내열성 Chitinase 생산균주의 분리 및 효소생산 특성

홍범식,윤호근,신동훈,조홍연 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.5

자연계 고온환경으로부터 내열성 chitinase 우수하고 반응산물로 N-acetyl-D-glucosamine 이량체(GlcNAc)_2를 생산하는 균주를 분리 선별하고 Bacillus licheniformis KFB-C14로 동정하였다. 선별균주의 효소생산 특성은 탄소원으로서 효소기질인 colloidal chitin이 첨가될 때만이 생합성이 유도되었으며 유도제의 첨가시기에 의해 효소생산이 크게 영향을 받았다. 각종 무기, 유기태 질소원 중 yeast extract가 활성과 비활성을 각각 약 2배 증가시켰으며 높은 친화도를 나타내었다. 균의 최대생육과 효소의 최대생산온도는 55℃이었다. 본 균주의 내열성 chitinase 생산에 미치는 최적배양조건은 1.2% colloidal chitin, 0.15% K_2HPO_4, 0.05% KH_2PO_4 0.01% MgSO_4·7H_2O, 0.1% yeast extract, pH 6.5의 배지를 55℃, 150rpm에서 40시간 회전진탕배양 하였을 때로 3.89 units/ml의 효소활성과 7.4 units/mg의 비활성을 나타내었다. A strain capable of producing thermostable chitinase suitable for chitooligosaccharide production was isolated from high temperature environment and identified as Bacillus licheniformis. The chitinase from Bacillus licheniformis KFB-C14 was only induced by addition of colloidal chitin into the basal medium as carbon source, showing the decrease of the chitinase production by supplemental addition of other carbon sources into the medium containing 1.0% colloidal chitin. Among organic and inorganic nitrogen sources, yeast extract was the most effective for the increase of total activity and specific activity, and had high affinity for the enzyme production. The optimum temperature of cell growth and thermostable chitinase production was 55℃. The optimum culture medium was composed of 1.2% colloidal chitin, 0.15% K_2HPO_4, 0.05% KH_2PO_4 0.01% MgSO_4·7H_2O, 0.1% yeast extract (pH 6.5). Bacillus licheniformis KFB-C14 produced the thermostable chitinase of 3.89 units per culture fluid and 7.4 units per mg protein under rotary shaking at 150 rpm for 40 hr.

Bacillus licheniformis KFB-C14가 생산하는 내열성 Chitinase의 정체 및 특성

홍범식,윤호근,신동훈,조홍연 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.5

Bacillus licheniformis KFB-C14가 생산하는 내열성 chitinase를 30~70% ammonium sulfate fractionation, DEAE-Toyopearl 650M, Butyl-Toyopearl 650M, TSK-Gel Toyopearl HW-55F에 의해 정제도 66배, 수율 21%로 전기영동적으로 균일하게 정제하였다. 정제 단백질은 gel permeation chromatography에 의해 86,000±2,000의 분자량을 나타내었으며, SDS 전기영동에 의해 밝혀진 본 효소의 subunit 구조는 monomer였다. 효소 단백질의 안정성을 검토한 결과 80℃에서 30분 열처리에 의해 56%, 37℃에서 20분간 40% ethanol과 ethyl acetate, 단백질 변성제 등의 처리시에도 50% 이상의 잔존활성을 나타냄으로써 공업적으로 유용성이 높은 안정한 단백질로 판명되었다. 효소반응의 최적 pH와 온도는 pH 6.0과 60℃이었고 Mn^2+ 이온에 의해 효소 활성이 저해되었으나 EDTA, N-ethylmaleimide, p-chloromercuribenzoate 등에 의한 활성감소는 관찰되지 않음으로써 금속효소 또는 thiol계 효소에 속하지 않음을 알 수 있었다. 본 효소는 colloidal chitin, 시판용 chitin에는 반응성이 높았으나 exo형 chitinase의 대표적인 기질인 p-nitrophenyl-2-aectamido-2deoxy-β-glucopyranoside, NN'-diacetylchitobiose에는 전혀 반응성을 보이지 않는 전형적인 endo형의 chitinase였다. 본 효소는 colloidal chitin으로부터 주로 (GlcNAc)_2를, 반응시간 경과에 따라 (GlcNAc)_1과 (GlcNAc)_3을 생성하는 반응성을 보였다. Chitinase (EC 3.2.1.14) from culture fluid of Bacillus licheniformis KFB-C14 was purified 66-folds to homogenity in overall yield of 21% by ammonium sulfate fractionation, DEAE-Toyopearl, Butyl-Toyopearl and TSK-Gel HW-55F column chromatography. The enzyme protein had a molecular weight of about 86,000 and was composed of one subunit. The enzyme was significantly stable not only at high temperature but also on treatment with organic solvents and protein denaturants such as SDS, urea and guanidine·HCl. The optimum temperature and pH for reaction was 60℃ and 6.0,respectively. The enzyme activity was inhibited by only Mn^2+ ion, but not inhibited by EDTA, N-ethylmaleimide and pCMB. The enzyme had high activity with colloidal chitin (V_max: 421) and commercial chitin (V_max :480), but not with typical substrates of exo type chitinase. The thermostable chitinase had an useful reactivity for producing functional chitooligosaccharide, showing the production of (GlcNAc)_1, (GlcNAc)_3, and (GlcNAc)_2 as major product.

Flavobacterium meningosepticum의 Nucleoside Oxidase와 Peroxidase 생산특성

최양문,조홍연,양한철 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

Flavobacterium meningosepticum로부터 nucleoside oxidase와 peroxidase의 최대생산 조건을 검토한 결과 탄소원으로는 surcose, 질소원으로는 Polypepton이 좋았으며, Fe^2+, Fe^3+이나 또는 이들 이온과 함께 Cu^2+을 배지에 첨가시 두 효소의 생산량은 증가하였다. 또한 2'-deoxyguanosine, 2'-deoxyadenosine, N^6-methyladenosine 및 1-methyladenosine의 첨가는 두 효소의 생산을 증가시켰으며 특히 N^6-methyladenosine과 1-methyladenosine의 첨가는 생산된 균체량은 적었으나 두 효소생산량은 증가시켰다. 배지조건 및 배양시간에 따른 효소생산의 양상을 검토한 결과 두 효소는 거의 유사한 경향을 보였고 효소생산에 미치는 환경인자들 중 산소의 충분한 공급이 필수적이었다. 이상의 결과들로부터 세포내에서 nucleoside가 nucleoside oxidase에 의해 산화될 때 생성되는 H_2O_2를 peroxidase가 분해할 가능성이 시사되었다. Optimal cultural conditions were investigated for the maximal productivity of nucleoside oxidase and peroxidase from Flavobacterium meningosepticum. Sucrose and Polypepton were the best as a carbon source and a nitrogen source. Fe^2+, Fe^3+ and Cu^2+ increased the activities of the two enzymes and were essential in medium containing peptone as a nitrogen source. Nucleoside derivatives such as 2'-deoxyguanosine, 2'-deoxyadenosine, N^6-methyladenosine and 1-methyladenosine were effective for the production of the two enzymes. Especially, the addition of N^6-methyladenosine and 1-methyladenosine decreased cell growth, but increased the two enzyme activities. High level of oxygen also was essential factor for formation and/or induction of these enzymes. From the summary of this study about optimal medium and environmental conditions, nucleoside oxidase was biosynthesized in proportion to peroxidase. These results suggested that the role of peroxidase should be degradation of H_2O_2 generated by nucleoside oxidase in the cell of Flavobacterium meningosepticum.

Flavobacterium meningosepticum이 생산하는 Nucleoside Oxidase의 효소학적 특성

최양문,조홍연,양한철 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.5

Flavobacterium meningosepticum의 cell free extract로부터 정제된 nucleoside oxidase의 분자량은 480,000이었으며, subunits 구조는 SDS-PAGE에 의해 81,000, 68,000, 32,000과 16,000의 분자량을 갖는 8개 이상의 polypeptide로 구성되어 있었다. 본 효소의 등전점은 5.1이었으며 visible absorption spectra의 결과 전형적인 hemoprotein의 양상을 나타내었으며 효소학적 방법에 의해 정량한 결과 nucleoside oxidase 1 mole당 3 mole의 FAD을 함유하고 있는 flavohemoprotein으로 확인되었다. 본 효소는 1 mM SnCl_2 와 PbCl_2에 의해 각각 94%와 90%의 효소활성이 저해되었으며, hemoenzyme 저해제(NaCN, NaN_3)와 flavoenzyme 저해제(acriflavine, quinacrine)에 의해서도 효소활성이 저해되었다. 효소반응의 최적온도는 50℃, 최적 pH는 7.0이었으며, 60℃ 이하의 온도와 pH 5.5~9.0 범위에서 효소단백질은 안정함을 나타내었다. The molecular weight of the purified nucleoside oxidase estimated by gel filtration column chromatography was 480,000 and the enzyme protein was composed of four nonidentical subnits (81,000, 69,000, 32,000 and 16,000). On the basis of the visible absorption spectra and the enzymatic determination of the purified enzyme, the enzyme was supposed as a hemoprotein and also a flavoprotein containing 3 moles of FAD per 1 mole of enzyme. The isoelectric point of the enzyme was pH 5.1. Addition of metal salts such as 1 mM SnCl_2 and PbCl_2 into an enzyme reaction solution inhibited the enzyme activity by 94 and 90%, respectively. The enzyme activity was also lost significantly by hemoenzyme inhibitors such as NaCN and NaN_3 and flavoenzyme inhibitor, acriflavine and quinacrine. The maximal nucleoside oxidase activity was observed at pH 7.0 and 55℃. The nucleoside oxidase was relatively stable in the range of pH 5.5~9.0 and below 55℃.

Flavobacterium meningosepticum 기원 Peroxidase의 정제 및 특성

최양문,조홍연,양한철 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.5

Peroxidase를 Flavobacterium meningosepticum의 cell free extract로부터 약 8% 수율로 80배 정제하였다. Peroxidase의 분자량은 Sephacryl S-300으로 측정한 결과 220,000이였으며, subunits는 SDS-FAGE에 의해 54,000의 동일한크기의 4개로 구성되어진 것으로 추정되었다. 본 효소의 visible absorption spectra는 410, 500 및 630 nm에서 최대흡수를, 환원형에서는 435와 500 nm에서 최대흡수를 나타내어 전형적인 hemoprotein으로 관찰되었으며, 등전점은 4.1이였다. 수소공여체로서 N-ethyl-n-(2-hydroxy-3-sulfopropyl)-m-toluidine (Toos)를 사용할 때 반응최적 온도는 50℃, 반응 최적 pH는 5.5였으며, carbonyl reagent 및 Hg^2+ 이온에 의해 효소활성저해를 받았다. Peroxidase was purified to homogeneity from cell free extract of Flavobacterium meningosepticum. The molecular weight of the enzyme estimated by gel filtration column chromatography was 220,000. A identical subunit (54,000) was detected on SDS-PAGE of the enzyme. From these results, the enzyme was supposed to have four identical subunits. On the basis of the visible absorption spectra of the purified enzyme, the enzyme was a typical hemoprotein. The isoelectric point of the enzyme was 4.1. On using N-ethyl-n-(2-hydroxy-3-sulfopropyl)-m-toluidine (Toos) as a hydrogen donor, the enzyme showed optimum activity at the pH 5.5 and 50℃. The enzyme activity was inhibited by carbonyl reagent and Hg^2+.

Corynebacterium sp. K-199가 생산하는 단백질성 생물용집제

김영준,최양문,조홍연,양한철 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

미생물응집제를 개발하기 위하여 자연계로부터 방선균 600여 균주와 보관균주를 대상으로 응집활성이 우수한 균주를 선별하고 동정한 결과 corynebacteium 속으로 동정되었다. 응집제 생산을 위한 최적조건은 2% glycerol, 0.4% peptone, 0.1% yeast extract, 0.3% K_2HPO_4, 0.1% CaCO_3, 0.05% NaCl, 0.1% MgSO_4·7H_2O, pH 7.5의 배지 100 ml을 500ml baffle flask에 넣고 96시간 회전진탕배양할 때였으며 상기의 조건으로 jar fermentor 배양시 96시간에서 780 units/ml의 응집활성을 나타내었다. 응집물질은 pronase 처리와 periodate 산화에 의해 각각 무처리구와 13%와 106%의 활성을 나타냄으로써 응집활성은 주로 단백질에 기이함을 알 수 있다. About 600 microorganisms isolated form soil, marsh, compost, etc. were examined for their flocculating ability in the kaolin suspension and swine wastewater. Among them, K-199 was the best producer of flocculant and was identified to be a species belong to the genus corynebacteium. The maximum production of the flocculant from Corynebacterium sp. K-199 was observed in culture medium containing 2% glycerol, 0.4% peptone, 0.3% K_2HPO_4, 0.1% CaCO_3, 0.05% NaCl, 0.1% yeast extract, 0.1% MgSO_4·7H_2O and initial pH 7.5 when cultured with rotary shaker controlled at 25℃ and 150 rpm. Under the optimal culture condition with jar fermentor, the maximum production was reached to flocculating activity of 780 units/ml after 4 days. From the results of the activity be fully maintained by periodate oxidization, it suggests that the activity is due to the protein.

Effects of Peach (Prunus persica L. Batch) Extracts on Glucose Uptake in L6 Myoblasts

Hong-Yon Cho,Jae-Hyung Mah,Hee-Seop Lee,Won-Chul Lim 한국식품영양과학회 2014 한국식품영양과학회 학술대회발표집 Vol.2014 No.10

Cloning and Expression of a Chitinase Gene from Thermoactinomyces vulgaris KFB-C100

( Hong Yon Cho ),( Young Hee Lim ),( Ho Geun Yoon ),( Hee Yun Kim ) 한국미생물생명공학회 1998 Journal of microbiology and biotechnology Vol.8 No.6

Inhibitory Effects of Peach (Prunus persica L. Batsch) Extracts on Melanogenesis in B16/F10 Melanoma Cells

Hong-Yon Cho,Jae-Hyung Mah,Su-Hee Ahn,Hyo-Eun Lee 한국식품영양과학회 2014 한국식품영양과학회 학술대회발표집 Vol.2014 No.10

Protective Effects of Peach Extracts against UVB-Induced Skin Damage in HS68 Cells

Hong-Yon Cho,Jae-Hyung Mah,Hyo-Eun Lee,Su-Hee Ahn 한국식품영양과학회 2014 한국식품영양과학회 학술대회발표집 Vol.2014 No.10