• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.36-42
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• 1996

The production of sorbitol by permeabilized cells of Zymomonas mobilis immobilized in $\kappa$-carrageenan was investigated. Cetyltrimethylammoniumbromide (CTAB) permeabilized cells were treated with glutaraldehyde prior to immobilization for cross-linking of enzymes, glucose-fructose oxidoreductase (GFOR) in cells. Rigidity of the immobilized beads was increased two-fold with 90$\%$ conversion efficiency by the additions of 40$\%$ (w/v) polyols (glycerol 25 g + propylene glycol 15 g) to 60$\%$ (w/v) distilled water containing 2.5$\%$ (w/v) $\kappa$-carrageenan as a final concentration, prior to immobilization. $\kappa$-Carrageenan beads entrapping permeabilized cells were dried to improve bead rigidity and storage stability. During s6mi-batch process for 72 h with dry beads, there was an improvement of the loss of enzyme activity (less than 10$\%$). In batch process, the kinetic results of $K_m.fructose$ value for the free cells, wet beads and dry $\kappa$-carrageenan beads were 71.7, 72.4 and 116.7 g/l, respectively. Higher productivity was obtained with two-stage continuous packed bed reactors with both wet and dry $\kappa$-carrageenan beads at 25.00 and 21.15 g/l/h, respectively, when measured at second stage.

• Journal of the Korean Wood Science and Technology
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• v.46 no.6
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• pp.670-680
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• 2018

Acanthophysium sp. KMF001, a wood rotting fungus, produces a strong crude enzyme complex that efficiently produces simple sugars from wood. The transcriptomic analysis of Acanthophysium sp. KMF001 identified 14 genes for putative glycoside hydrolases. Among them, isotig01043 was expressed heterogeneously in Escherichia coli BL21(DE3), and the expressed protein exhibited an endo-${\beta}$-1,4-xylanase activity which showed the optimum reaction at pH 5.0 and $30^{\circ}C$. The enzyme kinetic values of $K_m$ and $V_{max}$ were 25.92 mg/ml and $0.628{\mu}mole/mg/ml$, respectively. The enzymatic characteristics of the expressed xylanase showed a typical fungal xylanase. However, the bioinformatics analysis suggested that the protein encoded by isotig01043 was a novel xylanase based on a low identity when it was compared with the closest protein in the NCBI database and a similar protein domain with GH16_fungal_Lam16A_glucanase, which had not been earlier suggested as a xylanase.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.837-845
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• 2016

A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.

• Korean Journal of Microbiology
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• v.29 no.4
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• pp.230-237
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• 1991

Citrate synthase 1 (mitochondrial) and citrate synthase 2 (nonmitochondrial) were purified from Saccharomyces cerevisiae. The physical and enzymatic characteristics of citrate synthase 2 were ananlyzed in comparison with citrate synthase 1. Both isoenzymes were shown to be dimeric proteins of identical subunits, and the molecular weights of the subunits were estimated to be 48.3kDa for citrate synthase 1 and 47.0kDa for citrate synthase 2, respectively. The optimal pH value for enzyme activity was pH 7.5 for both isoenzymes. However, the optimal temperature for the activity was strikingly different; while the activity of citrate synthase 1 reached its peak at 65.deg.C, that of citrate synthase 2 was maximal at 40.deg.C. Citrate synthase 2 showed much lower thermal and pH stability than citrate synthase 1. In addition, citrate synthase 2 was affected much more by the metal ions such as $Zn^{2+}$ , $Mn^{2+ , and $Co^{2+} than citrate synthase 1. Among the several possible regulatory metabolites tested, ATP showed the strongest inhibitory effect on both enzymes. ADP and NADH were found to have greater effect on citrate synthase 2 than on citrate synthase 1. Kinetic analysis revealed that citrate synthase 2 has approximately 7- and 3.5-fold lower affinity to acetyl CoA and to oxaloacetate, respectively, than citrate synthase 1.

• Bulletin of the Korean Chemical Society
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• v.1 no.1
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• pp.10-17
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• 1980

Penicillin amidohydrolase was partially purified from the fermented broth of Bacillus megaterium, and was immobilized on nylon fiber. The surface area of nylon fiber was increased by roughening it with fine sand and activated by acid treatment. The free amino groups on the nylon fiber exposed by such treatment were then utilized to immobilize the penicillin amidase. Enzymatic properties of penicillin amidohydrolase immobilized on the nylon fiber by covalent bonding and cross linking with glutaraldehyde were studied and compared with those of soluble enzyme. The optimal pH and temperature profile of immobilized enzyme showed only slightly broader peaks, and the values of kinetic constants, $K_m$, $K_{ia}$, and $K_{ip}$, of the immobilized enzyme are only slightly greater than those of the soluble enzyme. These results suggest that the mass transfer effect on the reaction rate for the penicillin amidase immobilized on nylon fiber is not so significant as the enzyme immobilized on some other support material like bentonite. The experimental results of batch reaction agreed well with the results of computer simulation for both the immobilized and soluble enzyme systems, confirming the validity of the rate equation derived which was based on the combined double inhibition by two reaction products.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.29 no.1
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• pp.64-70
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• 1996

Kinetic properties of typsins purified from dark-fleshed fish (anchovy, mackerel, yellowfin tuna, and albacore) were examined and analyzed on $benzoyl-_{D,L}-arginine-p-nitroanilide\;(BAPNA)$. The values of Km' and $k_{cat}$ of the purified trypsins from the four dark-fleshed fish were found to be $49.3{\mu}M$ and $90.9\;min^{-1}$ for anchovy, $53.7{\mu}M$ and $61.2min-^{-1}$ for mackerel A, $96.5{\mu}M$ and $76.6min^{-1}$ for mackerel B, $62.8{\mu}M$ and $46.6min^{-1}$ for yellowfin tuna, and $98.3{\mu}M$ and $47.7min^{-1}$ for albacore, respectively. The values of $K_i$ on $tosyl-_L-lysine$ chloromethyl ketone (TLCK) were determined to be $20.90{\mu}M$ for anchovy trypsin, $2.86{\mu}M$ for mackerel trypsin A, $3.90{\mu}M$ for mackerel trypsin B, $0.96{\mu}M$ for yellowfin tuna trypsin, and $1.82{\mu}M$ for albacore trypsin. Thus yellowfin tuna trypsin was the most sensitive to TLCK among all trypsins. The activities and catalytic efficiency of the trypsins purified from the temperate zone fish, anchovy and mackerel, were higher than those of the trypsins purified from yellowfin tuna and albacore which migrate widely from the tropic zone to the temperate zone.

• Tuberculosis and Respiratory Diseases
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• v.42 no.6
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• pp.838-845
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• 1995

The determination of ADA(adenosine deaminase) activity in pleural fluid is useful in differental diagnosis of pleural effusion. The conventional method of determining ADA activity used by Giusti was influenced by contamination of ammonia. Additionally, because Giusti's method was mannual method a determining the ADA activities in sample, was not easily automated. In 1993, Oosthuizen HM with collegues developed simple kinetic method for determining ADA activity. It was reliable and suiable method for automation. In this study, we have measured ADA activity in 162 patients with various pleural effusion by Hitachi 747 autoanalyser using the Oosthuizen kinetic method for the purpuse of determination of new diagnostic cut-off value for the tuberculous effusion and evaluation of the correlation between the conventional method and new automated method. This new method of an enzymatic reaction involves 2, 6-dichlorophenolindophenol dye(DICP), adenosine, xanthine oxidase(XO), and nucleoside phosphorylase(NP). The results were as follows: 1) The mean pleural ADA activity of the tuberculous effusion was $52.53{\pm}16.43\;U/L$ and significantly higher than that of other groups(p<0.001). If the diagnostic cut-off value of pleural ADA activity for tuberculous effusion is above 30 U/L, the sensitivity is 96% and the specificity is 90%. 2) The mean pleural to serum ADA activity ratio of the tuberculous effusion was $2.29{\pm}0.96$ and it was also significantly higher than that of other pleural groups(p<0.001). If the diagnostic cut-off value of pleural to serum ADA activity ratio is 1.5, the sensitivity is 80% and the specificity is 88% in the diagnosis of tuberculous pleural effusion. 3) The new kinetic method is correlates well to Giuisti's conventional method(r=0.971). In conclusion, the new kinetic method described is easily automated and seems to be suitable for the routine determination of ADA activity.

• Journal of the Korean Society of Food Science and Nutrition
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• v.11 no.3
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• pp.21-27
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• 1982

A kinetical approach for the non-enzymatic browning reaction of the dried file-fish was studied. The reaction rates revealed a tend to increase with increasing water activity and showed the maximum at $0.75\;a_w$ The activation energies obtained from the Arrhenius plot ranged 12.5 to 16.5 Kcal/mole. From these energies of activation, the $Q_{10}$ values at $45^{\circ}C$ showed 1.9 to 2.3 and both activation energy and $Q_{10}$ values were reduced with increase in $a_w$ Shelf-lives, the time to reach an 0.15 O.D./g solid at which severe brown color change could be de ectable, decreased rapidly as the temperature and water activity increase. A storage study under a square-wave fluctuating temperature condition (at 35 and $55^{\circ}C$ periodically with 7 days interval), the rate constants at all water activities used in the experiments were higher than those at $45^{\circ}C$, the mean temperature of the cycle which affects other kinetic parameters including activation energies, $Q_{10}$ values and finally the shelf-lives. The data obtained from the fluctuating temperaure storage study will be used in prediction of shelf-life. The shelf lives assessed at $25^{\circ}C$ from the accelerated shelf-life tests ranged from 179 daysat $0.75\;a_w$ to 302 days at $0.44\;a_w$.

• Korean Journal of Food Science and Technology
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• v.30 no.6
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• pp.1426-1431
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• 1998

The research was undertaken to characterize enzymatic properties of Streptomyces albus amylase expressed in recombinant Bacillus subtilis. Molecular weight and pI of the purified enzyme were estimated to be 50 kD by SDS-PAGE and 4.3 by isoelectric focusing. The optimum temperature and optimum pH were $45^{\circ}C$ and 6.0, respectively. D-and Z-value were estimated to measure thermostability of the purified enzyme. The Z-value was estimated $17.7^{\circ}C$, which is lower than typical amylase. Maltotetraose was produced as a major component from soluble starch in the early state of reaction but gradually degraded to maltose. Thin layer chromatography was also performed to analyze the reaction products. The parameters involved in Michaelis-Menten enzyme kinetics were found to be the maximum velocity of 0.37 mM/min and the Michaelis constant of 0.13%, respectively.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.292-300
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• 2012

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters $K_m$, $V_{max}$, and $k_{cat}$ for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at $50^{\circ}C$, but the optimal temperature of activity was $37^{\circ}C$. It also showed maximal and optimal activities at pH 9.0. The values of $K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$ were $8.9{\pm}0.967{\times}10^{-3}$ M, $128{\pm}2.8$ U/mg protein, $106{\pm}2s^{-1}$, and $1.2{\pm}0.105{\times}10^4M^{-1}s^{-1}$, respectively. The L-asparaginase activity was reduced in the presence of $Mn^{2+}$, $Zn^{2+}$, $Ca^{2+}$, and $Mg^{2+}$ metal ions for about 52% to 31%. In addition, we found that $NH_4{^+}$, L-Asp, D-Asn, and ${\beta}$-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobial-type asparaginase II family.