Earticle

In order to apply metabolic engineering principles to systematic design of ethanol production process, there are several crucial factors that have to be considered. Among the several crucial factors, the ability to describe the dynamic kinetics quantitatively is most crucial. Application of metabolic engineering principles to systematic design of ethanol production process depend on quantitative dynamic kinetics of enzymes that are related to ethanol biosynthesis network. Goal of this investigation is the development of an essential dynamic model for ethanol biosynthesis of Saccharomyces cerevisiae and the characterization of simulation results to probe its characteristic behavior.1) We have collected data for enzymes of central metabolism and ethanol biosynthesis using BRENDA, KEGG, BioCyc and literature and in the network model, the effects of inhibitors to amino acid biosynthesis model is included as an appropriate mathematical form. In silico analysis of kinetic models of cellular metabolism is simulated by COPASI program to identify metabolic flux analysis and metabolic control analysis.2-4) We developed MATLAB programs for parameter estimation and parameter sensitivity. Parameter sensitivities represent fundamental aspects of flux control as they summarize the extent of systemic flux control exercised by the activity of a single enzyme in the pathway. It will be a great help to make optimization pathway to ethanol.

Growth advantage phenotype of E. coli is a desirable trait for many biotechnological processes. In this study, we isolated recombinant E. coli strains which grow better than a wild type strain through serial subculture. Specifically, we constructed an E. coli genomic library that enables overexpression of open reading frames inside the insert. After transforming the library into E. coli DH5α strain, we performed serial subcultures in order to isolate the transformants exhibiting grwoth advantage phenotypes. Final ODs of the cultures increased gradually after each round of the serial subculture. We finished the serial subculture and isolated individual colonies after 25 times of transfer colonies grew better than E. coli DH5α strain containing a control plasmid in terms of final OD. This result suggests that overexpression of endogenous E. coli gene could confer growth advantage phenotypes into E. coli strain.

Elastin-like polypeptides (ELPs) have the transition temperature (), so-called “lower critical solution temperature” (LCST), which leads to the phase transition; they are aggregated above like a gel and dissolved below like a sol.1) This thermal property of ELPs is applicable to the several fields such as inverse transition cycling (ITC) purification, dip-pen nanolithography (DPN), and drug delivery system (DDS), etc. Using the biological method, we synthesized genes of two different ELP sequences and combined two of these sequences to produce block copolymers. To study the change of thermal properties, two different sequences, and , were designed and combinatorial polypeptides were expressed in E.coli. The thermal properties of the expressed polypeptides are being investigated according to their chain length and sequence variation.

Antimicrobial peptides (AMPs) are agents of the most ancient form of defense systems. With the increasing concerns about the antibiotics- resistant bacteria, the AMPs have attracted attention as alternative antibacterial agents. To date, however, few effective high-throughput approaches have been developed for designing and screening peptides. In this work, we attempted to express biologically active antimicrobial peptides in a cell-free protein synthesis system. Through numerous approaches to enhance the efficiency of translation as well as to increase the stability of the expressed peptides, preparative amounts of active antimicrobial peptides were successfully expressed in the developed system.

CoenzymeQ10 is a promising nutraceutical supplement that is currently produced using chemical and biotechnological methods from plant materials. While wildtype E. coli produces CoQ8 as an electron carrier in the electron transport pathway, direct production of CoQ10 from glucose is possible in recombinant E. coli expressing dps gene coding for decaprenyl diphosphate synthase. However, the amount of accumulated CoQ10 in the recombinant E .coli strain is not high enough for commercial production. Prior studies reported that overexpression of endogenous genes (dxs and idi) enhanced the production of lycopene in E. coli by increasing metabolic fluxes in the isoprenoid synthesis pathway. We hypothesized that overexpression of dxs and idi could increase CoQ8 synthesis in E. coli because both lycopene and CoQ8 synthesis require precursors from the isoprenoid synthesis pathway. As such, we investigated the effects of dxs and idi overexpression on CoQ8 production in E. coli. Specifically, we constructed four expression cassettes containing dxs, idi, dxs+idi, idi+dxs under the control of a strong promoter. As expected, CoQ8 production in E. coli significantly increased after introduction of the expression cassettes.

Pancreatic carboxypeptidase B (CPB) is a zinc metalloenzyme that hydrolyzes the C-terminal amino acids from alimentary protein and peptides with a preference for C-terminal Lys or Arg residues [1]. The cDNA for the human pancreatic procarboxypeptidase B (hproCPB) was amplified by PCR and subcloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the high level expression of heterologous proteins in Pichia pastoris. The recombinant plasmid was named as pPIC9-hproCPB (9.2kb). By digestion with SacI, the linearized pPIC9-hproCPB was transformed into P. pastoris strain GS115 and integrated into its genome. In the fermentor batch culture of transformed cell on BMY medium, the activity of CPB after removal of pro region by trypsin treatment reached about 1.3 unit/ml. The fed-batch cultivation employing a carbon source with a methanol-to-glycerol ratio of 1:0.5 gave about 26.1 unit/ml of hproCPB at 60 h. No enzyme activity was found in the intracellular fraction. This result is corresponded to 20-fold higher level of enzyme activity than that of batch culture.

Thioredoxin-1 (TRX) is a redox-active protein with multiple intracellular and extracellular functions1-2). TRX has recently been shown to be responsible for the onset of various diseases. However, at present, the mechanism and pathway by which extraceullular TRX influences cellular behavior remain unknown3-4). The aim of this study was to determine the binding partner of exogenous TRX. We first found that exogenous TRX (tagging with hexa-histidine) internalized into cell. We also used His-TRX pull down experiment, immunoprecipitation, LC-MS/MS, and ESI Q-TOF MS/MS to identify membrane proteins involved in the entrance of TRX. As a result, several membrane proteins including GRP78 were found to bind exogenous TRX, which may play a crucial role in TRX internalization and signal transduction. Our studies are in progress to understand the molecular mechanisms of the TRX entry under the inflammatory response and oxidative stress.

Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 x 104 ATFs that consist of 3 ZF DNA binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure, and cold shock. Genes associated with the selected phenotypes were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the phenotypic engineering of and can facilitate functional genomic studies in microorganism.

Cancer that is one of serious diseases for human causes angiogenesis, metastasis, and abnormal cell divisions including genetic change (mutagenesis). Thus, lots of researches have been investigated to inhibit cancer. Human 90K (h90K) protein has very crucial role for inhibition of cancer metastasis. h90K is a glycoprotein having 7 N-glycosylation sites. In this work, we performed expression of recombinant h90K glycoprotein in Drosophila S2 cell system. We constructed stably-transfected S2-h90K cells by antibiotic selection and confirmed h90K expression using Western Blot analyses with and without PNGase F treatment. We also identified glycosylation patterns of recombinant h90K glycoprotein using HPLC and MALDI-MS.

The sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2199 bp sesquiterpene cyclase encoding gene from germacradienol synthase gene cluster named as Sp-gS whose putative function is Germacradienol/Geosmin synthase. The amino acid sequence of Sp-gS shows 66 % identity with SAV2163 (geoA) gene from Streptomyces avernitilis MA-4680 and which produces 4,8a-dimethyl-decahydronaphthalen-4a-ol (geosmin). The gene encoding sesquiterpene cyclase from Streptomyces peucetius ATCC 27952 has been amplified, cloned and expressed in E. coli BL21 (DE3).

The dihydrochalcomycin gene cluster from S. sp. KCTC0041BP contains two glycosyltransferases assigned as gerT1 and gerT2 where gerT2 encodes for the protein which functions as dTDP-chalcosyltransferase and is responsible to the attachment of dTDP-chalcose to the macrolide ring at C-5 position in the dihydrochlacomycin biosynthetic pathway. We have identified, characterized the functions of gerT2 in the biosynthesis gene cluster and disrupted using the insertional in-frame deletion method in order to know the biosynthetic pathway in 16-membered macrolide. Complementation of gerT2 has been carried out by using desVII from heterologous host S. venezuelae ATCC15439. These results suggest that the desVII can functionally replace gerT2 for the glycosylation to proceed in vivo in presence of its auxillary protein DesVIII.

Hydrogenase is one of key enzymes in biological hydrogen production using various microorganisms. After it was found in Escherichia coli by Stephenson and stickland in 19311 at the first time, tremendous efforts have been done to elucidate its biological role and biochemical property. However, many things remain uncertain because its formation and maturation process follows very complicated steps and gene transcription and expression is controlled by complex regulation. Up to now, it has been known that hydrogenase 1 and 2 have only hydrogen uptake activity different to hydrogenase 32. In this study full operon of hya, encoding hydrogenase 1 of E. coli, was cloned and effect of overexpression of hydrogenase 1 in E. coli on biological hydrogen production was checked. Recombinant E. coli BL21 expressing hydrogenase 1 produced highly enhanced molecular hydrogen compared to wild type strain which produces little amount of hydrogen.

Mannheimia succiniciproducens MBEL55E is a non-motile, non-spore-forming, mesophilic, and capnophilic gram-negative bacteria isolated from Korean Bovine. M. succiniciproducens can produce a large amount of succinic acid as a major fermentation product while producing succinic, acetic, and lactic acids as the major end-products. However, cloning genes directly into M. succinciproducens is not reported so far. Therefore we constructed a shuttle vector pME19 which replicates stably in both Escherichia coli and M. succiniciproducens using the replicon of pMVSCS1. cre gene was cloned into the shuttle vector and its recombinase activity was verified in M. succiniciproducens.

Streptomyces are the well known gram positive bacteria which contribute a large number of secondary metabolites including a number of antitumor drugs. Streptomyces peucetius ATCC 27952 is reported to be only an organism till date to produce anticancer chemotherapeutic doxorubicin. Genome analysis of this strain shows the presence of a large number of gene clusters responsible for doxorubicin production. We describe here the construction of disruption vector by using pKC1139 to delete doxorubicin biosynthetic gene clusters from S. peucetius ATCC 27952.

This study presents an in silico method for improving an organism on the basis of the flux sum (Φ), metabolite availability, and more particularly to a method for screening key metabolites that increase production yield of a useful substance, the method comprising defining the metabolite utilization of an organism for producing a useful substance as flux sum and perturbing the flux sum, as well as a method for improving an organism producing a useful substance, the method comprising deleting and/or amplifying genes associated with said screened key metabolites. [This work was supported by the Korean Systems Biology Project of the Ministry of Science and Technology (M10309020000-03B5002-00000). Further supports by the LG Chem Chair Professorship and KOSEF through the CUPS are appreciated].

Due to dwindling of fossil fuel, microbial production of bio-fuel from organic byproducts has acquired significance in recent years. Ethanol has been trusted as an alternate fuel for the future. Zymomonas mobilis ferments sugar into ethanol with two different enzymes, alcohol dehydrogenase and pyruvate decarboxylase.2) By inserting Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II in E. coli, E. coli was able to ferment sugars into ethanol.1,3) This study Zymomonas mobilis (containing pdc and adhB genes) were used as the construction sources of genes and plasmids. Expression vectors carrying pdc and adhB genes were constructed by using pET-32a vectors. Ethanol productivity in Escherichia coli strain seemed to be affected by the extent of expresstion of pdc gene along with adhB genes. By successful gene mutation we could establish a new Escherichia coli strain which can produce ethanol efficiently.

The heterologous expression of microbial DNA extracted directly from environmental samples (environmental DNA, eDNA) in easily cultured hosts can provide access to natural products produced by previously uncultured bacteria. A gene encoding N-acyltyrosine synthase (NAS) from eDNA was cloned and sequenced. The NAS gene was cloned into an expression plasmid pIBR25 and integration plasmid pSET152 ermE* to construct pINAS and pSNAS recombinant plasmids then transformed in Streptomyces strains (S. lividans TK24 and S. venezuelae YJ028) by PEG-mediated protoplast transformation. The bioassay, HPLC analysis, GC-mass and analysis ESI-Mass analysis are to be carried out for identification of the isolated compound from transformants.

Stability of mRNA is an important factor that directly affects the efficiency of cell-free protein synthesis. Although the half-life of mRNA in a cell-extract does not generally exceed several miniutes, in this study, we demonstrate that an appropriately designed mRNA molecule can serve as functional template for greatly extended reaction periods in a continuous-exchange cell-free protein synthesis (CECF) system. When a reaction mixture for CECF reaction was programmed with in vitro-transcribed mRNA, continuous accumulation of protein product was observed at least for 20 hours, indicating that the mRNA molecules were effectively protected from nucleolytic enzymes present in the cell-extract. We believe that this astonishing result was due to the blockage of mRNA with continuously recycling ribosomes. We also expect that the presented results will significantly contribute to the development of efficent tools for deciphering genetic information.

Bacteria form biofilms by adhering to biotic or abiotic surfaces, causing several problems such as reducing the transport of mass and heat, increasing resistance to antibiotics, and shortening the lifetime of modules in bioindustrial fermentors. To overcome these problems, a biofilm-deficient Escherichia coli BD123 was constructed by deleting genes involved in curli biosynthesis and assembly 8(csgG-csgC), colanic acid biosynthesis and assembly 8(wcaL-wza), and type I pili biosynthesis 8(fimB-fimH). BD123 almost remained as planktonic cells under laboratory conditions. BD123 became more sensitive to antibiotics than the wild-type E. coli MG1655: the growth of BD123 was inhibited even with one fourth of the antibiotics needed for the growth inhibition of MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655 and the production of recombinant proteins and secretion of protein expressed was about 16% and 25% greater in BD123, respectively. These results indicate that the biofilm-deficient strain has several significant advantages as a host strain for biotechnology.

Vibrio vulnificus is a halophilic and highly human-pathogenic bacterium, showing very high mortality rate when infected. In order to facilitate the drug development process for this, we undertook in silico analysis to identify specific drug targets in the genome-scale metabolism of V. vulnificus. With a newly sequenced and annotated genome of V. vulnificus, we first reconstructed its genome-scale metabolic network consisting of 946 reactions and 766 metabolites. Subsequently, we employed constraints-based flux analysis, an optimization-based simulation technique, to validate the model in comparison with experimental data, and identify essential genes comprising the metabolic network. This study is a first step for the rational design of biomedical experiments based on the computational approach. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. M10309020000-03B5002-00000).Further supports by LG Chem Chair Professorship, Microsoft and IBM SUR program are appreciated.]

Sphingolipids are important structural components of eukaryotic membranes and are thought to provide rigidity to these structures. Along with this architectural role, sphingolipids and their precursor molecules are major participants in the regulation of cell proliferation and function.1) Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Though the effectiveness of ceramide is not understood fully, ceramide has become a widely used ingredient in cosmetics and pharmaceutical industries. Here we investigated the effect of three genes, Lac1, Lag1, and Sur2. In ceramide pathway, a very long chain fatty acyl-CoA is linked by and amide bond to DHS or PHS to generate dihydroceramide or phytoceramide. This step is catalyzed by Lac1 and Lag1. DHS is hydroxylated at C4 to yield PHS, which is the primary sphingoid base bound in most fungal and plant ceramide. This reaction is catalyzed by Sur2. The genes were cloned on pYES2 vectors. S. cerevisiae was cultured in a batch fermentor and analyzed by HPLC using ELSD after cell lysis followed by solvent extraction.2)

More than 70% of our planet’s surface is covered by oceans and life on earth has its origin in the sea. In recent years, marine natural products are used to foods, minerals, dietary fiber, and medicine. Especially, marine natural product has yielded a considerable number of drug candidates. Thus, we have constructed marine natural product library through collection of a number of marine natural products in the east sea. In the present study, we are screened the marine natural products that are capable of vasodilatation on vasoconstrictor-, such as endothelin-1, angiotensin II, thromboxane A2, and depolarization-induced isometric constriction of rabbit basilar artery using organ bath. Also, we are investigated that vasodilatative mechanisms of marine natural products and selectivity on rabbit basilar and carotid artery onto cell and tissue levels. As the results, vasodilatation of marine natural products are entirely mediated to L-type Ca2+ channel inhibition and partially contributed to vasodilatative effects on endothelin-1-induced vasoconstriction. Marine natural products are selectively dilated basilar artery than carotid artery over the 10 folds.

More than three hundreds microorganisms to hydrolysis carboxylmethylcellulsoe (CMC) were isolated from the seashore of the Kyungsang province in Korea. Among them, twelve microorganisms showed relatively higher activity of cellulase. Activity expressed as unit of cellulase by twelve microorganisms ranged from 35 to 110 unit with the liquid medium containing 2.0%(w/v) glucose, 0.25% yeast extract, 0.5% K2HPO4, 0.1% NaCl, 0.02% MgSO4·7H2O and 0.06% (NH4)2SO4 at 30°C for 72 hr under aerobic conditions. The best carbon source and nitrogen sources for the production of cellulase by P.aquimaris LBH-10 were found to be rice bran and peptone. CMC, Filter paper, Avicel and pNPG activities of the cellulase produced by P.aquimaris LBH-10 with 2.0%(w/v) rice bran and 0.5%(w/v) peptone were 929.97, 109.04, 94.37 and 4.38 unit for 72hr respectively.

The advent of in silico genome-scale model developed various algorithms for the gene targeting. In FBA, the flux distribution is quantified by linear optimization on the basis of the information about the stoichiometry of metabolic reactions and mass balances around the metabolites under pseudo-steady state, or stationary, assumption. minimization of metabolic adjustment (MOMA) and regulatory on/off minimization (ROOM) reflect the bacterial characteristics by minimizing metabolic adjustment for knockout condition. However, these algorithms optimize only the limited objective function. To improve a strain for biochemical production, the organism should be investigated from diverse sides simultaneously: for instance, biomass formation, biochemical production, and waste formation. In this respect, we propose a new approach called the flux scanning with compromised objective fluxes (FSCOF) that optimizes multi-objective functions. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. M10309020000-03B5002-00000). Further supports by LG Chem Chair Professorship, Microsoft and IBM SUR program are appreciated.]

The attachment of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. Sialyltransferase is responsible for the terminal sialylation by transferring a donor sugar nucleotide, cytidine monophospho (CMP)-sialic acid, to a galactose residue of oligosaccharide. Sialic acid is activated to the nucleotide sugar CMP-sialic acid by CMP-sialic acid synthase. To increase the sialylation of recombinant glycoprotein, human 2,3-sialyltransferase (ST) and CMP-sialic acid synthase (CMP-SAS) were engineered into Chinese hamster ovary (CHO) cells which produce human erythropoietin (EPO). When the 2,3-ST was expressed in CHO cells, the sialylation of EPO glycans increased, compared with the control. However, when both 2,3-ST and CMP-SAS were co-expressed in CHO cells, the sialylation increased slightly compared with the expression of 2,3-ST alone, even though the intracellular pool of CMP-sialic acid increased significantly.

The diversity of marine bacterial communities has been extensively investigated in many studies using the traditional culture methods. Such techniques are known for their selectivity and are not considered representative of the extent of the bacterial community. The proportion of cells which can be cultured is estimated to be 0.1% of the total population and few data are available concerning how closely they reflect the actual composition of these communities. Comparison of the base sequences of 16S rRNA has become a powerful tool to infer the evolutionary pathways of procaryotes. Recently, a procedure that uses 16S rRNA sequence homology was proposed for defining and enumerating the components of a mixed natural population of bacteria.. It should allow the definition of a population, both quantitatively (species diversity) and qualitatively (phylogenetic relatedness), without culturing1). In this study, we investigated the bacterial communities associated with the marine water in the East Sea. Marine water samples were collected in Damupo, Pohang, Korea and the environmental DNA was directly extracted from the sea water. Bacterial 16S rRNA gene library of 200 clones was generated using bacterial universal primers and 16S rDNA sequences were analyzed phylogenetically. The library was dominated by marine uncultured bacteria, gamma-Proteobacteria, and alpha-Proteobacteria. Ninety-four percent of the cloned sequences was highly related to the known bacteria (Rhodobacter, Bacillus, Acidobacteria), while some of the cloned sequences showed less affiliation with known taxa (< 90% sequence similarity) and may represent novel taxa.

A bacterial strain producing a high concentration of red pigment isolated from sediment that had been collected East China Sea, Which was recorded as CH-34. It was identified as Zooshikella sp., as based on the biochemical properties and 16S rRNA gene sequence. CH-34 was grown in marine broth were incubated (120rpm, 25 ) for 10 days. They produced pigments with ℃ maximum absorption at 540 nm, which indicated the presence of prodigiosin, a well-known red pigment previously detected in Serratia marcescens1). This study was investigated the antimicrobial activity of the culture broth(ZB), and bacterial cell(ZC). ZC and ZB extracts according to extraction solvents and fractions3). The antimicrobial activities of ZC and ZB extracts was tested against 21 microorganisms which were fish and human pathogens bacterial species. The extracts showd high activity against Streptococcus iniae, Streptococcus parauberis, Staphylococcus aureus Propionibacterium acnes, Streptococcus mutans and had poor effect on Serratia marcescens, Proteus mirabilis. Among the various solvent layers, the ZC methanol extraction (ZCEM) and ZC ether soluble fraction (ZCFE) showed strong antimicrobial activities.

A putative EHase (EEH1) was identified by analyzing open reading frames of Erythrobacter litoralis HTCC2594, the phylogenetic analysis of the putative gene showed that eeh1 was similar to microsomal EHase. The eeh1 gene was cloned and the recombinant (rEEH1) was purified. Kinetic resolution of SO and ECH using the purified EEH1 of E. litoralis HTCC2594 was performed. Optically active (S)-SO with an enantiomer excess (ee) higher than 97% was obtained from its racemate with a yield of 10% (theoretical 50%). It was not high, compared to the activities of the pervious report. On the other hand, the hydrolysis of the (R)- ECH proceeds at a much higher rate than the hydrolysis of the (S)- ECH. Enantiopure (S)-ECH could be obtained with an ee value higher than 97% with a yield of 32%. Enantiopure ECH is a valuable epoxide intermediate for preparing optically active pharmaceuticals1). EEH1 from E. litoralis HTCC 2594 can be applied to bioprocess for the production of optically pure epoxides in the pharmaceutical industry. [This work supported by KORDI in house program (PE97803) and the Marine and Extreme Genome Research Centre Program, Ministry of Marine Affairs and Fisheries, Republic of Korea.]

Recently, a genetically engineered succinic acid overproducing mutant, M. succiniciproducens LPK7, was developed based on full genome sequence. 2-DE and LC-MS/MS were used to analyze proteome of the mutant cells at the exponential and stationary phases. The results were compared with those of the wild type strain (MBEL55E) to elucidate the global physiological and metabolic changes responsible for succinic acid overproduction. Comparative proteomic analyses between the MBEL55E and the mutant strain showed the apparent differences in 87 and 69 protein spots at the exponential and stationary phases, respectively. As the mutant cells grow, the expression levels of 58 proteins also changed. Several features, indispensable for further improving the succinic acid producers by rational metabolic engineering, will be described in detail. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (2005-01294). Further supports by the LG Chem Chair Professorship, IBM SUR program, Microsoft, and by the KOSEF through the Center for Ultramicrochemical Process Systems are appreciated.].

Bacillus subtilis subsp. subtilis A-53 was isolated from the seashore of the Kyungsang province in Korea.1) The highest production of cellulase was obtained when concentrations of rice hull and yeast extract were 5.0% (w/v) and 0.0% (w/v). Optimal initial pH of the medium and temperature were 6.8 and 37℃. Under these conditions, optimal agitation speed and aeration rate in a 7L bioreactor were found to be 400 rpm and 1.0 vvm.2) The gene coding cellulase in B. subtilis subsp. subtilis A-53 was isolated and cloned. The deduced amino acid sequence of the cellulase showed high identity to cellulase from other Bacillus species. E. coli JM109 was used as a host strain for cloning and maintenance of plasmid. The production of cellulase by B. subtilis subsp. subtilis A-53 and its transformant from rice bran as a carbon source was 68.7(U/ml) and 868.8(U/ml),respectively.