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Alkaline Protease 를 생산하는 Bacillus subtilis 의 원형질체 융합
홍범식,양한철,성하진,최양미,이태경 한국농화학회 1989 Applied Biological Chemistry (Appl Biol Chem) Vol.32 No.4
To improve alkaline protease producing strain by protoplast fusion, a strain of Bacillus sp. was treated with 100㎍/㎖ NTG (N-methyl-N'-vitro-N-nitrosoguanidine) for 45 minutes and mutants of Bacillus subtilts Arg^-, Try^-, His^- and Ade^- were isolated. The frequency of protoplast formation was about 99%, when teas of exponential phase were treated with 200㎍/㎖ lysozyme at 42℃ for 10∼30 minutes. In a regeneration medium containing 0.3M sodium succinate, 2.0% polyvinylpyrrolidone, 0.5% casamino acid, 10mM MgCl₂ and 20mM CaCl₂, regeneration frequency cf the isolated Bacillus subtilis strains was 25.2%. The fusion frequency between mutant, was from 2.1×10^(-5) to 8.1×10^(-5) under optimum condition.
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.
Fe-Sn系 燒結合金의 諸性質에 미치는 Sn粉의 粒度 및 配合量의 影響
홍범식,김윤채 三陟大學校 1997 論文集 Vol.30 No.1
Effects of particle size and content of tin powder on the properties of iron-tin sintered alloy for porous bearing were investigated in comparison with those of iron powder compact. The results obtained are as follows; 1) The effect of particle size of tin powder : When the particle size becomes finer, the compact shrinks a little and pore-structure becomes finer and then, the strength increases considerably. 2) The effect of tin powder content : When content of tin powder increases, the compact expands a little and also the pore-structure grows a little. But the strength decreases especially with increasing of the tin content above 1.5wt&. Therefore, it is desirable that the particle size is -350mesh and the content is about 1.0wt%.