• Microbiology and Biotechnology Letters
• /
• v.19 no.1
• /
• pp.100-108
• /
• 1991

Molecular characteristics and improving methods for thermal stability of enzyme have been considered. Intrinsic and extrinsic stabilizing mechanisms are two governing principles for enhanced thermal stability of enzyme in molecular basis. Factors contributing to the former and the latter mechanisms may be involved in the enhanced thermal stability of enzyme complementarily. Also, the methods for improving thermal stability of enzyme which comprise reaction in organic solvent system, chemical modification, immobilization, sequential unfolding and refolding, gene manipulation techniques and enzyme-antibody complexing are reviewed.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.3 no.1
• /
• pp.32-34
• /
• 1998

Phytase from Aspergillus species is a very heat unstable enzyme which inactivates to a great extent during the thermal processing of animal feed formulation. Various protein stabilization additives were tested to improve its heat stability. Among them, a basic amino acid, L-arginine remarkably increased the thermal stability of phytase in an aqueous solution state.

• Journal of Applied Biological Chemistry
• /
• v.66
• /
• pp.512-518
• /
• 2023

To address inherent weaknesses such as low mechanical strength and limited enzyme loading capacity in conventional chitosan or alginate beads, an additional step involving the exchange of anionic surfactants with hydroxide ions was employed to prepare porous chitosan hydrogel capsules for enzyme immobilization. Consequently, excellent thermal stability and long-term storage stability were confirmed. Furthermore, coating the porous chitosan hydrogel capsules with polydopamine not only improved mechanical stability but also exhibited remarkable enzyme immobilization efficiency (97.6% for M1-D0.5). Additionally, it was demonstrated that the scope of application for chitosan hydrogel beads, prepared using conventional methods, could be further expanded by introducing an additional step of polydopamine coating. The enzyme immobilization matrix developed in this study can be selectively applied to suit specific purposes and is expected to be utilized as a support for the adsorption or covalent binding of various substances.

• Korean Journal of Microbiology
• /
• v.13 no.2
• /
• pp.59-63
• /
• 1975

When the enzyme preparations were at various temperatures for 1 hour, the thermal stability for the enzyme was maximum at $30{\circ}C.$ The optimum temperature for the enzyme activity was at $40{\circ}C.$ When the enzyme preparations were exposed to various pHs for 22 hours, the enzyme stability was maximum at pH 3.8, and it was decreased gradually as the pH rose up to 4.8, above which the stability was greatly restored. When the exposure period was extended from 22 to pH's 3.0 and 5.9, but the stability tended to rise at pH's below 3.0 and above 5.9. The optimum pH for the enzyme activity was obtained at 4.8.

• The Korean Journal of Food And Nutrition
• /
• v.12 no.3
• /
• pp.265-270
• /
• 1999

The stabilization of Aspergillus sp. $\alpha$-amylase was attained by modification with periodate-oxidized sol-uble starch. The pH stability of modified enzyme was increased at pH 3~4 and 9~11 in the presence of $\alpha$-cyclodextrin($\alpha$-CD) compared with that of native enzyme. Thermal stability of the modified enzyme was increased. After treatment at 6$0^{\circ}C$ for 30min the activity remained 20% for the enzyme modified at pH 9.7 in the presence of $\alpha$-CD and tested in the presence of $\alpha$-CD 10% for the enzyme modified at pH 9.7 in the presence of $\alpha$-CD 0% for the native enzyme. The native enzyme and modified enzyme showed one peak in HPLC. The substrate specificity of the modified enzyme was not changed in HPLC analysis of reaction product.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.2
• /
• pp.332-339
• /
• 2010

The lipase from Mucor racemosus NRRL 3631 was partially purified by fractional precipitation using 60% ammonium sulfate, which resulted in a 8.33-fold purification. The partially purified lipase was then immobilized using different immobilization techniques: physical adsorption, ionic binding, and entrapment. Entrapment in a 4% agar proved to be the most suitable technique (82% yield), as the immobilized lipase was more stable at acidic and alkaline pHs than the free enzyme, plus 100% of the original activity was retained owing to the thermal stability of the immobilized enzyme after heat treatment for 60 min at $45^{\circ}C$. The calculated half-lives (472.5, 433.12, and 268.5 min at 50, 55, and $60^{\circ}C$, respectively) and the activation energy (9.85 kcal/mol) for the immobilized enzyme were higher than those for the free enzyme. Under the selected conditions, the immobilized enzyme had a higher $K_m$ (11.11 mM) and lower $V_{max}$ (105.26 U/mg protein) when compared with the free enzyme (8.33 mM and 125.0 U/mg protein, respectively). The operational stability of the biocatalyst was tested for both the hydrolysis of triglycerides and esterification of fatty acids with glycerol. After 4 cycles, the immobilized lipase retained approximately 50% and 80% of its original activity in the hydrolysis and esterification reactions, respectively.

• 한국생물공학회:학술대회논문집
• /
• 2003.04a
• /
• pp.445-449
• /
• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.32 no.1 s.55
• /
• pp.29-33
• /
• 2006

This article describes an enzyme stabilization method that allows the use of enzymes irrespective of environmental factors, especially heat, while maintaining their activity for a long time. We have designed enzyme microcapsules that consist of papain enzyme cores, poly(propylene glycol) interlayers, and poly(${\epsilon}-caprolactone$) walls. By confocal laser scanning microscopy measurements and the thermal stability of papain-loaded microcapsules, it is demonstrated that the papain is surrounded by a hydrophobic polyol layer and stabilized by the exclusive volume effect. In our study, improved thermal stability can be obtained by using more hydrophobic long-chained polyols, which is understood to be attributed to the effective formation of a hydrophobic polyol layer between the papain and the polymer wall by means of conformational anchoring in the interface.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.5
• /
• pp.738-744
• /
• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

• Journal of Life Science
• /
• v.11 no.3
• /
• pp.230-234
• /
• 2001

The cyclodextrin glucanotransferase(CGTase) gene of Bacillus macerans was expressed in Saccharomyces cerevisiae and the recombinant CGTase was partially purified from the yeast culture supernatant. The optimal pH and temperature of the CGTase were found to be 6.0 and 5$0^{\circ}C$, respectively. The pH and temperature stabilities of the recombinant enzyme were significantly enhanced and the half life at 55$^{\circ}C$ was about 60 hr. When the recombinant CGTase was reacted with 5% soluble starch, the conversion yield of total cyclodextrin (CD) from starch was estimated to be 41% at 48 hr, whereas the wild type enzyme showed the yield of 12%. This improvement of conversion yield and thermal stability of CGTase may be useful for the development of low-cost CD production process.