STUDIES ON THE PEROXIDASE ACTIVITY OF RABBIT HEMOGLOBIN

주한승,김승수 한국생화학분자생물학회 (구 한국생화학회) 1987 추계학술대회집 Vol.- No.-

서해 갯벌로부터 유용 신 물질탐색 및 응용성

주한승,최장원,장정순,Ju, Han-Seung,Choe, Jang-Won,Jang, Jeong-Sun 한국미생물학회 2003 微生物과 産業 Vol.29 No.2

Bacillus clausii I-52의 형질 전환에 의한 Alkaline Protease의 생산성 향상 및 Pilot-scale 생산 연구

주한승,박동철,최장원 경상대학교 농업생명과학연구원 2013 농업생명과학연구 Vol.47 No.1

Plasmid types, pRB374-BCAP2 and pLip-BCAP2 carrying B. clausii alkaline protease (BCAP) were constructed, introduced into B. clausii I-52, and protease productivities were compared with B. clausii C5 (pHPS9-BCAP), which the BCAP gene was integrated into the chromosomal DNA. The protease yield was increased by 15% for B. clausii R6 (pRB374-BCAP2) and 61% for B. clausii C5 (pHPS9-BCAP) in an optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, K2HPO4 0.4%, Na2HPO4 0.1%, NaCl 0.4%, MgSO4⋅7H2O 0.01%, FeSO4⋅7H2O 0.05%, liquid maltose 2.5%, Na2CO3 0.6%) under the fixed culture condition (37℃, 48 h, 1 vvm, 650 rpm). The B. clausii L7 (pLip-BCAP2) transformant showed no significant difference. B. clausii C5 showing the highest protease yield could produce 105,685 U/mL from 300 L pilot-scale fermentation (37℃, 30 h, 1 vvm, 250 rpm). BCAP 유전자를 지니는 pRB374-BCAP2와 pLip-BCAP2를 B. clausii I-52에 도입 후, 염색체 integration에 의해 구성된 pHPS9-BCAP 형질전환체(B. clausii C5)와 alkaline protease 발현율을 비교하였다. 최적화 배지(대두박 2%, 밀가루 1%, 구연산나트륨 0.5%, K2HPO4 0.4%, Na2HPO4 0.1%, NaCl 0.4%, MgSO4⋅7H2O 0.01%, FeSO4⋅7H2O 0.05%, 물엿 2.5%, 탄산나트륨 0.6%)에서 액침배양(37℃, 48 h, 650 rpm, 1 vvm) 시, pRB374- BCAP2 및 pHPS9-BCAP 형질전환체 각각은 15% 및 61% 정도 alkaline protease 생산이 증가하였다. 그러나 pLip-BCAP2 형질전환체에서는 변화가 없었다. 최고의 활성 균주인, B. clausii C5를 300 L 규모 pilot-scale 액침 배양(37℃, 30 h, 250 rpm, 1 vvm) 시, alkaline protease 생산은 105,685 U/mL로 측정되었다.

토끼 Hemoglobin 의 Peroxidase 활성에 관한 연구

주한승,임주원,김승수 ( Han Seung Joo,Joo Won Lim,Soung Soo Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

Rabbit hemoglobin was purified by using ammonium sulfate fractionation, CM-Sephadex chromatography, hydroxyapatite chromatography and Sephadex G-100 gel filtration. The purified hemoglobin oxidized various synthetic and naturally occurring phenolic compounds in the presence of H₂O₂. The substrate specificity studies indicate that rabbit hemoglobin has about 10 times lower K_m values for guaiacol (0.39 mM) and o-dianisidine (0.09 mM) when compared to those of plant peroxidases. Among natural substrates tested, rabbit hemoglobin catalyzes the oxidation of caffeic acid, chlorogenic acid and indole-3-acetic acid quite rapidly compare to other naturally occurring phenolic compounds such as scopoletin, esculetin, ferulic acid and p-coumaric acid. In general, phenolic compounds having methoxy group appears to have high affinity to rabbit hemoglobin as revealed by their very low K_m values. Chemical modification studies indicate that p-chloromercuribenzoate inactivates the peroxidase activity of rabbit hemoglobin, whereas N-ethylmaleimide or iodoacetamide does not. Furthermore, the peroxidase activity of rabbit hemoglobin is rapidly inactivated by diethylpyrocarbonate, a histidyl selective reagent, unlike plant peroxidases.

Inactivation of the Peroxidase Activity of Rabbit Hemoglobin by Diethylpyrocarbonate

주한승,김승수,Joo, Han-Seung,Kim, Soung-Soo 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6

토끼 헤모글로빈의 peroxidase 활성은 histidine에 비교적 특이하게 반응하는 diethylpyrocarbonate에 의하여 불활성화 되었다. 50 mM sodium phosphate 완충용액 (pH 6.0)을 사용하였을 때 $30^{\circ}C$에서 modification되는 속도상수는 $220M^{-1}min^{-1}$이었으며, N-carbethoxylhistidyl 유도체의 생성으로 인한 242 nm에서의 흡광도 증가가 동시에 관찰되었다. 그러나 280nm에서의 흡광도 변화는 관찰되지 않았으며 토끼 헤모글로빈의 peroxidase 활성은 N-acetylimidazole, phenylglyoxal 및 N-ethylmaleimide 등에 의하여 활성이 저하되지 않았다. Hydroxylamine은 peroxidase 활성의 inhibitor로 작용하기 때문에 diethylpyrocarbonate에 의하여 불활성화된 토끼 헤모글로빈의 peroxidase 활성을 회복시키지 못했지만, 기질인 guaiacol은 diethylpyrocabonate에 의한 불활성화를 약간 감소시켰다. Tsou의 통계적 분석방법에 의하여 peroxidase 활성이 완전히 사라질 때 헤모글로빈 1분자당 11개의 histidine 잔기가 modification되었으며 그 중 1개의 histidine만이 peroxidase 활성에 필수적인 것으로 밝혀졌다. 또한 5 mM $H_2O_2$로 처리한 토끼 헤모글로빈의 경우 diethylpyrocarbonate에 의한 불활성화가 촉진되고, diethylpyrocarbonate로 modification된 apohemoglobin이 native 헤모글로빈으로 재조합하지 못하는 것으로 보아 essential histidine이 heme 근처의 distal histidine일 것으로 추정된다. Diethylpyrocarbonate inhibits the peroxidase activity of rabbit hemoglobin with a second order rate constant of $220M^{-1}min^{-1}$ at pH 6.0 and $30^{\circ}C$. The increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives and other modification studies suggest that the modification of histidine residues is responsible for the loss of activity. However, treatment of inactivated hemoglobin with hydroxylamine does not restore catalytic activity due to the inhibitory effect of hydroxylamine itself on the peroxidase activity of hemoglobin. The substrate guaiacol have partially reduced the diethylpyrocarbonate dependent inactivation, and a reconstitution experiment using diethylpyrocarbonate treated apohemoglobin shows that it is not reconstituted to hemoglobin having full activity. The decrease of absorption at Soret region (406 nm) upon diethylpyrocarbonate modification of native hemoglobin and the acceleration of inactivation by preincubation of the diethylpyrocarbonate inactivated hemoglobin with $H_2O_2$ (5 mM) implies that the essential residue is distal histidine as previously proposed in case of cytochrome C peroxidase.

Bacillus clausii I-52로부터 alkaline protease 유전자의 클로닝 및 발현

주한승,최장원 경상대학교 농업생명과학연구원 2011 농업생명과학연구 Vol.45 No.6

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases. 인천 연안의 심하게 오염된 갯벌로부터 강력한 세포외 알카리성 단백질 분해효소를 생산하는 호알카리성 Bacillus clausii I-52를 분리하였으며, 이 균주로부터 알카리성 단백질 분해효소의 유전자를 cloning하여 서열 분석을 하였다. Chromosome 서열이 완전히 밝혀진 Bacillus subtilis의 서열을 기초로 하여 알카리성 단백질 분해효소 및 promoter를 포함하도록 primer를 고안하여 PCR을 수행하여 2,277 bp의 DNA 단편을 얻었으며 BLAST 분석 결과 29 개의 아미노산으로 이루어진 signal peptide, 77 개의 아미노산으로 이루어진 propeptide 및 275 개의 아미노산을 갖는 활성형의 BCAP으로 구성된 총 381 개의 아미노산을 코딩하는 1,143 bp의 open reading frame을 확인하였다. 활성형 BCAP의 N-말단 아미노산은 Ala이며, 분자량 및 pI 값은 각각 27698.7 Da과 6.30으로 계산되었다. 아미노산 상동성을 분석한 결과, B. subtilis 유래의 nattokinase precursor 및 B. subtilis BSn5 유래의 subtilisin E precursor와 99%의 서열 상동성을 나타내어 B. clausii I-52 유래의 BCAP은 subtilisin 계열의 단백질 분해효소임을 확인하였다. E. coli BL21(DE3)에서 발현한 재조합 BCAP는 N-Suc-Ala-Ala-Pro-Phe-pNA 를 효율적으로 분해하였다. Refolding한 재조합 BCAP은 전형적인 serine protease inhibitor인 PMSF에 의하여 강하게 효소 활성이 억제됨으로써 serine protease 계열의 단백질 분해효소임을 알 수 있었다.

Diethylpyrocarbonate 에 의한 토끼 헤모글로빈의 Peroxidase 활성의 불활성화

주한승,김승수 ( Han Seung Joo,Soung Soo Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

Diethylpyrocarbonate inhibits the peroxidase activity of rabbit hemoglobin with a second order rate constant of 220 M^(-1)min^(-1) at pH 6.0 and 30℃. The increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives and other modification studies suggest that the modification of histidine residues is responsible for the loss of activity. However, treatment of inactivated hemoglobin with hydroxylamine does not restore catalytic activity due to the inhibitory effect of hydroxylamine itself on the peroxidase activity of hemoglobin. The substrate guaiacol have partially reduced the diethylpyrocarbonate dependent inactivation, and a reconstitution experiment using diethylpyrocarbonate treated apohemoglobin shows that it is not reconstituted to hemoglobin having full activity. The decrease of absorption at Soret region (406 nm) upon diethylpyrocarbonate modification of native hemoglobin and the acceleration of inactivation by preincubation of the diethylpyrocarbonate inactivated hemoglobin with H₂O₂ (5 mM) implies that the essential residue is distal histidine as previously proposed in case of cytochrome C peroxidase.

Studies on the Peroxidase Activity of Rabbit Hemoglobin

주한승,임주원,김승수,Joo, Han-Seung,Lim, Joo-Won,Kim, Soung-Soo 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6

토끼 erythrocyte lysate으로부터 hemoglobin을 $(NH_4)_2SO_4$ 분별침전, CM-Sephadex 크로마토그래피, hydroxyapatite 크로마토그래피, Sephadex G-l00 겔 크로마토그래피 방법을 이용하여 순수 정제하였다. 정제한 토끼 hemoglobin은 peroxidase 활성을 나타냈으며 guaiacol과 o-dianisidine을 기질로 사용하였을 경우 식물 peroxidase보다 10배 가량 낮은$K_m$값을 나타냈다. 여러 phenol 화합물들을 기질로 사용하였을 때 토끼 hemoglobin은 caffeic acid, chlorogenic acid, indole-3-acetic acid를 빠르게 산화하였으며, disubstituted phenol 화합물과 methoxylated phenol 화합물들이 rabbit hemoglobin에 대하여 친화력이 큰 것으로 나타났다. Chemical modification 실험결과 토끼 hemoglobin은 iodoacetamide와 N-ethylmaleimide에 의하여 활성이 억제되지 않지만 p-chloromercuribenzoate에 의해서는 활성이 현저히 억제되었다. 또한 식물 peroxidase와는 달리 hemoglobin의 peroxidase 활성은 diethylpyrocarbonate에 의하여 급격히 불활성화 되었다. Rabbit hemoglobin was purified by using ammonium sulfate fractionation, CM-Sephadex chromatography, hydroxyapatite chromatography and Sephadex G-100 gel filtration. The purified hemoglobin oxidized various synthetic and naturally occurring phenolic compounds in the presence of $H_2O_2$. The substrate specificity studies indicate that rabbit hemoglobin has about 10 times lower $K_m$ values for guaiacol (0.39 mM) and o-dianisidine (0.09 mM) when compared to those of plant peroxidases. Among natural substrates tested, rabbit hemoglobin catalyzes the oxidation of caffeic acid, chlorogenic acid and indole-3-acetic acid quite rapidly compare to other naturally occurring phenolic compounds such as scopoletin, esculetin, ferulic acid and p-coumaric acid. In general, phenolic compounds having methoxy group appears to have high affinity to rabbit hemoglobin as revealed by their very low $K_m$ values. Chemical modification studies indicate that p-chloromercuribenzoate inactivates the peroxidase activity of rabbit hemoglobin, whereas N-ethylmaleimide or iodoacetamide does not. Furthermore, the peroxidase activity of rabbit hemoglobin is rapidly inactivated by diethylpyrocarbonate, a histidyl selective reagent, unlike plant peroxidases.

Bacillus clausii I-52의 Chromosomal Integration에 의한 Alkaline Protease의 생산성 향상

주한승,박동철,최장원 경상대학교 농업생명과학연구원 2012 농업생명과학연구 Vol.46 No.1

To increase productivity of a strong extracellular alkaline protease (BCAP), stable strains of Bacillus clausii I-52 carrying another copy of BCAP gene in the chromosome were developed. Integrative vector, pHPS9-fuBCAP carrying BCAP promoter, ribosome binding site, signal sequence and active protease gene was constructed and transferred into B. clausii I-52, and integration of the constructed plasmid into chromosome was identified by PCR. An investigation was carried out on BCAP production by B. clausii I-52 and transformant C5 showing the highest relative activity of alkaline protease using submerged fermentation. Maximum enzyme activity was produced when cells were grown under the submerged fermentation conditions at 37℃ for 48 h with an aeration rate of 1 vvm and agitation rate of 650 rpm in a optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, K2HPO4 0.4%, Na2HPO4 0.1%, NaCl 0.4%, MgSO47H2O 0.01%, FeSO47H2O 0.05%, liquid maltose 2.5%, Na2CO3 0.6%). A protease yield of approximately 134,670 U/ml was achieved using an optimized media, which show an increase of approximately 1.6-fold compared to that of non-transformant (83,960 U/ml). When the stability of transformant C5 was examined, the integrated plasmid pHPS9-fuBCAP was detected in the transformant after cultivation for 8 days, suggesting that it maintained stably in the chromosomal DNA of transformant C5. 인천 연안 갯벌에서 분리한 호알카리성 Bacillus clausii I-52로부터 세포외 알카리성 단백질 분해효소 (BCAP)의 발현 및 생산성을 증가시키기 위하여 BCAP promoter, ribosome 결합 서열, 신호서열, 전구체 서열 및 활성형 BCAP 유전자를 cloning한 재조합 plasmid pHPS9-fuBCAP을 penicillin-protoplast 법으로 B. clausii I-52의 염색체 DNA에 integration 하였고, 도입된 plasmid pHPS9-fuBCAP 유전자는 PCR에 의해 확인하였다. 가장 높은 단백질 분해효소 상대 활성을 보이는 선별된 transformant C5를 생산 최적화 배지(대두박 2%, 밀가루 1%, 구연산나트륨 0.5%, K2HPO4 0.4%, Na2HPO4 0.1%, NaCl 0.4%, MgSO47H2O 0.01%, FeSO47H2O 0.05%, 물엿 2.5%, 탄산나트륨 0.6%)에서 액침 배양법(배양온도, 37℃; 배양 시간, 48 h; 교반 속도, 650 rpm; 통기 속도, 1 vvm)으로 배양하여 단백질 분해효소를 발현 및 분비시켰을 때, BCAP 발현 양(134,670 U/ml)은 wild-type(83,960 U/ml)에 비하여 약 1.6 배 증가하였으며, 비활성도(91,611.5 U/mg 단백질)는 wild-type(71,760 U/mg 단백질)에 비하여 약 1.3 배 증가하였다. 또한, B. clausii I-52 염색체 DNA에 integration된 pHPS9-fuBCAP plasmid는 단백질 발현과 함께 8일간의 계대배양 동안에 안정하게 유지되고 있음을 확인하였다.

Molecular Cloning and Functional Expression of a Fibrinolytic Protease Gene from the Polychaeta, Periserrula leucophryna

주한승,나경수,Hee Sung Park,최장원 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.1

Full-length cDNA encoding a fibrinolytic protease (PLFP) from the cDNA library of the polychaete,Periserrula leucophryna was cloned, sequenced and expressed in Escherichia coli. The entire cDNA of the PLFP clone was 921 bp (CDS: 41-837), including a coding nucleotide sequence of 798 bp, a 5'-untranslaed region of 40 bp, and a 3'-noncoding region of 83 bp. The ORF encoded a 265-amino acid polypeptide precursor consisting of a 36-residue signal sequence and a 229-residue mature polypeptide. The sequence alignment results of PLFP revealed sequence similarity with several fibrinolytic enzymes. Sequence analysis revealed a conserved catalytic triad of His78, Asp126 and Ser219 residues, suggesting that PLFP is a serine protease. Mature PLFP had an apparent molecular weight of approximately 25 kDa and was produced in inclusion bodies when expressed in E. coli. Substrate specificity results that recombinant PLFP was active towards Arg-X or Lys-X and did not hydrolyze substrates with nonpolar amino acids at the P1 site. Recombinant PLFP was strongly inhibited by typical serine protease inhibitors, further indicating that PLFP is a member of the serine protease family. PLFP was able to dissolve artificial plasminogenfree fibrin, and its fibrinolytic behavior was similar to that of plasmin. Taken these results together, PLFP is a new member of the fibrinolytic enzyme family with selective specificity on fibrin, and the availability of PLFP offers an attractive alternative approach for thrombolysis therapy because rPLFP is believed to have advantages over currently used plasminogen activators, that is, lower price and lower side effect.