Penicillium verruculosum으로부터 Cellobiohydrolase의 정제 및 특성에 관한 연구

조남철,김강화,전순배,정기철,Cho, Nam-Chul,Kim, Kang-Hwa,Chun, Soon-Bai,Chung, Ki-Chul 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.5

효소생성유도물질로서 cellobiose octaacetate를 포함하는 P. verruculosum 배양상등액으로부터 소단위체 분자량이 60,000(I) 과 66,000(II) 인 두 종류의 cellobiohydrolase와 KG-flock을 함유하는 배지로부터 소단위체 분자량이 76,000(III)인 또 하나의 cellobiohydrolase를 분리정제하여 그들의 일반적 특성을 검토하였다. 이들의 자연상태의 분자량은 소단위체 분자량의 2~3배 였으며 모두 당단백질로서 당함량은 cellobiohydrolase I, II 및 III가 각각 8.6%, 4.2% 그리고 8.5%였다. 세 효소 모두 pH 4.5~5.0과 온도 $50{\sim}60^{\circ}C$에서 높은 활성도를 나타냈으며 Avicel, cotton, 여지 등의 결정성 섬유소 뿐만 아니라 CMC, PNPC 등에도 분해활성도를 나타내었다. Cellobiohydrolase I, II 및 III의 Avicel 분해 비활성도는 각각 0.07, 0.10, 그리고 0.07 unit per mg. of protein이었으며 그 분해생성물은 거의 cellobiose였다. PNPC 기질에 대한 효소반응에 있어서 포도당에 의해서는 활성도가 억제되지 않은 반면 10~20mM의 cellobiose에 의해 50% 이상의 억제효과를 보였다. Three cellobiohydrolase I, II and III, from culture solution of Penicillum verruculosum grown in liquid media containing cellobiose octaacetate or KC-flock, as the sole carbon source has been extensively purified and characterized. Molecular weights of the purified enzymes, I, II and III, were estimated as 60,000, 66,000 and 76,000 on 10% polyacrylamide gel containing sodium dodecyl sulfate and have 8.6, 4.2 and 8.5% of carbohydrate contents, respectively. The three enzymes showed hydrolytic activities towards crystalline celluloses such as Avicel, cotton and filter paper and soluble cellulose such as carboxymethylcellulose. Specific activities of cellobiohydrolase, I, II and III against Avicel as a substrate were 0.07, 0.10 and 0.07 unit per mg. of protein, respectively. The optimum pHs and temperatures of three enzymes were 5.0 and $50^{\circ}C$. All enzyme produced extensively cellobiose from Avicel, and the enzymatic hydrolysises of ${\rho}$-Nitrophenyl-${\beta}$-D-cellobioside were inhibited by cellobiose, but not by D-glucose.

Penicillium verruculosum 으로부터 Celobiohydrolase 의 정제 및 특성에 관한 연구

조남철,김강화,전순배,정기철 ( Nam Chul Cho,Kang Hwa Kim,Soon Bai Chun,Ki Chul Chung ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.5

Three cellobiohydrolase I, II and III, from culture solution of Penicillum verruculosum grown in liquid media containing cellobiose octaacetate or KC-flock, as the sole carbon source has been extensively purified and characterized. Molecular weights of the purified enzymes, I, II and III, were estimated as 60,000, 66,000 and 76,000 on 10% polyacrylamide gel containing sodium dodecyl sulfate and have 8.6, 4.2 and 8.5% of carbohydrate contents, respectively. The three enzymes showed hydrolytic activities towards crystalline celluloses such as Avicel, cotton and filter paper and soluble cellulose such as carboxymethylcellulose. Specific activities of cellobiohydrolase, I, II and III against Avicel as a substrate were 0.07, 0.10 and 0.07 unit per mg. of protein, respectively. The optimum pHs and temperatures of three enzymes were 5.0 and 50℃. All enzyme produced extensively cellobiose from Avicel, and the enzymatic hydrolysises of p-Nitrophenyl-β-D-cellobioside were inhibited by cellobiose, but not by D-glucose.

Penicillium verruculosm 의 Endo - β - 1 , 4 - Glucanase 의 정제 및 특성

김연희,조남철,김강화,최원기,전순배,이용규,정기철 ( Yeon Hi Kim,Nam Chul Cho,Won Ki Choi,Kang Hwa Kim,Soon Bai Chun,Yong Kyu Lee,Ki Chul Chung ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.2

Two endoglucanase(EG, 1,4-β-D-glucan glucanohydrolase, EC 3.2.1.4) were purified from culture broth of P. verruculosum by ammonium sulfate precipitation, and HPLC-phenyl, HPLC-DEAE and HPLC gel filtration column chromatography. Purified enzymes were estimated to have molecular weight of 40 kDA(EG II), and 58 kDa(EG III) by SDS-PAGE and of 120 kDa(EG II), 140 kDa(EG III) by HPLC gel filtration. The optimal pH and temperature for these enzymes were found to be 4∼5 and 50-60℃, respectively. EG II and EG III hydrolyze carboxymethylcellulose but can not hydrolyze p-Nitrophenyl-α-D-glucoside and crystalline cellulose such as cotton, Avicel, filter paper. The hydrolytic products of CMC catalyzed by the enzyme were cellobiose, glucose and oligosaccharides. From the similarity in amino acid composition and elution profiles of tryptic peptides of EG II and EG III and cross reactivity of antibodies against the enzymes, it is suggested that amino acid sequences of these two EGs are very similar.

Penicillium verruculosum으로부터 D-Xylanase Ⅱ의 정제 및 특성

조남철,강영태,이태훈,정기철,김강화 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

Penicillium verruculosum으로부터 D-xylanase(1,4-β-D-xylan xylanohydrolase ; EC 3.2.1.8) II를 분리 정제하여 일반적 특성을 조사하였다. 정제된 효소의 분자량은 SDS-PAGE 상에서 22,000으로 추정되며 xylan 이외에 인공기질인 ρ-nitrophenyl-β-D-xylopyranoside, ρ-nitrophenyl-β-D-glucopyranoside, ρ-nitrophenyl-β-D-cellobiopyranoside 뿐만 아니라 Avicel, cotton, filter paper, carboxymethylcellulose와 같은 섬유소를 가수분해하지 못했다. 정제된 효소의 xylan에 대한 비활성도는 단백질 ㎎당 270 units였으며 효소활성도에 대한 최적 pH와 최적 온도는 각각 3.0∼4.0과 50∼60℃였고 50℃에서는 pH 3.0∼10.0의 비교적 넓은 범위에서 24시간 동안 효소활성도가 안정하게 유지되었다. 정제된 효소를 xylan과 반응시켰을 때 분해산물로서 xylobiose를 비롯하여 여러가지 xylooligosaccharide들이 생성되었으며 그 중 xylobiose가 가장 많이 생성되었다. 그러나 xylose는 이들에 비해 매우 소량 생성되었으며 이러한 xylan 분해양상은 앞서 P. verruculosum으로부터 정제되어 보고된 D-xylanase I과 동일하였다. Xylanase(1,4-β-D-xylan xylanohydrolase; EC 3.2.1.8) II was purified from Penicillium verruculosum by suing the techniques of two anion exchange chromatographies, and gel filtration. The molecular weight of this enzyme was about 22,000 as determined by SDS-electrophoresis. The enzyme showed hydrolytic activity toward xylan but did not catalyze hydrolysis of ρ-nitrophenyl-β-D-xylopyranoside, ρ-nitrophenyl-β-D-glucopyranoside, ρ-nitrophenyl-β-D-cellobiopyranoside, and celluloses such as Avicel, cotton, filter paper, carboxymethylcellulose. Specific activity of the xylanase against xylan as a substrate was 270 units per mg of protein. The optimum pH and temperature of the enzyme were pH 3.0∼4.0 and 50∼60℃, respectively, and the enzyme was stable in the wide pH range from 3.0 to 10.0 at 50℃ for 24 hrs. The purified enzyme split xylan to yield xylooligosaccharides containing xylobiose, and xylobiose was major product, while xylose was produced very little. Those properties were similar to that of D-xylanase I showing a M.W 35,000 from the same fungus, P. verruculosum.

Penicillium verruculosum의 Avicelase 생성에 대한 Cellobiose Octaacetate 와 Avicel 및 KC - flock의 영향

조남철,김강화,전순배,정기철 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.4

Penicillium verruculosum 섬유소 분해효소의 유도물질인 KC-flock, avicel 및 cellobiose octaacetate(COA)를 유일한 탄소원으로 포함하는 배지에서 21일간 배양하면서 배양여액 중의 섬유소 분해효소 각 성분의 활성도와 전기영동상의 단백질 양상을 검토하였다. 배양여액 중의 총단백질 생정 및 섬유소 분해활성도의 유도효과는 COA 배지가 다른 두 배지의 3배 이상이었다. 배양기간의 증가에 따라 배양여액 중의 총단백질량이 증가되었는데 CMC 분해활성이나 β-glucosidase 활성도의 증가보다는 avicel 분해활성도의 증가가 총단백질량의 증가와 유사했다. 전기영동상의 단백질 양상은 배양 초기에는 각 배지의 경우 달랐으나 배양기간이 증가함에 따라 비슷해졌으며, 60,000 내지 75,000의 분자량을 갖는 단백질들이 배양여액 중의 전체 단백질의 거의 대부분을 차지했다. 각 배양여액 중의 단백질을 이온교환수지를 이용한 고속 액체크로마토그라피로 분리한 각 분획 중의 섬유소 분해효소 각 성분의 활성도와 전기영동상의 단백질 양상으로부터 배양여액 중의 단백질의 거의 차지하는 60∼75kDa의 단백질들의 avicel 분해활성과 관계가 있으며 이러한 단백질은 적어도 3종류가 있음을 추정할 수 있었다. During the cultivation of Penicillium verruculosum in the media containing cellobiose octaacetate(COA), avicel, or KC-flock as an inducer and as a sole carbon source for 21 days, cellulolytic activity and SDS-PAGE pattern of proteins in the culture broth were investigated. Protein concentration and cellulolytic activity were highest in the COA medium. As cultivation period was increased, protein content and avicel hydrolytic activity of culture broth were increased as similar extent but neither β-glucosidase nor CMC hydrolytic activity was correlated to protein content. When crude proteins from the culture broth were separated on DEAE column by HPLC, distribution of avicel-hydrolytic activities were well correlated with that of major proteins. From those results it was suggested that three major proteins having 60 K, 68 K, and 76 K of Mr. were acicel-hydrolytic enzymes.