• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.34-45
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• 2012

A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.

• Korean Chemical Engineering Research
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• 제49권4호
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• pp.387-392
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• 2011

생촉매인 효소의 기질선택성은 다양한 분야에서 유용하게 이용되고 있다. 그 중에서도 효소결합면역흡착검사(enzyme-linked immunosorbent assay, ELISA)는 항원항체의 결합을 항체와 공유결합된 효소의 반응으로 나타냄으로써 다양한 항원들의 진단을 가능케 했다. 하지만 기존의 효소결합면역흡착검사는 하나의 항체당 하나의 효소가 결합된 형태이기 때문에 감도(sensitivity)의 증가 폭에 그 한계가 있으며, 이를 극복하기 위한 방안으로 하나의 항체당 결합된 효소의 수를 증가시킴으로써 혁신적인 감도의 향상을 가져오는 연구가 진행되었다. 최근 나노바이오촉매(nanobiocatalyst, NBC) 접근방식을 이용한 효소활성의 안정화는 효소결합면역흡착검사의 감도 향상뿐만 아니라 그 성능의 안정성을 확보할 수 있는 연구결과로 이어지고 있다. 본 총설에서는 일반적인 효소결합면역흡착검사의 기본적인 원리와 감도향상을 위한 연구, 그리고 성능안정성(performance stability)을 향상시키기 위한 나노바이오촉매-결합면역흡착검사(Nanobiocatalyst-Linked Immunosorbent Assay, NBC-LISA)에 대하여 살펴보고자 한다.

• 한국식품영양학회지
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• 제18권1호
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• pp.4-10
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• 2005

과요오드산-산화가용성전분은 Aspergillus awamori a-glucosidase의 pH 안정성을 증가시켰다. 40℃에서 두시간 항온시킨 결과, 과요오드산-산화가용성전분이 존재하지 않을 때의 효소는 pH 3∼7, 존재할 때의 효소는 pH 3∼9, 50℃에서 과요오드산-산화가용성전분이 존재하지 않을 때의 효소는 pH 3∼6, 존재할 때의 효소는 pH 3∼8 범위에서 안정하였다. 60℃에서는 과요오드산-산화가용성전분의 존재여부에 관계없이 효소는 pH 3∼6 범위에서 안정하였으나 pH 5와 6에서 과요오드산-산화가용성전분이 존재하면 효소의 잔존활성은 존재하지 않을 때보다 20% 더 높았다. 과요오드산으로 변형한 효소는 pH 9에서 활성이 70% 남았으나 변형하지 않은 효소는 남지 않아서 변형으로 안정성이 증가된 것으로 나타났다. 변형효소는 50℃에서 12%, 80℃에서 7%의 활성이 남았으나 변형시키지 않은 효소는 50℃에서 8%가 남고, 70℃이상에서는 남지 않았다. HPLC 분석 결과 pH 2 이하 및 9 이상에서는 효소의 서브유니트가 분리되고, 변성 중합되었다. 변형하지 않은 효소는 산성과 알칼리성 pH에서 변성되어 단백질의 구조가 무너졌지만 과요오드산-산화가용성전분이 존재하면 변성되지 않았다.

• 미생물학회지
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• 제25권3호
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• pp.221-228
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• 1987

Nocardioides sp. J-275L이 생산하는 세포의 adenine deaminase를 황산 암모늄 분획 , DEAE- Cellulose column c chromatography, DEAE-Sephadex A-50 column chromatography 및 Sephacryl S-200 superfine gel filtration 등의 파 정에 의해 5.2%의 수율로서 3889.5배로 부분정제하였으며, 본 효소의 분자량은 Sephacry] S-200 superfine gel에 의해 39, O 000 부근으로 측정되었다. 본 효소의 안정 pH와 온도는 pH7.와 $40^{\circ}C$로 나타났으며, 15%의 glycerol이 효소를 안정화시키는데 효과적이었다. 효소의 활성 최적 pH와 온도는 pH 7. 5 $40^{\circ}C$였다. 효소의 ademne에 대한 Km 값은 $7.4\times 10^{-5}$M로 측정되었으며, 검토된 punne a analogue 중에서 6-chloropurine. 2,6-diaminopurine, 6-bromopurine, 4-aminopyrazolo[3,4-dJ pyridine, 8-azaadenine 이 기질로 이용되었다. 본 효소는 0.1mM의 $Cu^{2+}, Fe^{3+}, Pb^{2+}, Hg^{2+}, Ag^{+}$ 등에 의해 활성이 크게 저해되었으며, $Pb^{2+}, Hg^{2+}, Ag^{+}$에 의해 저해된 효소의 활성은 EDTA에 의해 회복되었다. 검토된 일반적인 효소 저해제 중에서 1mM의 $\alpha$,$\alpha$'-dipyridyl, pentachlorophenol, and pCMB 등에 의해 효소의 활성 이 크게 저해되는 것으로 나타났다.

• 대한유전성대사질환학회지
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• 제14권1호
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• pp.37-41
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• 2014

Fabry disease is an X-linked disease caused by deficiency of the lysosomal enzyme alpha-galactosidase A. Affected males present anhydrosis, acroparesthesia and angiokeratoma, and subsequently cardiac, cerebral and renal complications are followed. Females and atypical variants show heterogeneous clinical symptoms. In 2001, two recombinant enzymes were approved for Fabry disease: agalsidase alpha and agalsidase beta. Since the introduction of enzyme replacement therapy (ERT), the number of long-term follow-up studies has been reported. Long-term ERT showed effectiveness on renal function in patients with chronic kidney disease, decrease or stabilization of left ventricular mass, and improvement of pain and quality of life. However, there were limited effects on cerebrovascular events and their mortality. Current literatures on the clinical effect of ERT have reported limited datain adult patients who have already advanced disease. Therefore, further study for pre-symptomatic patients and atypical variants is needed to verify the impact of ERT. This review summarized recent progresses in ERT and limitations of long-term effect of ERT in patients with Fabry disease.

• Journal of Microbiology and Biotechnology
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• 제20권7호
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• pp.1077-1085
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• 2010

With an emphasis on its thermal behavior with different pHs and salts, the kinetic and thermodynamic parameters of the purified polygalacturonase (PG) from E. carotovora subsp. carotovora (Ecc) BR1 were studied, as the characterization of an enzyme is significant in the context of burgeoning biotechnological applications. The thermodynamic parameters for polygalacturonic acid hydrolysis by the purified PG were ${\Delta}H^*$=7.98 kJ/mol, ${\Delta}G^*$=68.86 kJ/mol, ${\Delta}S^*$=-194.48 J/mol/K, ${\Delta}G_{E-S}$=-1.04 kJ/mol, and ${\Delta}G_{E-T}$=-8.96 kJ/mol. In addition, its turnover number ($k_{cat}$) was 21/sec. The purified PG was stable within a temperature range of $20-50^{\circ}C$ and was deactivated at $60^{\circ}C$ and $70^{\circ}C$. The thermodynamic parameters (${\Delta}H^*$, ${\Delta}G^*$, ${\Delta}S^*$) for the irreversible inactivation of the PG at different temperatures ($30-60^{\circ}C$) were determined, where the effectiveness of various salts and different pHs (4-8) for the thermal stability of the PG were also characterized. The efficacy of various salts for the thermal stability of the PG was in the following order: $MgCl_2$ > $BaCl_2$ > KCl > $CaCl_2$ >NaCl. Therefore, the present work presents the biochemical, substrate hydrolysis thermodynamics and the thermal stabilization parameters of the PG from Ecc.

• BMB Reports
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• 제40권3호
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• pp.315-324
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• 2007

The novel $\alpha$-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of $Ca^{2+}$ and EDTA. Therefore, KRA acts as a $Ca^{2+}$-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of $Ca^{2+}$ and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its Tm. The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis $\alpha$-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the $\alpha$-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.

• 한국물환경학회지
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• 제25권1호
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• pp.84-89
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• 2009

Our nation's water resources remain susceptible to contamination by phenolic agrichemicals. These compounds can be toxic to a variety of organisms including humans. Their disposal is restricted in many countries with strict limits for acceptable concentrations in drinking water. Enzyme-mediated in situ stabilization has been advocated as an approach for the treatment of phenolic compounds in soils and groundwater. This study reports the development of a new approach to quantify the activity of the HRP enzyme in aqueous systems. The method is based on the coupled processes of energy transfer and enhanced chemiluminescence using a luminol-$H_2O_2$-HRP system. In this study, the effects of solution pH, ionic strength and aqueous concentrations of HRP, $H_2O_2$ and enhancer were evaluated on the p-iodophenol-enhanced, HRP-catalyzed chemiluminescence reaction intensity in Tris-HCl buffer. All assay components were found to affect the maximum chemiluminescene intensity. The calibration curve for HRP showed the linear relationship with maximum light intensity.

• 한국식품영양과학회지
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• 제24권3호
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• pp.355-361
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• 1995

Lipoprotein lipase(LPL) is an acylglycerol hydrolase and is the extrahepatic enzyme responsible for the hydrolysis of triglyceride-rich plasma lipoproteins. LPL has been isolated from bovine milk by affinity chromatography on heparin-sepharose in 2M NaCl, 5mM barbital buffer, pH 7.4. Para-nitrophenyl butyrate(PNPB) was used as a substrate for the determination of LPL activity. Molecular weight of LPL was 55KD on 10% SDS-PAGE. When the effects of heparin on LPL activation were compared, LPL activity of heparin added group increased approximately 5 times higher than that of heparin non-added groups. These results indicated that heparin involved in the stabilization of LPL structure that led to increase enzyme activity. Furthermore, LPL activity increased about 4 times compared to the absence of heparin at various pH. LPL was stabilized when heparin was added either low or high salt concentrations. With the presence of heparin, NaCl concentration did not affect LPL activity at pH range 6∼9.

• Journal of Microbiology and Biotechnology
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• 제29권5호
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• pp.749-757
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• 2019

Nitrilase is a valuable hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh conditions of industrial reaction processes. To solve this problem, a nitrilase from Acidovorax facilis 72W was inserted into an Escherichia coli-Bacillus subtilis shuttle vector for spore surface display. Western blot, enzyme activity measurements and flow cytometric analysis results all indicated a successful spore surface display of the CotB-nit fusion protein. In addition, the optimal catalytic pH value and temperature of the displayed nitrilase were determined to be 7.0 and $50^{\circ}C$, respectively. Moreover, results of reusability tests revealed that 64% of the initial activity of the displayed nitrilase was still retained at the $10^{th}$ cycle. Furthermore, hydrolysis efficiency of upscale production of cyanocarboxylic acid was significantly higher in the displayed nitrilase-treated group than in the free group expressed by E. coli (pET-28a-nit). Generally, the display of A. facilis 72W nitrilase on the spore surface of Bacillus subtilis may be a useful method for immobilization of enzyme and consequent biocatalytic stabilization.