Cellulomonas sp. YE-5가 생산하는 Cellulase의 특성

최동철,김동섭,유주현,오두환 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.2

Cellulomonas sp. YE-5가 생산하는 cellulase를 분리, 정제하여 효소의 특성을 알아보았다. Avicelase, CMCase, β-glucosidase의 반응 최적온도는 각각 40, 45, 40℃이었고, 반응 최적 pH는 5.5, 6.0 그리고 6.0이었다. 효소의 열안정성은 30∼70℃에서 6시간 처리하였을 때 avicelase와 β-glucosidase는 50℃ 이상에서 거의 실활하였고, CMCase는 50℃에서 약 40%의 활성을 유지하였다. 효소의 안정성에 미치는 pH의 영향은 25℃에서 24시간 처리하였을 때 avicelase와 CMCase는 pH 5.O∼9.0 사이에서 안정하였으며, β-glucosidase는 pH 5.O∼8.0 사이에서 안정하였다. 효소활성에 미치는 금속이온의 영향은 Mn^2+와 Co^2+에 의해 avicelase의 활성이 약간 증가하였고, Cu^2+와 Zn^2+에 의해서는 세 효소가 크게 저해를 받았다. 효소의 기질친화력(K_m)은 avicel에 대한 avicelase가 4.76 ㎍/㎖, CMC에 대한 CMCase I이 27.80㎍/㎖, CMCase Ⅱ가 16.40 ㎍/㎖이었으며, PNPG에 대한 β-glucosidase는 3.51 mM이었다. Enzymatic properties of avicelase, carboxymethyl cellulase (CMCase) and β-glucosidase produced by Cellulomonas sp. YE-5 were studied. Optimal temperature and pH of avicelase were 40℃ and 6.0, and those of CMCase and β-glucosidase were 45℃ and 6.5. Avicelase and CMCase were stable between pH 5.0 and 9.5, and β-glucosidase was stable between pH 5.5 and 8.0. Avicelase and β-glucosidase were inactivated when incubated at 35℃ for 6 hrs, and CMCase was at 40℃ for 6 hrs. All cellulases were strongly inhibited by Cu^2+ and Zn^2+ K_m values of avicelase for avicel, CMCase I and CMCase Ⅱ for CM-cellulose, and β-glucosidase for p-nitrophenyl-β-D-glucoside (PNPG) were 4.76, 16.4, 16.4 ㎍/㎖ and 3.51 mM, respectively.

합성(合成) 배지(培地)에서 느타리속(屬)이 생산(生産)하는 섬유소(纖維素) 분해효소(分解酵素)에 관한 연구(硏究)(제2보) -비타민류(類), 무기(無機) 염류(鹽類)와 배양(培養) 조건(條件)의 영향(影響)-

홍재식,이종배,고무석,김정숙,이극로,정기태,Hong, Jai-Sik,Lee, Jong-Bae,Koh, Moo-Seok,Kim, Jeong-Sook,Lee, Keug-Ro,Jung, Gi-Tae The Korean Society of Mycology 1986 韓國菌學會誌 Vol.14 No.1

Pleurotus spp.중 섬유소(纖維素) 분해효소(分解酵素) 생산력(生産力)이 가장 강한 Pleurotus sajor-caju JAFM 1017을 합성배지(合成培地) 상에서 vitamin류(類), 무기염류(無機鹽類)와 배양조건(培養條件)의 영향을 검토(檢討)한 결과(結果)는 다음과 같다. 섬유소(纖維素) 분해효소(分解酵素) 생산(生産)은 folic acid와 thiamine-HCI에 의해 촉진되었고 $KH_2PO_4$와 $MgSO_4$의 최적농도(最適濃度)는 각각 0.2%, 0.04%(w/v)이었으며 그 밖의 무기염류(無機鹽類)는 효과(效果)가 없었다. 효소생산(酵素生産)에 최적(最適)인 배양온도(培養溫度)와 배지(培地) pH는 avicelase가 $25^{\circ}C$, 5.5이었고, CMCase는 $30^{\circ}C$, 5.0이 였으며, ${\beta}-glucosidase$는 $30^{\circ}C$, 6.5이었다. The production of cellulolytic enzymes by Pleurotus sajor-caju JAFM 1017 was stimulated by folic acid and thiamine-HCl. Among the inorganic salts, optimum concentrations of $KH_2PO_4$ and $MgSO_4{\cdot}7H_2O$ were 0.2% (w/v) and 0.04% (w/v), respectively, but other inorganic salts were not effective for the production of the enzymes. The optimum culture temperature and pH for the production were $25^{\circ}C$ and 5.5 for avicelase, and $30^{\circ}C$ and 5.0 for CMCase, and $30^{\circ}C$ and 6.5 for ${\beta}-glucosidase$, respectively.

우분으로부터 Bacillus subtilis CH-10의 분리 및 균주가 분비하는 Cellulase의 특성에 관한 연구

김태일,한정대,전병수,하상우,양창범,김민균,Kim, Tae-Il,Han, Jung-Dae,Jeon, Byoung-Soo,Ha, Sang-Woo,Yang, Chang-Bum,Kim, Min-Kyun 한국미생물학회 1999 미생물학회지 Vol.35 No.4

우분으로부터 cellulase를 생산하는 미생물을 congo red 염색과 활성측정을 통해 선발하여 cellulase 활성이 우수한 균을 분리하였다. 분리균은 생화학적, 형태학적, 균체 지방산 조성을 근거로 Bacillus subtilis CH-10으로 동정하였다. 분리균이 분비하는 효소학적 특성 중 효소생산 조건은 초기 pH7.5 및 배양돈도 50${\circ}C$ 그리고 48시간 배양이 가장 적합하였다. 조효소액에서 CMCase 최적 온도는 75${\circ}C$이었고, 온도안정성은 50${\circ}C$까지 70%의 효소활성을 유지하였다. 조효소액에서 CMCase 최적 pH는 7.5이었고, pH 안정성은 pH7.5~9.0 영역에서 70%의 효소활성을 유지하였다. 분리균을 CMC 배지에서 37${\circ}C$, 24시간 배양시 나타난 CMCase와 Fpase 활성은 각각 1.13 U/㎖와 0.16U/㎖ 였으나 avicelase와 ${\beta}$-glucosidase의 활성은 검출되지 않았다. CMC-SDS-PAGE 방법으로 3개의 효소활성 band를 확인하였고, Cel 1 및 2 그리고 Cel 3의 분자량은 각각 약 39 및 41 그리고 57kDa이었다. A bacterium producing the extracellular cellulase was isolated from cattle feces and screened as cellulase activity was excellent upon congo red straining method and activity measurements. Isolate was identified as Bacillus subtilis CH-10 on the basis of morphological and biochemical properties as well as cellular fatty acids composition. The enzyme which the isolate secretes had the optimum initial pH and temperature for its induction was 7.5 and 50${\circ}C$, respectively. The maximum CMCase activity in crude enzyme solution was observed at pH 7.5 and 75${\circ}C$ and was stable for pH 7.5 to 9.0 to maintain 70% activity. When the isolate was cultured in CMC media at 37${\circ}C$ for 24 hrs, CMCase and FPase activity was 1.13 U/㎖and 0.16U/㎖, respectively whereas Avicelase and ${\beta}$-glucosidase activity was not detected. When crude supernatant was used for zymogram, three major bands, cel 1, cel 2 and cel 3, were detected approximately 39, 41 and 57 KDa, respectively on CMC-SDS-PAGE.

Bacillus cellulyticus K-12 Crystalline Cellulose-Degrading Avicelase Gene and Expression in Escherichia coli

Kim, Cheorl Ho,Shim, Woo Man,Kim, Dong Soo 한국식품영양학회 1993 韓國食品營養學會誌 Vol.6 No.4

We have cloned the Bacillus cellulyticus K-12 avicelase(Avi, E.G.3.2.1.4) gene(ace A) in E. coli. This was accompanied by using the vector pT7T3U19 and Hind Ⅲ -HindⅢ libraries of Bacillus cellulyticus K-12 chromosomal inserts created in E. coli. The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the anti-avicelase(immunoscreening). Positive clones(Ac-3, Ac-5, and Ac-7) contained the identical 3.5kb HindⅢ fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constituvely using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide which showed a CMCase activity with an Mr of 54000 was detected.

Avicelase 생산성 Trichoderma sp. HK 47의 분리 및 동정

박헌국,이계호 한국식품영양학회 1993 韓國食品營養學會誌 Vol.6 No.3

In order to obtain a good microorganism capable of degrading microcrystaline cellulose (avicel), the screening test was carried out from soil and brown-rot wood. 8 strains which had good avicel-hydrolyzing activity were isolated. Among them, HK 47 which exhibited the highest avicel hydrolyzing activity was identified as Trichoderma sp. HK 47. Maximum avicel-hydrolyzing enzyme production from Trichoderma sp. HK 47 was obtained with the optimum medium contained carboxymethylcellulose 1.5% as carbon source, NaNO_3 0.75% as nitrogen source, KH_2PO_4 0.5%, MgSO_4·7H_2O 0.1%, Tween 80 0.005% (V/V) during stationary cultivation at pH 6.0, 30℃. In this case, the production of avicel-hydrolyzing enzyme was 0.028U/㎖.

Penicillium verruculosum의 Acicelase 생성에 대한 Cellobiose Octaactate와 Avicel 및 KC-flock 의 영향

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

Penicillium verruculosum 섬유소 분해효소의 유도물질인 KC-flock 및 cellobiose octaacetate(COA)를 유일한 탄소원으로 포함하는 배지에서 21일간 배양하면서 배양여액 중의 섬유소 분해효소 각 성분의 활성도와 전기영동상의 단백질 양상을 검토하였다. 배양여액의 중의 총단백질 생성 및 섬유소 분해활성의 유도 효과는 COA 배지가 다른 두 배지의 3배 이상이었다. 배양기간의 증가에 따라 배양여액 중의 총단백질량이 증가되었는데 CMC 분해활성이나 $\beta$-glucosidase 활성의 증가보다는 avicel 분해활성도의 증가가 총단백질량의 증가와 유사하다. During the cultivation of Penkillium uerrmulosum 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 $\beta$-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 avicel-hydrolytic enzymes.

Clostridium thermocellum JW20가 생성하는 섬유소분해 효소복합체(cellulosome) 구성단백질의 특성에 관한 연구

최상기 한국미생물학회 2000 미생물학회지 Vol.36 No.3

The cellulosome of Clo.~tr~rlil~m tl\ulcornererfnocellum consistmg of 26 dfferent polypeptides contains calcium. The polypeptides dissociated when calcium was removed. Most of dockerill region in the catalytic polypeptides cleavcd during dmociation. The dissociated polypeptides were well separated by MonoQ column chromatography into CipA containing fraction, a fraction still complexed wit11 91 kDa (CelK-a). 60 IiDa and 57 kDa polypeptides, and fractious contailling mainly single polypeptide of 46 kDa (CelA-a) or 71 1d)a polypeptide (CelS-trj Most or the fractions hydrolyzed c~ystalliue cellulose The purified 71 kDa polypeptide was strictly dependent on calcium for crystalline cellulose hydvolyzing activities a1 $60^{\circ}C$~$70^{\circ}C$ but 46 kDa polypeptide was not. 46 M)a polypeptide digested cellodextri~~ as cellobiose or cellotriose unit, and glucose was produced together with cellobiose and cellotriose froln cellotetraosc. It seems that cellulosome produces final product, cellobiose, through coordinated ~qulation of activities of vannus subunits. Clostridium thermocellum이 생성하는 섬유소분해 효소복합체인 cellulosome은 26개 의 서로 다른 단백질로 구성되어 있으며 그 구성물질로서 calcium을 포함하고 있다. 견고한 구조의 이 복합체에서 구성단백질을 분리하여 그 기능을 연구할 목적으로 이 복합체를 해체 (dissociation)하려 시도하였다. 이 복합체는 calcium을 제거하였을 대 해체되었다. 해체된 구성 단백질들은 MonoQ column chromatogrphy에 의해 구조단백질인 CipA를 포함한 분획, 91 kDa(CelK-tr), 60 kDa 과 57 kDa 단백질로 구성된 분획과 주로 46 kDa(CelA-tr), 또는 71 kDa(CelS-tr) 단백질을 포함하는 분획들로 크게 분리되었다. 대부분의 분획들은 crystalline cellulose 분해 활성을 보였다. 순수 분리된 71 kDa 단백질은 $60^{\circ}C$~$70^{\circ}C$에서 섬 유소 분해시 calcium에 의존적이었으나 46 kDa 단백질은 그렇지 않았다. 46 kDa 단백질은 cellodextrin을 celloviose 및 cellotriose 단위로 절단하며 cellotetraose 로부터 glucose가 생 성되는 것이 관찰되었다. Cellulosome의 섬유소분해 최성 산물이 cellobiose인 것을 고려할 때 개개 구성단백질의 활성이 이 효소 복합체내에서 조절되어 있음을 알 수 있다.

Purification and Characterization of a Thermophilic Cellulase from a Novel Cellulolytic Strain, Paenibacillus barcinonensis

Asha, Balachandrababu Malini,Revathi, Masilamani,Yadav, Amit,Sakthivel, Natarajan The Korean Society for Microbiology and Biotechnol 2012 Journal of microbiology and biotechnology Vol.22 No.11

A novel bacterial strain, MG7, with high cellulase activity was isolated and identified by morphological characteristics and molecular phylogeny analysis as Paenibacillus barcinonensis. Maximum production of cellulase by MG7 was observed at pH 7.0 and $35^{\circ}C$. The enzyme was purified with a specific activity of 16.88 U/mg, the cellulase activity was observed in a zymogram, and its molecular mass (58.6 kDa) was confirmed by SDS-PAGE. The purified enzyme showed maximum activity at pH 6.0 and $65^{\circ}C$ and degraded cellulosic substrates such as carboxy methyl cellulose (CMC), Avicel, filter paper, and ${\beta}$-glucan. The enzyme showed stability with 0.5% concentration of various surfactants. The $K_m$ and $V_{max}$ of cellulase for CMC and Avicel were found to be 0.459mg/ml and 10.46mg/ml/h, and 1.01 mg/ml and 10.0 mg/ml/h, respectively. The high catalytic activity and its stability to temperature, pH, surfactants, and metal ions indicated that the cellulase enzyme by MG7 is a good candidate for biotechnological applications.