• BMB Reports
• /
• v.30 no.4
• /
• pp.262-268
• /
• 1997

The thermophilic and thermostable alkaline phosphatase was purified to near homogeneity from the osmotic lysis of Thermus caldophilus GK24, The purified enzyme had an apparent molecular mass of 108, 000 Da and consisted of two subunits of 54,000 Da. lsoelectric-focusing analysis of the purified enzyme showed a pi of 7.3. The enzyme contained two Cys residues, and its amino acids composition was quite different from that of Thermus aquaticus YT-1 alkaline phosphatase and Escherichia coli alkaline phosphatase, The optimum pH and temperature of the enzyme were 11.0-11.5 and $80^{\circ}C$ respectively. The enzyme was stable in the pH range of 9.0-12.0 at $25^{\circ}C$ for 36 h. and the half-life at $80^{\circ}C$ (pH 11.0) was 6 h. The enzyme was activated by $MgCl_2$ and inhibited by EDTA. With ${\rho}-nitrophenyl\;phosphate\;({\rho}NPP)$ as the substrate, the enzyme had a Michaelis constant $(K_m) $of $3.6{\times}10^{-5}M$, The enzyme preferentially hydrolyzed the phosphomonoester bond of AMP in ribonucleotides and glycerophosphate.

• Journal of the Korean Chemical Society
• /
• v.63 no.4
• /
• pp.260-265
• /
• 2019

Alcohol dehydrogenase (ADH) (EC 1.1.1.1) was selected as the enzyme which will be immobilized on ultrafiltration membrane by fouling with different transmembrane pressure of 1, 2 and 3 bars. ADH will catalyze formaldehyde (CHOH) to methanol ($CH_3OH$) and simultaneously oxidized nicotinamide adenine dinucleotide (NADH) to $NAD^+$. The concentration of enzyme and pH are fixed at 0.1 mg/ml and pH 7.0 respectively. The objective of the study focuses on the effect of different transmembrane pressure (TMP) on enzyme immobilization in term of permeate flux, observed rejection, enzyme loading and fouling mechanism. The results showed that at 1 bar holds the lowest enzyme loading which is 1.085 mg while 2 bar holds the highest enzyme loading which is 1.357 mg out of 3.0 mg as the initial enzyme feed. The permeate flux for each TMP decreased with increasing cumulative permeate volume. The observed rejection is linearly correlated with the TMP where increase in TMP will cause a higher observed rejection. Hermia model predicted that at irreversible fouling with standard blocking dominates at TMP of 3 bar, while cake layer and intermediate blocking dominates at 1 and 2 bar respectively.

• Fibers and Polymers
• /
• v.6 no.3
• /
• pp.181-185
• /
• 2005

Silk fibroin (SF) nanofibers were prepared by electrospinning and their application as an enzyme immobilization support was attempted. By varying the concentration of SF dope solution the diameter of SF nanofiber was controlled. The SF nanofiber web had high capacity of enzyme loading, which reached to $5.6\;wt\%$. The activity of immobilized a-chymotrypsin (CT) on SF nanofiber was 8 times higher than that on silk fiber and it increased as the fiber diameter decreased. Sample SF8 (ca. 205 nm fiber diameter) has excellent stability at $25^{\circ}C$ by retaining more than $90\%$ of initial activity after 24 hours, while sample SF11 (ca. 320 nm fiber diameter) shows higher stability in ethanol, retaining more than $45\%$ of initial activity. The formation of multipoint attachment between enzyme and support might increase the stability of enzyme. From these results, it is expected that the electrospun SF nanofibers can be used as an excellent support for enzyme immobilization.

• Journal of Microbiology and Biotechnology
• /
• v.2 no.3
• /
• pp.161-165
• /
• 1992

The gene encoding the bacteriolytic enzyme cell wall peptidoglycan hydrolase from alkalophilic Bacillus sp. was cloned in E. coli using pBR322 as a vector. A recombinant plasmid, designated pYTR451, was isolated and the size of the cloned HindIII fragment was found to be 4.8 Kb. The cell wall hydrolysis activity of an extract of the E. coli harboring the recombinant plasmid pYTR 451 was detected by SDS- polyacrylamide gel containing 0.2% (w/v) purified cell wall of Bacillus sp. The molecular weight of the enzyme was estimated to be about 27, 000 corresponding to the molecular weight of the Bacillus sp. bacteriolytic enzyme. The recombinant plasmid was found to contain the fragment originated from Bacillus sp. YJ-451 chromosomal DNA by Southern hybridization.

• Korean Chemical Engineering Research
• /
• v.54 no.2
• /
• pp.171-174
• /
• 2016

Enzyme fuel cells were composed of enzyme cathode and PEMFC anode. Enzyme cathode was fabricated by compression of a mixture of graphite particle, laccase as a enzyme and ABTS as a redox mediator, and then coated with Nafion ionomer. Open circuit voltage (OCV) were measured with variation of cathode manufacture factors, to find optimum condition of enzyme cathode. Optimum pressure was 4.0 bar for enzyme cathode pressing process. Highest OCV was obtained at 95% graphite composition in enzyme cathodee. Optimum glucose concentration was 0.4 mol/l in cathode substrate solution.

• Journal of Microbiology and Biotechnology
• /
• v.6 no.1
• /
• pp.19-25
• /
• 1996

Chitosanase from Bacillus sp. HW-002 was purified with CM-cellulose column chromatography, and HPLC with DEAE- TSK gel and YMC-pack Diol 120. The purified enzyme appeared as a single band on SDS-polyacrylamide gel. The molecular weight of the enzyme was estimated to be about 46 kDa on SDS-polyacrylamide gel, and was estimated to be about 23 kDa by GFC. The optimal pH of chitosanolytic activity was about pH 5.5-6.0, and the purified enzyme was most stable at pH 5.0. The optimal temperature of chitosanolytic activity was $65^{\circ}C$ and the enzyme was stable at $45^{\circ}C$ for 1 h. Chitosan was the most favorable substrate among various $\beta$-glucan. UVmax of the purified enzyme was 195 nmand was not noted around 280 nm. The main product of enzyme reaction with chitosan was chitobiose.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.11 no.1
• /
• pp.32-37
• /
• 2006

In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria, Ochrobactrum anthropi SY509, which was isolated from soil samples. O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to $70^{\circ}C$. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membranebound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.

• The Journal of Korean Institute for Practical Engineering Education
• /
• v.2 no.1
• /
• pp.35-41
• /
• 2010

An enzyme-catalized reaction is usually characterized by a very large increase in the rate and high specificity. Kinetics of simple enzyme-catalized reactions are often referred to as Michelis-Menten kinetics. A chemical that interferes with an enzyme's activity is called inhibitor. There are two types of enzyme inhibitions (viz. reversible and irreversible). If an inhibitor attaches to the enzyme with weak bonds, such as hydrogen bonds, the inhibition is usually reversible. Many enzyme reactions are also inhibited reversibly by their corresponding products. The rate of substrate disappearance together with the rate of product formation may be written by nonlinear differential equations. In the present study, numerical analyses of simple enzyme kinetics and inhibited enzyme kinetics are reported for the purpose of undergraduate education in engineering.

• Microbiology and Biotechnology Letters
• /
• v.13 no.2
• /
• pp.115-118
• /
• 1985

Rifamycin B oxidase converts rifamycin B to rifamycin S using oxygen as cosubstrate. Humnicola spp. (ATCC 20620) was treated with acetone and the cell powder was immobilized with cellulose acetate. The properties of the immobilized enzyme was examined. The optimum pHs of the immobilized and the free enzymes were 7.2. The optimum temperature of the immobilized enzyme was at 50-55$^{\circ}C$, which was 5$^{\circ}C$ higher than that of the free enzyme. The activities of the immobilized enzyme appeared less sensistive with respect to the changes of temperature and pH as compared to those of the free enzyme. Twenty percent of the enzyme activity was recovered when the enzyme was immobilized in 3mm beads. The storage stability was good below 4$0^{\circ}C$, but the activity decreased very rapidly above 5$0^{\circ}C$. The physical strength of the beads was good and was suitable as packing material in a three-phase enzyme reactor.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.3
• /
• pp.465-471
• /
• 2008

Highly active, stable, and magnetically separable immobilized enzymes were developed using carboxymethyl cellulose (CMC) and diethylaminoethyl cellulose DEAE-C; hereafter designated "DEAE" as supporting materials. Iron oxide nanoparticles penetrated the micropores of the supporting materials, rendering them magnetically separable. Lipase (LP) was immobilized on the surface of the supporting materials by using cross-linked enzyme aggregation (CLEA) by glutaraldehyde. The activity of enzyme aggregates coated on DEAE was approximately 2 times higher than that of enzyme aggregates coated on CMC. This is explained by the fact that enzyme aggregates with amine residues are more efficient than those with carboxyl residues. After a 96-h enantioselective ibuprofen esterification reaction, 6% ibuprofen propyl ester was produced from the racemic mixture of ibuprofen by using DEAE-LP, and 2.8% using CMC-LP.