Hg^{2+}\;and\;Fe^{3+}$ inhibited chitinase activity. The chitobiase was purified by Sephacryl HR-l00 gel chromatography and DEAE-Sephadex A-50 ion-exchange chromatography with 33.5 purification folds and $4.3\%$ yield. The purified enzyme showed a single band with MW 63 kDa. The optimum pH and temperature of the purified chitobiase were 7.0 and $37^{\circ}C$, respectively. And this enzyme was stable in the range of pH 6.0 to 9.0 and at the temperature below $37^{\circ}C$. The enzyme activity was increased by $Mn^{2+}$, but it was inhibited by $Ag^+$.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.331-338
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• 2012

A novel gene coding for an endo-${\beta}$-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The ${\beta}$-mannanase showed an identity of 90.2-92.9% ${\leq}95%$) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified ${\beta}$-mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDI-TOF mass spectrometry. The recombinant ${\beta}$-mannanase had an optimum temperature of $45^{\circ}C$ and optimum pH of 6.5. The enzyme was stable at temperatures up to $50^{\circ}C$ (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions ($Hg^{2+}$, $Pb^{2+}$, and $Co^{2+}$) substantially inhibited the recombinant ${\beta}$-mannanase. The chemical additives including detergents (Triton X-100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the ${\beta}$-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.214-221
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• 1998

A Brevibacterium ammoniagenes gene coding for glucose/mannose-specific enzyme II ($EII^{Glc}$) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing an Escherichia coli mutation affecting a ptsG gene, and the complete DNA nucleotide sequence was determined. The cloned gene was identified to be a ptsG, which enables the E. coli transportment to use glucose more efficiently than mannose as the sole carbon source in an M9 minimal medium. The ptsG gene of B. ammoniagenes consists of an open reading frame of 1,983 nucleotides putatively encoding a polypeptide of 661 amino acid residues and a TAA stop codon. The deduced amino acid sequence of the B. ammoniagenes $EII^{Glc}$ shows, at $46\%$, the highest degree of sequence similarity with the Corynebacterium glutamicum EII specific for both glucose and mannose. In addition, the $EII^{Glc}$ shares approximately $30\%$ sequence similarities with sucrose-specific and ${\beta}$-glucoside-specific EIIs of the several bacteria belonging to the glucose-PTS class. The 161-amino-acid C-terminal sequence of $EII^{Glc}$ is also similar to that of E. coli enzyme $IIA^{Glc}$, specific for glucose ($EIIA^{Glc}$). The B. ammoniagenes $EII^{Glc}$ consists of three domains; a hydrophobic region (EIIC) and two hydrophilic regions (EIIA, EIIB). The arrangement of structural domains, IIBCA, of the $EII^{Glc}$ is identical to those of EIIs specific for sucrose or ${\beta}$-glucoside. While the domain IIA was removed from the B. ammoniagenes $EII^{Glc}$ the remaining domains IIBC were found to restore the glucose and mannose-utilizing capacity of E. coli mutant lacking $EII^{Glc}$ activity with $EIIA^{Glc}$ of the E. coli mutant. $EII^{Glc}$ contains a histidine residue and a cysteine residue which are putative phosphorylation sites for the protein.

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.545-551
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• 1989

Thermostable alkaline protease of alkalophilic Bacillus sp. No. 8-16 has been purified, and the properties of the enzyme investigated. The characteristic point of the organism used is especially good growth in alkaline and thermal condition. The alkaline protease of the strain No. 8-16 was purified from crude enzyme by acetone precipitation, CM-cellulose ion exchange chromatography, Sephadex G-100 and Sephadex G-75 gel filtration. Through the series of chromatograpies, the enzyme was purified to homogeneity with specific activity of 37 fold higher than that of the crude broth. Characteristics of the purified enzyme were as follow; $K_m$ value for the enzyme was 1.3 mg/ml, the alkaline protease showed a maximal activity at 7$0^{\circ}C$ and from the pH 6.0 through 12.0, and stable for 1 hr. at 6$0^{\circ}C$. The moleclar weight of the enzyme was estimated to be 33,000 by Sephadex G-100 gel filtration. The activity of the alkaline protease was inhibited by iodoacetic acid and Ag$^+$, Hg$^+$, PMSF (phenylmethylsulfonyl fluoride), and activated by $Ca^{2+}$ and Mn$^{2+}$.

• Korean Journal of Food Science and Technology
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• v.21 no.4
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• pp.569-574
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• 1989

These studies were conducted to investigate the purification and characterization of Kiwifruit protease, and the results obtained were as follows The protease was purified by ammonium sulfate fractionation, Sephadex G-100 filtration and DEAE-Sephadex A-50 column chromatography and purified enzyme gave a single protein band on polyacrylamide gel electrophoresis The specific activity of purified enzyme was 30,10 units/mg protein and the yield was 7.48. The purified enzyme showed a high affinity for casein and hemoglobin. The optimal pH and temperature for enzyme activity were 7.0 and $45^{\circ}C$, respectively. The enzyme activity was strongly inhibited by $HgCl_2,\;MnSO_4$. However. the enzyme was activated by cysteine and EDTA. The Michaelis constant for casein was calculated to be 50.5mg/ml according to the Line weaver-Burk method, and its molecular weight was determied as 23,500 by polyacrylamide gel electrophoresis.