• Biomolecules & Therapeutics
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• 제17권1호
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• pp.104-110
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• 2009

Phenylketonuria (PKU) is an inherited metabolic disorder caused by mutations in the phenylalanine catabolic enzyme, phenylalanine hydroxylase (PAH). The use of phenylalanine ammonia-lase (PAL) by oral and parenteral routes as a therapeutic drug for PKU has been severely limited due to inactivation by intestinal proteolysis and immune reactions. PEGylation was applied to PAL to reduce the degrees of antigenicity and proteolytic inactivation. Kinetic experiments with native PAL and pegylated PALs were performed, and pH stability, temperature stability, and protease susceptibility were evaluated. Enzyme linked immunosorbent assay (ELISA) was carried out to measure the immune complex between pegylated PALs and antiserum that had been extracted from a PAL-immunized mouse. Pegylated PAL, especially branched pegylated PAL (10 kDa, 1:32), was more active for phenylalanine and more stable in pancreatic proteases than native PAL. Native PAL was optimal at pH 8.5, corresponding to the average pH range of the small intestine; the same finding was noted for pegylated PALs. All linear and branched pegylated PALs had low reactivity with mouse antiserum, especially the 1:16 formulation with linear 5-kDa PEG and the 1:32 formulation with branched 10-kDa PEG. Therefore, we suggest the 1:32 formulation with branched 10-kDa PEG as the most promising formulation for enzyme replacement therapy.

• BMB Reports
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• 제32권3호
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• pp.247-253
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• 1999

Chicken egg ovalbumin is a non-inhibitory member of the serpin (serine protease inhibitors) family whose members share a common tertiary fold. In the present study, we succeeded in high-level production of a disulfide-free form of refolded recombinant ovalbumin. Conformational characterization of the recombinant ovalbumim revealed that it is well-folded, following two-state unfolding transition with the midpoint of transition at 4.7 M at $25^{\circ}C$. This value is very close to that of the reduced form of authentic ovalbumin. The recombinant ovalbumin can serve as a model molecule of non-inhibitory serpins in comparative studies with inhibitory members of the serpin family.

• 미생물학회지
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• 제29권2호
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• pp.80-84
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• 1991

We cloned and expressed hepatitis B viral core antigen (HBcAg) gene in E. coli using $P_{L}$ promoter system. For optimal expression of the gene, we undertook the studies on the effects of the distance between Shine-Dalgarno (SD) sequence and start codon, copy number of repressor gene, induction temperature, and the stability of the core antigen. The results demonstrated that the induction at 37.deg.C was more efficient than at 42.deg.C, and the 11 base pairs (bp) distance between SD sequence and start codon of HBcAg gene was more efficient than the 15 bp distance in E. coli. The copy number of cI857 repressor gene did not influence on the expression of HBcAg, and the expression level of HBcAg in mutant type (low protease activity) and wild type strains was almost the same. The produced core antigen appeared to be HBcAg not HBeAg judged by two different radioimmunoassat (RIA) kits. This result suggested that the antigen was stable in E. coli.i.

• 통합자연과학논문집
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• 제4권3호
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• pp.222-226
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• 2011

Cyclic AMP receptor protein(CRP) is involved in the activation of many genes corresponding to catabolite enzymes in Escherichia coli. In this study, mutant CRP(S128A) was used to elucidate the effect of Ser 128 on the cAMP-induced structural change. Based on the protease digestion and thermal analysis, serine 128 in CRP affects the cAMP binding capability and then structural change of CRP protein. In addition, CD spectra in near UV region revealed that S128A CRP retained the sensitive conformation to thermal effect relative to that of wild-type CRP, in spite of identical Tm values in the absence of cAMP.

• 한국축산식품학회:학술대회논문집
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• 한국축산식품학회 2005년도 제36차 추계 학술발표대회
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• pp.329-333
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• 2005

ACE inhibition activity of peptides was measured after 2 months of storage at $4^{\circ}C$ under condition of pH 6.0, 6.5, 7.0, 7.5, 8.0. and the ACE inhibitory activity were changed only slightly. After 2 months of chilled storage ($4^{\circ}C$), no dramatic change and significance was found. This indicates that acidic, neutral, weak alkali conditions did not affect ACE inhibitory activity of those peptides. Among peptide 1134, 1152, and 1155, peptides from thermolysin + protease A hydrolysates, inhibition activity of peptide 1134 and 1152 was decreased significantly at $60^{\circ}C$, however, they showed stable inhibition activity from $70^{\circ}C$ to $100^{\circ}C$ (P<0.001). Also, chromatogram of peptide 1134, 1152, and 1155 was shown that retention time of peptide of $60^{\circ}C$ was not correspond to the retention time of the rest of peptides. This indicated that temperature may change the inhibitory activity and profile of peptides.

• 한국식품영양학회지
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• 제9권2호
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• pp.143-150
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• 1996

The studies was carried out to investigate ripening degree and functional properties of EMC produced with pancreatic protease and palatase ML. During production of EMC, the amounts of free amino acid and free fatty acid were increased with increasing the reaction time. The amount of total nitrogen(T-N) and water soluble nitrogen(WSN) were increased with increasing time. EMC had contents of 1.79eA T-N and 0.52o WSN at 60 min of hydrolysis time. SRI and FRI value had also a similar correlationship. On the gel filtration, 2 kinds of soluble proteining capacity was also shown in alkali solution. Furthermore the foaming stability had the same result as that of the solubility. The water absorption of EMC showed the highest level at pH 4.0 and 5.0.

• 한국미생물·생명공학회지
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• 제52권1호
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• pp.76-87
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• 2024

Halophilic bacteria are promising reservoirs for halotolerant enzymes that have gained much attention in biotechnological applications due to their remarkable activity and stability. In this study, 62 halophilic bacterial strains isolated from a solar saltern were screened for the production of various extracellular enzymes. The results revealed that 31 strains (50%) were positive for amylase production while 26 strains (41.9%) were positive for protease. Further, 22 strains (35.48%) exhibited β-glucosidase activity and only 17 (27.41%) demonstrated lipase activity. Of the investigated halophiles, ten strains growing in the presence of ≥15% NaCl (w/v) were selected and identified based on their 16S rRNA gene sequences as Halomonas meridiana, Salinivibrio costicola, Virgibacillus oceani, Virgibacillus marismortui, Marinobacter lipolyticus, Halobacillus karajensis, Salicola salis, Pseudoalteromonas shioyasakiensis, Salinicoccus amylolyticus, and Paracoccus salipaludis. Therefore, the present study highlights the diversity of the culturable halophilic bacteria in a Mediterranean solar saltern, harboring various valuable halotolerant enzymes.

• KSBB Journal
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• 제21권5호
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• pp.394-400
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• 2006

이 연구의 최종 목표는 방향성 분자진화기술(directed molecular evolution)을 이용한 음이온에 안정한 papain의 개발이다. 음이온 안정성 papain 생산을 위한 유전자의 방향성 분자진화 방법 개발이 이루어졌으며 분자진화된 음이온 안정성 papain의 스크리닝 방법 개발됐다. 분자진화된 papain의 아미노산 서열 및 특성 분석과 분자진화된 재조합 papain의 생산방법 확립되었다. 분자진화된 재조합 papain의 formulation 및 제품 적용화 기술 개발이다. 연구 결과 Papain petidase IV 유전자의 확보 및 발현 균주 개발하였고 음이온 안정성 papain 유전자를 얻기 위한 분자진화의 방법 개발 및 조건 확립하였다. 분자진화 방법으로 error prone PCR 방법 확립, DNA shuffling을 통한 mutagenesis 방법 확립, staggered extension process 방법 확립 및 분자 진화된 음이온성 안정성 papain의 효율적 스크리닝 방법 개발하였다. Skim milk agar plate 이용, 활성 및 안정성이 뛰어난 개량형 papain을 Filter paper방법을 이용하여 screening 방법을 개발하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.180-184
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• 2003

선별된 7종의 균주는 넓은 범위의 산과 담즙산에 대해 기존에 보고된 결과에 비하여 높은 내성을 나타냈고, 장내세균인 E. coli에 대해서도 우수한 항균력을 나타냈고, 항생제 tetracycline, nisin, kanamycin, ampicillin. streptomycin에 대해서도 높은 내성을 보였다. 또한 7종의 균주는 어느 정도의 산을 생성하고 있었고, 네 가지 효소 amylase, protease, lipase, cellulase에 대해서도 높은 활성을 나타냈다. 균주 특성실험 결과, 7종의 균주는 그람음성균으로 모두 구형, 비운동성이고 균체의 색은 노란색이거나 흰색을 띠고 있고, 다양한 유기물에 대하여 분해력을 지니고 있었다.

• 한국토양비료학회지
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• 제21권4호
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• pp.380-385
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• 1988

Zeolile에 의한 Urease의 활성(活性)과 특성(特性)에 미치는 영향(影響)을 구명(究明)하기 위하여, Zeolite의 Urease 흡착양상(吸着樣相)과 흡착(吸着)된 Urease의 여러가지 성질(性質)들을 조사(調査)하였다. Zeolite의 존재하(存在下)에서 용액중(溶液中)에 유리(遊離)된 Urease는 흡착(吸着)이 최대(最大)에 도달(到達)할 때까지 Zeolite에 흡착(吸着)되며, pH 7.0에서의 최대흡착량(最大吸着量)은 11.3mg urease/100mg Zeolite이었다. Zeolite에 의한 Urease의 흡착(吸着)은 주로 정전기적(靜電氣的) 결합(結合)에 의하여 Zeolite의 외표면(外表面)에서 이루어지며, 흡착량(吸着量)의 70%이상(以上)이 30분(分) 이내(以內)에 흡착(吸着)되었다. 흡착(吸着)된 Urease는 Free Urease에 비(比)하여 활성(活性)이 89.6%나 감소(減少)되었으며, Km치(値)는 3배(倍) 이상(以上)이나 증가(增加)된 34.4mM이었다. 흡착(吸着)된 Urease의 최적(最適) pH는 6.5~7.0으로 Free Urease의 7.0에 비하여 그 범위(範圍)가 넓어졌으며, Protease에 대한 저항성(抵抗性)은 약(弱)하여 졌다. 또한, 기질특이성(基質特異性)과 열(熱)에 대한 안정성(安定性)에는 변화(變化)가 없었다.