Molecular Cloning and Expression of Cellulase of Gene of Pseudomonas sp. in Escherichia coli

Chung, Young Chul,Kim, Yang Woo,Kang, shin Kwon,Rho, Jong Su,Sung, Nack Kie 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.6

Cellulase 복합체와 xylanase를 동시에 분비하는 Pseudomonas sp. LBC 505와 CYC 10의 cellulase 유전자를 pUC19를 사용하여 E. coli에 클로닝시켰다. Congo red 염색시 노란색 환을 형성하는 대장균 형질전환체에서 0.7kb-와 4.6kb-HindⅢ 단편을 함유한 재조합 플라스미드 pLC1과 pLC2를 각각 분리하였다. DNA hybridization 실험에서 pLC1과 pLC2는 Pseudomonas sp. LBC 505와 CYC 10 유래임이 각각 밝혀졌고, lmmunoassay 실험에서도 유사성이 인정되었다. pLC1을 함유하고 있는 대장균은 cellulase의 24%를 세포외로 분비하였고, 효소활성은 모균주에 비해 1.4배 증가하였다. pLC1과 pLC2의 효소학적 성질도 모균주와 동일하였으며, 기질특이성과 HPLC로 유리당을 분석한 결과, 클로닝된 유전자는 endo type인 것으로 나타났다. The genes for cellulases of Pseudomonas sp. LBC505 and CYC10, potent cellulase complex-producing strains, were cloned in Escherichia coli with pUC19. Recombinant plasmids pLC1 and pLC2 were isolated from transformants producing cellulase by congo red staining, and their genes cloned were 0.7 kb and 4.6 kb HindⅢ fragments, respectively. The inserts of pLC1 and pLC2 were hybridized to chromosomal DNAs digested with HindⅢ from Pseudomonas sp. LBC505 and CYC10, respectively. Immunodiffusion assays revealed that pLC1-and pLC2-encoded cellulase showed similarity with that of host strains. About 24% of cellulase activity was observed in the extracellular fraction of E. coli carrying pLC1, and its activity was higher about 1.4 times than that of LBC505. The enzymatic properties of pLC1 and pLC2 encoded cellulase were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases cloned were endocellulase.

고지탈묵용 Cellulase 및 Xylanase 생산

김욱한,손광희,복성해,오세균 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.5

효소탈묵용 cellulase와 xylanase 생산을 위한 조건을 조사한 결과 배지초기 pH, Tween 80, 포자접종량, 질소원의 종류 및 탄소원의 종류에 따라 효소 생산량이 크게 변하였다. Cellulase 생산을 위한 최적조건으로는 pH 5.O∼6.5, Tween 80 0.02%, 포자현탄액(1×10^7㎖) 접종량 0.5∼l.0%, 질소원으로 면실박, 탄소원으로 옥분을 사용하였을 때 가장 효과적이었다. 또한, xylanase 생산을 위한 최적조건으로는 pH 6.5, Tween 80 0.Ol%, 질소원으로 corn steep liquor, 탄소원으로는 신문고지를 사용하였을 때 효과적이었으며, 이때 포자현탁액의 접종량은 cellulase 생산의 경우와 동일하였다. 한편, cellulase와 xylanase의 동시생산을 위해서는 플라스크 규모에서 질소원으로 면실박 0.5%, 탄소원으로 신문고지 1.0%와 옥분 2.0%를 첨가하였을 때 효과적이었으며, 이때 생산된 배양액의 cellulase의 활성은 6.11∼7.22IU/㎖, xylanase의 활성은 97.7IU/㎖이었다. 그러나 fermentor 규모에서는 동일 배지조성에서 효소생산이 다소 감소하였으며, 특히 xylanase 경우 배양액의 효소활성이 낮아서 이에 대한 보완이 요구된다. The optimal conditions for cellulase and xylanase production by Trichoderma reesei 28217 were studied for enzymatic deinking of old newspaper. The amounts of cellulase and xylanase from the strain was varied by initial medium pH, Tween 80, inoculum size of spore suspension, and carbon and nitrogen sources. The optimal conditions for cellulase production were pH 5.O∼6.5, 0.02% of Tween 80, 0.5∼1.O% of inoculum size of spore suspension (1×10^7/㎖), cotton-seed meal as nitrogen source, and corn flour as carbon source. On the other hand, the optimal conditions for xylanase production were pH 6.5, 0.01% of Tween 80, corn steep liquor as nitrogen source, and disintegrated old newspaper as carbon source. The inoculum size for xylanase production was the same as for cellulase production. The concomitant production of cellulase and xylanase in shake flask culture was efficiently induced in the medium containing 0.5% cottonseed meal as nitrogen source and 1.O% old newspaper and 2.O% corn flour as carbon sources. In this case the activities of cellulase and xylanase produced were 6.11∼7.22 IU/㎖ and 97.7 IU/㎖, respectively. However, the cellulase production in 5ℓ fermentor scale was slightly decreased compared with that in flask scale. Moreover, xylanase production was severely reduced in a fermentor scale. The study for the reason of decreased enzyme production in fermentor is further needed.

토양 유래 Cellulase 생산균주의 Screening 및 Bacillus subtilis NCE3 유래 Cellulase 특성 규명

박창수 ( Chang-su Park ) 한국키틴키토산학회 2017 한국키틴키토산학회지 Vol.22 No.1

자연환경의 토양시료로부터 cellulase를 생산하는 균주를 단리하기 위하여 Carboxymethylcellulose (CM-cellulose)와 try-pan blue를 첨가하여 제작한 LB-Agar 배지상에서 screening을 진행한 결과, 균주 유래 cellulase 활성으로 인해 배지상에서 명확한 활성환을 형성하는 총 7종류의 균주를 단리하였다. 단리한 균주들의 배양 상층액을 조효소액으로 활용하여 cellulase활성을 검토한 결과 CM-cellulose에 대하여 가장 높은 효소활성을 보인 1 균주를 선택하여 본 균주 유래의 16S rRNA유전자 염기서열 분석을 통하여 균주 동정을 진행한 결과 본 연구에서 단리한 균주는 Bacillus subtilis와 약 99%의 높은 상동성을 보였다. 이 결과로부터 본 연구에서 단리한 균주를 B.subtilis NCE3으로 명명하였다. B. subtilis NCE3 유래 cellulase의 최적 pH 및 온도를 검토한 결과, 본 균주 유래 cellulase는 pH 5.0과 50°C에서 가장 높은 효소 활성을 나타내었으며, 30, 40, 50, 60, 그리고 70°C에서 각각 1003, 236, 105, 43, 그리고 23분의 half-life를 나타내었다. 기질특이성 검토에 있어서는 CM-cellulose에서 가장 높은 효소 활성을 나타내었으며, Alkali swollen cellulose에 대해서도 명확한 효소활성을 나타내었다. 하지만, cellulose 고유의 결정구조를 보유하고 있는 α-Cellulose, Sigma Cellulose, 그리고, Avicel에 대해서는 매우 낮은 5% 미만의 효소 활성을 나타내었다. B. subtilis NCE3유래 cellulase는 2% CM-cellulose 기질에 대하여 가장 높은 효소 활성을 나타내었으며, 효소 반응 2시간 이내에 2% CM-cellulose에 대해 최대 효소 분해 활성을 나타내는 것으로 확인되었다. In the screening of cellulolytic microorganisms from environment soil, several strains were selected as cellulose production microorganisms due to their ability of forming active zone on LB-Agar plate containing carboxymethylcellulose (CM-cellulose) with trypan blue. Among the strains, one strain was selected because of the highest cellulase activity for CM-cellulose. This strain showed 99% of identity with Bacillus subtilis in the analysis of 16S rRNA gene sequence therefore we named this strain as Bacillus subtilis NCE3. Under the conditions pH 5.0 and 50°C, the cellulase from B. subtilis NCE3 exhibited the highest enzyme activity for CM-cellulose. And the half-lives of cellulase from B. subtilis NCE3 was calculated 1003, 236, 105, 43, and 22 min at 30, 40, 50, 60, and 70°C, respectively. In the investigation of substrate specificities for several substrates such as CM-cellulose, Alkali swollen cellulose, a-cellulose, Sigma Cellulose and Avicel, the cellulase from B. subtilis NCE3 has the highest activity for CM-cellulose. When the concentrated enzyme was used in the degradation of CM-cellulose, the highest enzyme activity was obtained at the 2% of CMcellulose.

토양에서 단리한 Bacillus sp. TH1 유래 Cellulase의 특성 규명

강동욱 ( Dong Wook Kang ),박창수 ( Chang-su Park ) 한국키틴키토산학회 2017 한국키틴키토산학회지 Vol.22 No.3

식품, 에너지, 제지 및 섬유등 다양한 현대 산업에 높은 활용성을 보유하고 있는 효소 cellulase를 생산하는 균주를 screening하기 위하여 토양시료를 이용하여 cellulase 생산균주를 단리하였다. Carboxymethylcellulose (CM-cellulose)를 기질로 첨가한 고체배지상에서 명확한 활성환을 형성하는 총 4종류의 cellulase 생산균주를 단리하여, 배양 상층액으로부터 가장 높은 cellulase 활성을 보인 1 균주를 선택하여 본 균주 유래의 16S rDNA유전자 염기서열 분석을 통하여 균주 동정을 진행하였다. 그 결과 본 연구에서 단리된 균주는 Bacillus sp. R-11590 (GenBank AJ438301)와 약 99%의 높은 상동성을 보였으며, 이 결과로부터 단리된 균주를 Bacillus sp. TH1으로 명명하였다. Bacillus sp. TH1 유래 cellulase는 균주 배양 48시간에서 가장 높은 cellulase 생산성을 보였으며, 최적 pH 및 온도를 검토한 결과, 본 균주 유래 cellulase는 pH 5.0과 50°C에서 가장 높은 효소 활성을 나타내었다. 그리고, pH 및 온도 안정성에 있어서는 각 pH 환경에서 30분간 처리하였을때, pH4.0~5.5 범위에서는 효소활성이 안정하였으며, 45°C까지는 30분간의 열처리에도 효소활성의 저하 없이 안정하게 효소활성을 유지하였다. CM-cellulose, Alkali swollen cellulose, Avicel, Sigmacell-cellulose, 그리고 Alpha-cellulose을 기질로 기질 특이성을 검토하였을 때 cellulose 결정구조를 보유하고 있지 않는 CM-cellulose와 Alkali swollen cellulose에 대해서는 명확한 효소활성을 보였으나, cellulose 결정구조를 보유하고 있는 Avicel, Sigmacell Cellulose, 그리고 α-Cellulose에 대해서는 효소 활성을 보이지 않았다. Bacillus sp. TH1 유래 cellulase는 CM-cellulose를 기질로 이용하였을 때 가장 높은 효소활성을 나타내었다. Alkali swollen cellulose를 기질로 이용하여 Bacillus sp. TH1 유래 cellulase의 효소 활성 특성을 검토한 결과 효소 농도 3.0 U/mL 그리고 기질 농도 1.0%일 때 가장 높은 Alkali swollen cellulose 분해 활성을 보였다. To screen cellulolytic microorganism, we carried out screening from environmental soil, which is sampled at Kyeongsans-si, Kyeonbuk, Korea. One strain, which is named as Bacillus sp. TH1, was isolated as a cellulolytic microorganism due to its ability of enzyme activity for carboxymethylcellulose (CM-cellulose). In the analysis of 16S rRNA gene sequence from the strain, the isolated strain showed 99% of identity with Bacillus sp. R-11590 (GenBank AJ438301) therefore we named the strain Bacillus sp. TH1. The cellulase from Bacillus sp. TH1 showed the highest activities at pH 5.0 and 50°C conditions. In the investigation of pH and temperature stability, the cellulase from Bacillus sp. TH1 stabled pH 4.0~5.5 range and until 45°C for 30 min perfectly. And the cellulase from Bacillus sp. TH1 for the various cellulosic substrates such as CM-cellulose, alkali swollen cellulose, Avicel, Alpha-cellulose, and Sigmacell-cellulose has the most high activity for CM-cellulose. In the degradation of swollen cellulose, the most proper enzyme and substrate concentrations were 3.0 U/ml of enzyme and 1.0% of CMcellulose, respectively.

토양으로부터 단리된 Bacillus subtilis KB1 유래 Cellulase 특성 규명 및 효소 유전자 규명

권은령 ( Eun Ryeng Kwon ),박창수 ( Chang Su Park ) 한국키틴키토산학회 2015 한국키틴키토산학회지 Vol.20 No.4

토양시료를 미생물 분리 시료로 이용하여 CM-cellulose를 기질로 함유한 배지상에서 명확한 cellulase 활성환을 형성함과 동시에 액체배양액으로부터 높은 cellulase 활성을 보이는 균주를 단리하였으며, 본 균주 유래의 16S rRNA 유전자 분석을 통하여 본 균주를 B. subtilis 로 동정함과 동시에 B. subtilisKB1으로 명명하였다. B. subtilis KB1 유래 cellulase는 pH 5.0과 50°C의 조건하에서 가장 높은 효소활성을 보였으며, 30, 40, 50, 60, 그리고, 70°C 에서의 효소 half-life는 각각 5,404,133, 37, 24, 그리고, 6분을 나타내었다. 그리고, 본 효소의 기질특이성 검토에서 전형적인 endo-type cellulase 기질인 CMcellulose에 대하여 가장 높은 효소활성을 보이는 것으로부터 endo-type cellulase임이 강하게 시사되었다. B. subtilis KB1균주 유래 cellulase 유전자를 cloning하여 유전자 배열을 규명한 결과 B. subtilis KB1 유래 cellulase 유전자는 아미노산499개를 암호화하는 1500 bp의 open reading frame (ORF)으로 이루어져 있었으며, 아미노산 배열로부터 추정되는 분자량은 55, 251 Da이었다. 그리고, 규명된 cellulase 유전자로부터 추정된 아미노산 배열을 이용하여 상동성을 검토한 결과 cellulase는 Glycoside hydrolase family (GH) 1에 속하는 cellulase와 높은 상동성을 나타내었다. We isolated a strain producing celluase from environmental soil. The strain was formed activity zone on the LB agar plate containing carboxymethylcellulose (CM-cellulose) stained with trypan blue as substrate with its cellulase activity. In the analysis of the 16S rRNA gene sequence, it was identified as Bacillus subtilis and was named as Bacillus subtilis KB1. The B. subtilis KB1 cellulase showed the maximum activity at pH 5.0 and 50°C. The half-lives of the enzyme at 30, 40, 50, 60 and 70°C were 5,404, 133, 37, 24, and 6 min, respectively. In the investigation of substrate specificity, B. subtilis KB1 cellulase showed the most high enzyme activity for the CM-cellulose substrate. To clone the cellulase gene from B. subtilis KB1, the cellulase gene was amplified from genomic DNA of the B. subtilis KB1 with designed primers based on the cellulase gene from Bacillus subtilis (GenBank: AGN52749) by PCR. In the analysis of the cellulase gene sequence, it was confirmed that the cloned cellulase gene was consisted of a 1,500 bp open reading frame (ORF) encoding a 499 amino acid protein with calculated molecular mass of 55,251 Da. The deduced amino acid sequences from cellulase gene showed high identity with glycosyl hydrolases family (GH) 1.

한국발효식품, 된장, 유래 Cellulase 생산균주의 Screening 및 Bacillus subtilis DJ3 유래 Cellulase 특성 규명

김상진 ( Sang Jin Kim ),최민수 ( Min Su Choi ),박창수 ( Chang-su Park ) 한국키틴키토산학회 2021 한국키틴키토산학회지 Vol.26 No.1

한국전통발효식품 된장을 cellulase 생산 균주 단리 시료로 이용하여 Carboxymethyl cellulose (CM-cellulose)를 기질로 제조한 LB 배지에 도말하여 37℃에서 24-48시간 배양한 결과 균주 유래 cellulase에 의해 배지상에서 명확하게 활성환을 형성하는 균주를 단리하였다. 단리한 균주를 CM-cellulose를 기질로 첨가한 액체 배지 접종하여 37℃에서 48시간 진탕배양 한 후 회수한 배양상층액을 조효소로 이용하여 cellulase활성을 검토한 결과 CM-cellulose에 대하여 가장 높은 효소활성을 나타낸 Bacillus subtilis 로 동정된 균주를 선택하여 본 균주를 Bacillus subtilis DJ3로 명명하였다. Bacillus subtilis DJ3 유래 cellulase의 효소 활성에 영향을 미치는 온도 및 pH에 대한 특성을 검토한 결과 본 효소는 pH5.0, 40℃에서 가장 높은 효소 활성을 보였으며 30분간의 각온도 및 pH 조건에서 처리하였을때 40℃까지 안정하였으며, pH는 4.0-6.0 범위에서 안정한 특성을 보였다. Bacillus subtilis DJ3 유래 cellulase의 기질 특이성을 검토하기 위하여 CM-cellulose, Alkali swollen cellulose, Sigmacell-cellulose, Alpha-cellulose 그리고 Avicel을 기질로 이용하여 효소 활성을 검토한 결과, 본 효소는 CM-cellulose에 대하여 5.32 U/mL로 가장 높은 효소 활성을 보였다. Bacillus subtilis DJ3유래 cellulase의 최적 기질인 CM-cellulose에 대한 분해 특성을 검토한 결과 효소반응의 최적 조건인 pH 5.0, 40℃ 조건에서 본 효소는 2%의 CM-cellulose 농도에서 가장 높은 효소 활성을 나타내었으며, 반응 80분 후에 최대 활성에 도달하는 특성을 보였다. 본 연구에서 단리한 Bacillus subtilis DJ3유래 cellulase는 endo-type cellulase의 전형적인 기질인 CMcellulose에 가장 높은 효소 활성을 나타내는 특성으로부터 endo-type cellulase임이 시사되었다. Six kinds of cellulolytic strains were selected as cellulose production microorganisms from the Korean fermented food, Doenjang. The selected strains formed active zone on LB-Agar plate containing carboxymethylcellulose (CM-cellulose) with trypan blue by their cellulase. Among the strains, one strain, Bacillus subtilis identified by the analysis of 16S rRNA gene, was selected because of the highest cellulase activity for CM-cellulose. We named this strain as Bacillus subtilis DJ3 from the result of strain identification. The B. subtilis DJ3 produced its cellulase maximally after cultivation of 48 h and the cellulase exhibited the highest enzyme activity at pH 5.0 and 40 ℃. In the effects of pH and temperature on the enzyme stability, the enzyme was stable up to 40℃ and pH 4.0-6.0 range after treatment for 30 min at each condition. In the investigation of substrate specificities, the cellulase from B. subtilis DJ3 has the highest activity for CM-cellulose for several substrates such as CM-cellulose, Alkali swollen cellulose, a-cellulose, Sigmacell Cellulose, and Avicel. In the efficient of substrate concentration for enzyme activity, the cellulase from B. subtilis DJ3 showed maximum activity on the 2% of CM-cellulose. When the 2% of CM-cellulose was used, the cellulase showed the highest degradation activity for CM-cellulose after 80 min. From the characterization of cellulosic substrates, the cellulase from B. subtilis DJ3 showed the highest enzyme activity for CM-cellulose, a typical substrate for endo-type cellulase. From this result, it was suggested that the cellulase from B. subtilis DJ3 is an endo-type cellulase.

Preparation of Functionalized Magnetic Silica Nanospheres for the Cellulase Immobilization

Wenjuan Zhang,Jianhui Qiu,Limin Zang,Eiichi Sakai,Huixia Feng 성균관대학교(자연과학캠퍼스) 성균나노과학기술원 2015 NANO Vol.10 No.1

Cellulase was immobilized on functionalized magnetic silica nanospheres using glutaraldehyde as a cross-linking agent. The morphologies, structures and magnetic properties of this immobilized cellulase were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, differential thermal analysis and vibrating sample magnetometry. The properties of immobilized cellulase were investigated, including the amount of immobilized cellulase and its relative activity, stability and reusability. The results indicated that immobilized cellulase exhibited better resistance to high temperature and pH inactivation in comparison to free cellulase. Moreover, immobilized cellulase with and without cross-linking agent were investigated and the former had greater amount of immobilized cellulase and better operational stability. The amount of immobilized cellulase with the cross-linking agent was 92 mg/g support. Furthermore, the activity of the immobilized cellulase was still 85.5% of the initial activity after 10 continuous uses, demonstrating the potential of this immobilized cellulase for large-scale biofuel production.

Pseudomonas sp.의 Cellulase 유전자의 대장균에의 클로닝 및 발현

정영철,김양우,노종수,성낙계,강신권 한국미생물 · 생명공학회 1990 한국미생물·생명공학회지 Vol.18 No.6

Cellulase 복합체와 xylanase를 동시에 분비하는 Pseudomonas sp. LBC 505와 CYC 10의 cellulase 유전자를 pUC19를 사용하여 E.coli에 클로닝시켰다. Congo red 염색시 노란색 환을 형성하는 대장균 형질전환에서 7.0Kb-와 4.6Kb-HindIII 단편을 함유한 재조합 플라스미드 pLC1과 pLC2를 가각 분리하였다. DNA hybridization 실험에서 pLC1 과 pLC2는 Pseudomonas sp. LBC 505와 CYC 10 유래임이 각각 밝혀졌고, Immunoassay 실험에서도 유사성이 인정되었다. pLC1을 함유하고 있는 대장균은 cellulas의 24를 세포외로 분비하였고, 효소활성은 모균주에 비해 1.4배 증가하였다. pLC1과 pLC2의 효소학적 성질도 모균주와 동일하였으며, 기질특이성과 HPLC로 유리당을 분석한 결과, 클로닝된 유전자는 endo type인 것으로 나타났다. The genes for cellulases of Pseudomonas sp. LBC505 and CYC10, potent cellulase complex-producing strains, were cloned in Escherichia coli with pUC19. Recombinant plasmids pLCl and pLC2 were isolated from transformants producing cellulase by Congo red staining, and their genes cloned were 0.7 kb and 4.6 kb HindIII fragments, respectively. The inserts of pLCl and pLC2 were hybridized to chromosomal DNAs digested with HindIII from Pseudomona~ sp. LBC505 and CYC10, respectively. Immunodiffusion assays revealed that pLC1-and pLC2-encoded cellulase showed similarity with that of host strains. About 24% of cellulase activity was observed in the extracellular fraction of E. coli carrying pLC1, and its activity was higher about 1.4 times than that of LBC505. The enzymatic properties of pLC1 and pLC2 encoded cellulase were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases cloned were endocellulase.

Biotechnological Advances and Trends in Engineering Trichoderma reesei towards Cellulase Hyperproducer

Hao Fang,Chaofeng Li,Jiajia Zhao,Chen Zhao 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.4

Cellulase has the biggest contribution to the high production costs of lignocellulose bioconversion and the substantial decrease of its production cost is the key to the commercialization of lignocellulosic biorefineries. Trichoderma reesei has the most robust cellulase among the candidates, which therefore is widely used for cellulase production in industry. This is not because of the size of its cellulase gene pool but its prodigious cargo of cellulase productivity. Still, T. reesei cellulase falls far short of perfection in real-world applications, especially for the composition. This review summarized the biotechnological advances in engineering T. reesei for enhanced cellulase production. Meanwhile, we proposed innovative ideas of systematically optimizing cellulase composition at the transcriptional level and improving cellulase production at the regulation level. Efficient genome editing is essential to achieving that target. Thus, the developments of the tools of multiple gene manipulations were discussed in detail here. This review provides ideas and/or inspirations to the future researches on T. reesei cellulase.

전통 장류에서 분리된 알칼리성 Cellulase 생성 Bacillus subtilis 4-1 균주의 효소학적 특성

백성열,이유정,윤혜주,박혜영,여수환 한국식품저장유통학회 2014 한국식품저장유통학회지 Vol.21 No.3

In this study, we isolated a cellulase-producing bacterium isolated from traditional Korean fermented soybean paste and investigated the effect of culture conditions on the production of cellulase. This bacterium, which was identified as Bacillus subtilis 4-1 through 16S rRNA gene sequence analysis, showed the highest cellulase activity when the cells were grown at 45℃ for 24 hours in the CMC medium supplemented with 1.0% of soluble starch and 0.1% yeast extract. The initial optimum pH of the medium was observed in the range of 5.0∼9.0. The optimal pH and temperature for the production of cellulase from B. subtilis 4-1 were pH 9.0 and 60℃ respectively. In addition, the enzyme showed significant activity in the temperature range of 20∼90℃, which indicates that B. subtilis 4-1 cellulase is an alkaline-resistance and thermo-stable enzyme. This enzyme showed higher activity with CMC as the substrate for endo-type cellulase than avicel or pNPG as the exo-type substrates for exo-type cellulase and β-glucosidase. These results suggest that the cellulase produced from B. subtilis 4-1 is a complex enzyme rather than a mono-enzyme.