• 약학회지
• /
• 제51권3호
• /
• pp.199-205
• /
• 2007

Thermostable asparaginase was purified to homogeneity from mesophilic Strepfomyces lincolnensis M-20 by 30${\sim}$70% ammonium sulfate precipitation and asparagine-Sepharose CL 6B affinity column chromatography, The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 47 kDa, whereas by its mobility on Sephacryl S-300 column was around 180 kDa, indicating that the enzyme at the native stage acts as tetramer, The purified enzyme showed a single band on acrylamide gel electrophoresis. The optimum pH and temperature were pH 9.5 and 55${\circ}$C, respectively. Chemical modification experiments of purified asparagines implied the existence cystein residue located at or near active site. Purified asparaginase retained the 85% of the initial activity after incubation at 90${\circ}$C for 30 min. A correlation between themostability and resistance to proteolysis of commercial asparaginase and purified asparaginase from Strepfomyces lincolnensis M-20 was investigated. Purified thermostable asparaginase was resistant to trypsin and chymotrypsin treatment, while the commercial asparaginase was not themostable and was susceptible to proteolytic treatment with trypsin and chymotrypsin.

• Molecules and Cells
• /
• 제41권11호
• /
• pp.933-942
• /
• 2018

Traditionally, small-molecule or antibody-based therapies against human diseases have been designed to inhibit the enzymatic activity or compete for the ligand binding sites of pathological target proteins. Despite its demonstrated effectiveness, such as in cancer treatment, this approach is often limited by recurring drug resistance. More importantly, not all molecular targets are enzymes or receptors with druggable 'hot spots' that can be directly occupied by active site-directed inhibitors. Recently, a promising new paradigm has been created, in which small-molecule chemicals harness the naturally occurring protein quality control machinery of the ubiquitin-proteasome system to specifically eradicate disease-causing proteins in cells. Such 'chemically induced protein degradation' may provide unprecedented opportunities for targeting proteins that are inherently undruggable, such as structural scaffolds and other non-enzymatic molecules, for therapeutic purposes. This review focuses on surveying recent progress in developing E3-guided proteolysis-targeting chimeras (PROTACs) and small-molecule chemical modulators of deubiquitinating enzymes upstream of or on the proteasome.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2008년도 International Meeting of the Microbiological Society of Korea
• /
• pp.23-25
• /
• 2008

Serum complement proteins comprise an important system that is responsible for several innate and adaptive immune defence mechanisms. There were three well described pathways known to lead to the generation of a C3 convertase, which catalyses the proteolysis of complement component C3, and leads to the formation of C3 opsonins (C3b, iC3b and C3d) that fix to bacteria. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial-binding C3 fragments recognized by leukocytes. The spleen clears microorganisms from the blood. Individuals lacking this organ are more susceptible to Streptococcus pneumoniae. Innate resistance to S. pneumoniae has previously been shown to involve complement components C3 and C4, however this resistance has only a partial requirement for mediators of these three pathways, such as immunoglobulin, factor B and mannose-binding lectin. Therefore it was likely that spleen and complement system provide resistance against blood-borne S. pneumoniae infection through unknown mechanism. To better understand the mechanisms involved, we studied Specific intracellular adhesion molecule-grabbing nonintegrin (SIGN)-R1. SIGN-R1, is a C-type lectin that is expressed at high levels by spleen marginal-zone macrophages and lymph-node macrophages. SIGN-R1 has previously been shown to be the main receptor for bacterial dextrans, as well as for the capsular pneumococcal polysaccharide (CPS) of S. pneumoniae. We examined the specific role of this receptor in the activation of complement. Using a monoclonal antibody that selectively downregulates SIGN-R1 expression in vivo, we show that in response to S. pneumoniae or CPS, SIGN-R1 mediates the immediate proteolysis of C3 and fixation of C3 opsonins to S. pneumoniae or to marginal-zone macrophages that had taken up CPS. These data indicate that SIGN-R1 is largely responsible for the rapid C3 convertase formation induced by S. pneumoniae in the spleen of mice. Also, we found that SIGN-R1 directly binds C1q and that C3 fixation by SIGN-R1 requires C1q and C4 but not factor B or immunoglobulin. Traditionally C3 convertase can be formed by the classical C1q- and immunoglobulin-dependent pathway, the alternative factor-B-dependent pathway and the soluble mannose-binding lectin pathway. Furthermore Conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-$R1^+$ spleen macrophages, and formation of C3 ligands. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway. We propose that in the SIGN-R1 mediated complement activation pathway, after binding to polysaccharide, SIGN-R1 captures C1q. SIGN-R1 can then, in association with several other complement proteins including C4, lead to the formation of a C3 convertase and fixation of C3. Therefore, this new pathway for C3 fixation by SIGN-R1, which is unusual as it is a classical C1q-dependent pathway that does not require immuno globulin, contributes to innate immune resistance to certain encapsulated microorganisms.

• 한국식품영양과학회지
• /
• 제28권2호
• /
• pp.438-443
• /
• 1999

A livetin solution(LS) containing yolk immunoglobulins(IgY) was separated by treating the egg yolk with natural gum, carrageenan. Carrageenan has been used as a food ingredient. Relative absorbance of IgY LS after proteolysis was investigated. IgY LS was fairly stable to pepsin digestion at pH 3.0. However, IgY LS appeared to be susceptible to pepsin digestion at pH 2.0, showing 18% of relative absorbance and complete breakdown H chain after 30 min exposure. Relative absorbance of IgY LS was considerably high after exposure to trypsin and chymotrypsin for 8 hr. IgY LS showed especially good stability to chymotrypsin digestion.

• Journal of Dairy Science and Biotechnology
• /
• 제38권2호
• /
• pp.89-98
• /
• 2020

본 연구의 목적은 한국 전통 음식 김치에서 분리한 유산균의 특성을 연구하기 위해 형태학적, 생화학적 특성을 조사하였다. 한국의 전통 발효 식품에서 젖산균을 확인하기 위해 분리된 균주의 그람염색을 수행한 후 Macrogen에서 16S rRNA 분석 결과, DKGF9(Lactobacillus plantarum), DKGF1(Lactobacillus paracasei ), DKGF8(Lactobacillus casei ), DK207(Lactobacillus casei ), DK211(Lactobacillus casei )이 확인되었다. 우리는 한국의 전통 발효 식품인 김치에서 분리된 5가지 LAB의 기본 생물학적 활성에 대한 실험을 수행했다. 37℃, 55℃, 65℃, 75℃에서 각각 5분, 15분 5균주의 내열성 확인 결과, 상업 균주인 Lactobacillus acidophilus LA-5의 내열성과 유사하거나 더 높음을 보여주었다. 장내부착능에서는 선발균주 모두 상용균주와 비교했을 때 10⁷ CFU/mL 이상으로 우수한 결합능을 보여주었고, KCTC(한국생명공학연구원 생물자원센터)에서 분양받은 Escherichia coli KCTC1682, Salmonella enterica KCTC2054, Bacillus cereus KCTC3624 3종을 활용한 항균활성 결과, 모든 균주는 상업용 균주인 L. acidophilus LA-5와 비교하여 유사하거나 더 높은 항균 활성을 나타냈다. 단백질분해능력 실험에서, 5개의 균주는 clear-zone의 직경이 24시간에서 72시간으로 갈수록 점차 증가하고, L. paracasei DKGF1이 가장 큰 직경을 갖고 있어 단백질분해능력이 가장 우수한 것으로 나타났다. 5개의 균주로부터 선택된 3개의 균주는 ABTS, DPPH, FRAP, Hydroxyl radical scanenging 활성을 포함하여 다양한 항산화활성 효과를 나타냈다. 결과적으로, 5가지 균주 중에서 우수한 기능성을 갖는 L. paracasei DKGF1이 잠재적인 프로바이오틱스 활성을 나타내며, 건강 관련 제품의 개발에 유용한 균주라고 판단된다.

• Journal of Microbiology and Biotechnology
• /
• 제24권2호
• /
• pp.160-167
• /
• 2014

Oligopeptidase B (OpdB) is a serine peptidase widespread among bacteria and protozoa that has emerged as a virulence factor despite its function has not yet been precisely established. By using an OpdB-overexpressing Escherichia coli strain, we found that the overexpressed peptidase makes the bacterial cells specifically less susceptible to several proline-rich antimicrobial peptides known to penetrate into the bacterial cytosol, and that its level of activity directly correlates with the degree of resistance. We established that E. coli OpdB can efficiently hydrolyze in vitro cationic antimicrobial peptides up to 30 residues in length, even though they contained several prolines, shortening them to inactive fragments. Two consecutive basic residues are a preferred cleavage site for the peptidase. In the case of a single basic residue, there is no cleavage if proline residues are present in the $P_1$ and $P_2$ positions. These results also indicate that cytosolic peptidases may cause resistance to antimicrobial peptides that have an intracellular mechanism of action, such as the proline-rich peptides, and may contribute to define the substrate specificity of the E. coli OpdB.

• 한국식품영양과학회지
• /
• 제26권6호
• /
• pp.1237-1245
• /
• 1997

Interspecific fusants were made from the cells of two strains of Lactobacillus genus, a streptomycin resistant Lactobacillus sp. JC-7 and a kanamycin resistant L. acidophilus 88. The morphological and physiological properties of the fusants were examined by determining bacteriocin productivity, acid-producing activity, ability of carbohydrates utilization and three important enzyme activities. The fusants produced a bacteriocin against indicator strains and fusant No. 1, 4 exhibited a larger inhibition zone compared to that of L. acidophilus 88. $\beta$-Galactosidase, phospho-$\beta$-galactosidase, lipase activities and resistance to NaCl of Lactobacillus sp. JC-7 were better than those of L. acidophilus 88. Fusant No. 3 and 7 exhibited excellent lipase activities. Protease activity and acid productivity of L. acidophilus 88 were better than those of Lactobacillus sp. JC-7. Proteasse activities of all fusants were higher than those of parental strains, and expecially fusant No. 5 and 7 exhibited excellent proteolysis ability.

• Journal of Microbiology and Biotechnology
• /
• 제20권3호
• /
• pp.460-466
• /
• 2010

A mutant of green fluorescent protein ($GFPmut3^*$) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of $GFPmut3^*$ was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear $GFPmut3^*$. The circular $GFPmut3^*$ was $5^{\circ}C$ more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase. The circular $GFPmut3^*$ also displayed increased relative fluorescence intensity. In addition, chemical stability of $GFPmut3^*$ against GdnHCl revealed more stability of the circular form compared with the linear form.

• 한국응용과학기술학회지
• /
• 제30권2호
• /
• pp.348-355
• /
• 2013

오징어 젓갈에 오징어 먹즙을 2% 및 4% 농도로 첨가하고 $10^{\circ}C$에서 8주일간, $20^{\circ}C$에서 32일간 숙성시키면서 아미노태 질소와 근육단백질 변화를 분석한 결과는 다음과 같다. 오징어 먹즙이 첨가되지 않은 오징어 젓갈의 아미노태 질소는 식염 농도가 낮고 숙성온도가 높을수록 숙성 후반까지 계속 유의성 높게 증가하여 숙성이 촉진되었으며 오징어 근육의 단백질 변화는 myosin heavy chain이 숙성 초반에 현저히 분해되지만 actin의 변화는 거의 없어서 protease에 강하였다. 오징어 먹즙을 첨가한 오징어 젓갈의 아미노태 질소 함량은 숙성후반까지 계속 증가하였으나 증가폭은 무 첨가군에 비하여 적었으며 오징어 근육 단백질 중 myosin heavy chain은 숙성 중반에 현저히 분해되었으며 식염농도가 높고, 온도가 낮은 먹즙 첨가군은 분해 속도가 느렸다.

• 생명과학회지
• /
• 제19권8호
• /
• pp.1047-1054
• /
• 2009

구강편평상피암종은 말기에서 종종 화학치료요법제들이 유도하는 세포자멸사에 저항성을 보인다. 박테리아의 독에 대한 진전된 이해는 암치료에 대한 새로운 치료전략으로 제기되어지고 있다. 본 연구는 Pseudomonas aeruginosa exotoxin A (PEA)가 세포자멸사 기작을 통해 항암제에 저항성을 보이는 YD-9 구강편평상피암종의 생존율을 현격하게 떨어뜨림을 설명하고 있다. 세포자멸사현상은 핵의 형태학적 변화와 DNA 분절 생성을 통해 입증되었다. PEA는 caspase-3, -6, -9 의 분절과 활성화를 일으켰다. 그리고 이러한 반응들은 caspase 의 기질에 해당하는 poly (ADP-ribose) polymerase (PARP), DFF45, 그리고 lamin A 의 단백질 분해를 야기했다. 사립체 막전위 감소, cytochrome c와 Smac/DlABLO의 사립체로부터 세포질로의 유리, 그리고 AIF의 사립체에서 핵으로 이동 등이 관찰되었다. p53, p21 그리고 $14-3-3{\gamma}$는 증가되는 반면 cyclin B와 cdc2는 감소되었다. 이상의 결과들을 종합해 보면 PEA는 caspase를 활성화시키고, 사립체에 변화를 야기시키고 더 나아가서 세포주기 유전자를 조절함으로써 항암제에 대한 강한 저항성을 보이는 YD-9 세포에서 세포자멸사를 유도한다.