Earticle

In a previous study, we have isolated 108 strains of Lactobacillus spp. from human. In this study, 22 strains showing antagonistic activity against avian pathogens Salmonella enteritidis and S. gallinarum were selected among them. One strain out of 22 strains which had high-leve of antimicrobial activity was identified as L. paracasei (KLB58) by 16S rRNA gene sequencing. The activity was heat stable and proteincaeous. The mode of action was shown to be bacteriostatic, and the antimicrobial substances were produced during the early stationary phase. In order to assess potential usage as probiotics for poultry KLB58 strain was tested for its ability to prevent Salmonella gallinarum infection in chickens. Chickens challenged with S. gallinarum showed markedly increased death rate compared with the control group. However, the death rates were significantly lower in chickens (13.3%) supplemented with KLB58 than in those (40%) not supplemented (P<0.05). Therefore, we suggest that L. paracasei KLB58 has potential probiotic effect for chickens. Further studies are underway to characterize the antimicrobial substance.

Cellulase have many industrial applications. Trichoderma reesei Rut C-30 produced high leveles of endo-β-1,4 glucanase and exo-β-1,4-glucanase. Cellulase production of Trichoderma reesei Rut C-30 with various initial concentrations of EM in medium producing cellulase was investigated. The conpositions of culture medium and condition are : Avicel 10g/l, Wheat Bran 50g/l, (NH4)2SO4 2.0g/l, Proteose Peptone 3.0g/l, Yeast Extract 0.5g/l, KH2PO4 4.0g/l, MgSO4·7H2O 0.3g/l, CaCl2·2H2O 0.3g/l, Tween-80 0.2ml/l, and 28℃ with 200rpm in shaking incubator for 6 days. Carboxymethyl cellulose(CMC) and filter paper(FP) activities were determined according to the method of the International Union of Pure and Applied Chemistry(IUPAC). The amount of reducing sugar liberated was determined by the dinitrosalicylic acid(DNS) method. One unit of enzyme activity was defined as the amount releasing 1 μmol of reducing sugar per minute. Adding EM temporarily makes cellulase activities increased. Cellulase activities are related to fungi and EM concentrations. As so cellulase activities are prone to influence of concentrations of contained EM.

Dengue virus is the cause of dengue fever epidemics in tropical areas around the world, leads to significant loss of life in these areas. There are four serotypes of dengue virus, which are genetically similar but not identical, and infection of one serotype will not provide immunity to the infection of another serotype. In fact, the fatal dengue hemorrhage fever and dengue shock syndrome are often caused by reinfection with a different serotype. Today, therefore, there has not been an effective vaccine to fight against this epidemic. Recently a component of the capsid proteins has been identified as an effective antigen for vaccine development, namely the domain III of capsid glycoprotein. In this study we explore the possibility of developing an oral recombinant vaccine against type II dengue virus. The domain II protein is fused with LTB, which acts as an adjuvant for oral administration route, and is expressed in Saccharomyces cerevisiae. The transformation was confirmed by colony PCR and the expression was confirmed by Northern, Western and ELISA analysis

To elucidate the function of an unknown regulator in Streptomyces, differences in phenotype and antibiotic production have been compared between a deletion mutant and wild type strain. However, these differences are easily hidden by complex media. Thus, we developed a multi-well method containing different nutrients along with bromothymol blue to determine the specific media conditions which can reveal such differences easily. By comparing the growth of wild type and mutant strains on the screened nutrient conditions, we were able to measure growth, organic acid production, and antibiotic production to elucidate the function of unknown regulator. As a result, a member of the MarR-like regulator family, SCO5405 (AbsC), was newly characterized. Deletion of SCO5405 increased the pH of the culture broth due to decreased production of organic acids such as pyruvate and α-ketoglutarate and increased extracellular ACT production in minimal media containing glucose and alanine. Therefore, this method could be a high-throughput method for the characterization of unknown regulators.

The induction stage of the unicellular alga, Haematococcus pluvialis, is constituted by two parts, encystment which is a morphological change from vegetative green cell to the cyst, and carotenogenesis that is to progress accumulating the economically valuable carotenoid, astaxanthin. And an effective induction requires the ROS (reactive oxygen species) generated by drastic environmental stresses like high light, high C/N ratio and high temperature. Therefore the microbe should be put on the pressure of transformation to encystment in the hard conditions. and such an intensive stresses lead to make the cell partially lethal by the burst of the oxidative damage so that a loss of the cell mass and the yield of astaxanthin. here we applied to combine an environmental stress (salt, sodium chloride) with an antioxidant (ascorbic acid) for the effective encystment to decrease the bio-factory loss caused by such an deadly damage, not for the case of carotenogenesis. The optimal condition to improve the carotenoid concentration was discovered at the 0.2%(w/v) NaCl with 0.1%(w/v) Ascorbic acid, cell mass 32% and total carotenoid concentration 23% enhanced respectively.

Leuconostoc citreum is the major lactic acid bacteria growing at an initial phase of kimchi fermentation. It grows fast at both aerobic and anaerobic conditions producing CO2, mannitol and dextran. In this study, high cell density culture of L. citreum was conducted in a fermentor equipped with an internal ceramic filtration system. The filter material was porous ceramic with pore size of 0.1 μm. The filter module consisted of 20 vertical cylindrical filter rods with inner diameter, outer diameter, and height of 6, 8, and 65 mm, respectively, and an upper frame of stainless steel. The total surface area of the filter module was ca. 330 cm2. The filter module was installed inside a 2.5-L jar fermentor. After 6 h of batch mode, filtration operation was started by feeding medium containing glucose, fructose, and yeast extract and at the same time by removing broth through the filter module. By using the internal filtration system, optical density at 660 nm increased to 70, which was 10 times higher than that in batch culture. The main co-products were mannitol and D-lactic acid. Mannitol and D-lactic acid concentrations increased to ca. 80 g/L and 40 g/L, respectively, by feeding fresh medium.

Vibrio harveyi is a pathogenic bacteria associated with vibriosis that is a common disease in marine aquaculture systems. Classified as generally recognized as safe (GRAS) strain of Korean fermented food, 34 strains were isolated from many Jeotgals (salted fish). Also 18 strains were isolated from the gut of Portunus trituberculatus. Growth inhibition assay showed that CFS (Cell-free supernatants) from the 24-h culture of these strains and competition by each microbe were able to inhibit the growth of Vibrio harveyi. The aim of this study is to control the water environment for culturing larvae of marine animals like the swimming crab and shrimp, using microorganisms. At present, experiments are being performed using 52 strains from Jeotgals and the gut of Portunus trituberculatus. We anticipate that it is possible that some bacterial strains will be effective for use a biological control agents in marine aquaculture systems.

Mechanical parameters for mass production of heteropolysaccharide-7 (PS-7) by Beijerinckia indica HS-2001 were optimized in pilot-scale bioreactors. The production of PS-7 and its conversion rate from 20.0 g/L glucose was 6.57 g/L and 33.0%. However, the maximal yield of total utilization (Yp/s + Yx/s) was 0.45 when the carbon source was 20.0 g/L sucrose. Higher inoculum size decreased the lag phase of cells and led to enhanced cell growth. Based on productivity and economic aspect, the optimal inoculum size for the production of PS-7 by B. ndica HS-2001 was decided to be 5.0% (v/v). The optimal agitation speed and aeration rate for cell growth of B. indica HS-2001 in a 100 L bioreactor were 300 rpm and 1.5 vvm, whereas those for the production of PS-7 were 250 rpm and 1.0 vvm. The optimal inner pressure for cell growth of B. indica HS-2001 in a 100 L bioreactor was 0.2 kgf/cm2, whereas that for the production of PS-7 was 0.4 kgf/cm2. The production of PS-7 by B. indica HS-2001 from 30.0 g/L sucrose with an optimized inner pressure of 0.4 kgf/cm2 was 9.02 g/L, which was 1.25 times higher than that without inner pressure in a 100 L bioreactor.

Nattokinase is a potent fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. The enzymes were discovered from various sources, such as Japanese natto, Korean Chungkookjang soy sauce, and Chinese douchi. Intravenous fibrinolytic agents such as urokinase and tissue plasminogen activator have been widely used in clinical practice for thrombolytic therapy. Microbial fibrinolytic enzymes have now attracted much more attention than typical thrombolytic agents because of the expensive prices and the undesirable side effects of the latter.In this study, Bacillus subtilis was cultivated to high cell density for nattokinase production. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on cell growth and nattokinase production. Among complex media, the highest cell growth (optical density monitored at 600 nm) and nattokinase activity were obtained with peptone supplementation. Amino acid analysis of the three complex media showed that glycine was richest in peptone. In fed-batch culture of B. subtilis, optimum ratio of peptone to glucose in feeding solution was 3:1. At this condition, cell concentration and nattokinase activity were 75 g/L and 15,000 unit/ml, respectively. By supplementing 2 g/L glycine in peptone-containing media, nattokinase activities increased compared without glycine supplementation

A wide range of enteropathogens cause serious diarrhoeal diseases in fattening piglets and account for food-related infections in humans. The objective of this study was to screen beneficial bacterial strains from the gastrointestinal tract of animal sources and lactic acid bacteria(LAB) for antimicrobial activity against diverse hazardous pathogens associated with pig growth period. In this work, 3 strains from porcine gut compartment and 6 strains from KCCM were cultured in MRS medium at 37℃ for 48hr. Using Kirby-Bauer method, 9 well-characterized strains belonging to Lactobacillus were studied for inhibition against Escherichia coli O-157, Escherichia coli K88, Staphylococcus aureus, Salmonella enteritidis, as well as Salmonella typhimurium. Potential antimicrobial strains were analyzed by studying their cell-free supernatants without pH neutralization. E. coli O-157 was effectively inhibited by L. acidophilus, L. rhamnosus, L. casei and L. reuteri LA26-1 strains. E. coli K88 and S. enteritidis were inhibited by L. acidophilus, L. rhamnosus, and L. plantarum. The findings of this study provide a basis to encourage feeding studies to evaluate the antimicrobial potential of promising strains in pig production.

The toxic effect of ionic liquids (ILs) on spectral features using Fourier Transform Infrared (FT-IR) absorbance spectroscopy was studied using a metabolically versatile bacterium Shewanella oneidensis MR-1. In this study, we focus on the ILs-induced biochemical changes in Shewanella oneidensis MR-1. The spectrum of MR-1 is quite complex and contains several bands arising from the contribution of different functional groups belonging to proteins, lipids, nucleic acids and others. The FT-IR spectra reveal significant differences in absorbance intensities in areas between control and ILs treated cells; this variation shows the alteration in biochemical contents like lipids and proteins in MR-1 cells due to ILs intoxication. Furthermore, subcellular fractionation was done to find out where the ILs were accumulating in the cells. Remarkably, the ILs could only be detected in the cytoplasmic fractions, and not in the cell membrane fractions neither the extracellular wash fractions.

The optimal conditions for the mass production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens by a recombinant E. coli JM109/DL-3 were established in this study using an orthogonal array method. Rice bran and tryptone were found to be the best carbon and nitrogen sources for the production of CMCase by E. coli JM109/DL-3. The optimal concentrations of rice bran and tryptone and the initial pH for the production of CMCase were 50.0, 2.5 g/L, and 7.5, respectively. The optimal concentrations of K2HPO4, NaCl, MgSO4·7H2O, and (NH4)2SO4 in the medium for the production of CMCase by E. coli JM109/DL-3 were 5.0, 1.00, 0.40, and 0.60 g/L. The optimal agitation speed and aeration for cell growth of E. coli JM109/DL-3 in 7 L bioreactors were 500 rpm and 1.0 vvm, whereas those for the production of CMCase were 400 rpm and 1.0 vvm. The optimal inner pressure for cell growth of E. coli JM109/DL-3 in 100 L bioreactors was found to be 0.8 kgf/cm2, whereas that for the production of CMCase was 0.6 kgf/cm2. The production of CMCase with an inner pressure of 0.6 kgf/cm2 was 1.36 times higher than that without an inner pressure.

The symbiosis of Effective Microorganisms (EM) is investigating. EM consists eighty different species and generally, there are three main genera of EM which are phototrophic bacteria, lactic acid bacteria, and yeast. Recently, EM is applied in various fields such as in industry, agriculture and application of natural health. Particularly, typical featured of EM has been already known can do antioxidant activity which furthermore can be used as an additive component for producing nutrient drinking water or health food. It mainly consisted three strains that were used in this study which were Lactobacillus plantarum, Saccharomyces cerevisiae, and Rhodobacter spheroides. These microorganisms in co culture condition are possible to work synergistically to do antioxidant activity. Thus the synergy of co cultured broth was evaluate as the antioxidant activity. The objectives of this study were to investigate the antioxidant activity of each microorganisms and to optimize the antioxidant activity of co-culture microorganisms. Optical density of cell was done by using Spectrophotometer at 600 nm and plate counting on selective pressure was used to analyze the cell growth. The scavenging activity was evaluated by measuring the decrease of absorbance of DPPH solution at 517nm after mixing with the supernatant of each cell culture. It was found those strains are capable of producing the antioxidant activity in culture condition.

In our previous study, recombinant E.coli harboring flavincontainingmonooxygenase(FMO) gene from Methylophaga aminisulfidivorans MPT produced indigo (853mg/L) and indirubin (≤5mg/L) in tryptophan medium (2g tryptophan, 10g NaCl, 5g yeast extract per liter). The goal of this study is to characterize the FMO and investigate the optimum culture conditions to enhance the amount of indirubin produced. The optimal pH andt emperature for FMO enzyme activity was found to be 8.5 and 45℃, respectively. The enzyme showed high affinity with trimethylamine (TMA). Vmax and Km values for trimethylamin were 19.3 nmol/min/mg protein and 23 μM, respectively. The optimum culture conditions determined by response surface method were as follows: 1g/L tryptophan, 5g/L yeast extract, 3mM cysteine, pH8.0 and temperature at 35℃. Under this condition, 15 times higher amount of indirubin (75.1mg/L) was produced.

Butyric acid producing bacteria have important role in medicinal, food, pharmaceutical and chemical industries. In this study, an anaerobic microorganism utilizing galactose as a sole carbon source was isolated from activated sludge, and that is capable of producing butyric acid. Based on genotypic investigation, the isolated microoranism is identified as Bacillus coagulans. Almost of Bacillus coagulans species was well known as a lactic acid producer, not a butyric acid producer. Interestingly, the isolated Bacillus coagulans can produce butyric acid, but not lactic acid. The optimal growth of the isolated Bacillus coagulans occurred 40℃ and pH 5.5 and the final concentration and yield of butyric acid was 17.43 g L-1 and 0.44 g g-1 in batch culture. Further study is required to investigate the utilization potential of seaweed hydrolysates by the isolated strain.

Chromohalobacter sp. strain HS-2 was isolated from salted fermented clams and analyzed for the ability to grow on benzoate, m- and p-hydroxybenzoate derivatives as the carbon and energy source. Our previous study found that the genes responsible for the (hydroxy)benzoate degradation of HS-2 were clustered within approx. 39 kb region, which is a distinctive organization that has not been identified from those of other benzoate catabolic genes. As an initial study of the benzoate pathways, m-hydroxybenzoate hydroxylase gene (mobA) and p-hydroxybenzoate hydroxylase gene (pobA) in the cluster were determined to be overexpressed in E. coli. The two genes were amplified from HS-2 genomic DNA, cloned into an expression vector, pET-28a(+), separately and then the cloned vectors were transformed into E. coli BL21 (DE3). The overexpression of mobA and pobA in E. coli, however, yielded significant amount of aggregated insoluble recombinant proteins. To increase solubility of recombinant MobA and PobA, the hydroxylase genes were co-expressed with heat-shock proteins (DnaJ-DnaK). The expressed MobA and PobA were analyzed by SDS-PAGE and specific bands with a molecular weight of about 72 kDa and 44 kDa were appeared, respectively. HPLC analysis revealed that the resting cells of E. coli BL21 (DE3) harboring mobA and pobA converted 50 mM of m- and p-hydroxybenzoate to 15 and 35 mM of protocatechuate, respectively, at 30℃.

The effects of raw glycerol on 1,3-propanediol (1,3-PDO) production were investigated with various microorganisms. Klebsiella strains were generally resistant to the tested 4 types of raw glycerol while the growth of Clostridium strains was variably inhibited depending on the type of raw glycerol. Fed-batch cultures with suspended cells of Klebsiella pneumoniae DSM 4799 revealed that 1,3-PDO production was more effective when utilizing raw glycerol than pure glycerol. In addition, more than 80 g/L of 1,3-PDO was produced using raw glycerol; this is the highest 1,3-PDO concentration reported thus far for K. pneumoniae using raw glycerol. Repeated fed-batch fermentation with cell immobilization in a fixed-bed reactor (FBR) was performed to enhance 1,3-PDO production. Production of 1,3-PDO increased with the cycle number due to the successful cell immobilization. Continuous fermentation with immobilized cells was also performed to investigate the effect of dilution rate and glycerol concentration on 1,3-PDO production.

In our previous study, indigo was produced by recombinant E. coli harboring a flavin-containing monooxygenase gene. Scale-up of batch, repeated batch and continuous fermentation for indigo production was performed in 5 to 3000L reactors containing tryptophan medium (2g tryptophan, 10g NaCl, 5g yeast extract, and 50mg ampicillin per liter) under the conditions as follows: working temperature 30℃, pH 7.0 and air supply 3vvm. The batch fermentation in a 3,000-L fermenter produced 911±22 mg L-1 of indigo from 2 g L-1 tryptophan as substrate (yield 45.5%). For continuous fermentation in a 5-L fermenter, volumetric productivity was found to be 230 mg L-1 h-1 for up to 110 h (final accumulated bio-indigo was 23 g) with a constant dilution rate (D) of 0.084 h-1 (constant feeding rate of 2.8 mL min-1 with medium containing 3 g L-1 tryptophan). Recombinant E. coli cells have the ability to withstand the high concentration of accumulated indigo toxicity in a batch fermentation. In a continuous fermentation, the recombinant cells exhibited high plasmid stability up to 110 h, after which time they lost plasmid. These two types of bio-indigo production system could be used for industrial application which could overcome the environmental problems with synthetic indigo and meet the natural indigo demands in the dye and textile industry.

Actinobacillus pleuropneumoniae is the causal agent of swine pleuropneumoniae, a disease resulting in morbidity and mortality of pigs and accordingly economic losses within the swine industry. Toxins (Apx) produced by A. pleuropneumoniae appear to be important virulence factors of swine pleuropneumoniae. Recently, in order to effective immune responses, various peptides and proteins for binding gangliosides. The role of gangliosides in mucosal immunization has attracted attention due to the emerging interest in oral vaccination. Ganglioside GM1 exists in abundance on the surface of the M cells of Peyer’s patch, a well-known mucosal immunity induction site. It was previously reported that peptide for GM1-specific peptides from a phage display library. The motif of Co1, expect to bind GM1 effectively. In the present study we have expression of ApxIIA toxin epitope of Actinobacillus pleuropneumoniae fused with Co1 ligand of a peptide enhancing mucosal and systemic immune response.

Polyhydroxyalkanoic acids (PHAs) and rhamnolipids considered as biotechnologically important compounds are simultaneously produced by Pseudomonas aeruginosa. Both are synthesized from common precursors, (R)-3-hydroxyfatty acids. To find the probable metabolic relationship between their syntheses, we investigated the PHA and rhamnolipids production in four pha (phaC1, phaC2, phaZ, and phaG), four rhl (rhlA, rhlB, rhlR, and rhlI) and rpoS mutant strains of P. aeruginosa PA14 and PAO1 grown in minimal medium containing 70 mM fructose or 30 mM decanoic acid. Higher PHA accumulation was found in the rhamnolipid-negative mutants than in the wild-type strains, suggesting that 3-hydroxyfatty acid precursors become more available for PHA synthesis when rhamnolipids synthesis is absent. However, compared to the wild-type strains, rhamnolipids production was not enhanced in the four pha mutants of P. aeruginosa PA14 and PAO1 which indicates that rhamnolipids production in P. aeruginosa could be tightly regulated at the transcriptional level by a quorum-sensing response. The metabolic pathways for PHA and rhamnolipid synthesis from medium-chain-length fatty acids were also investigated using octanoic-1-13C acid. 13C-NMR analysis revealed that the monomer-unit (R)-3-hydroxyoctanoate-1-13C being converted from the octanoic acid substrate was effectively incorporated into PHA. In the rhamnolipid synthesis, the (R)-3-hydroxyoctanoate-1-13C is suggested to be firstly converted to (R)-3-hydroxydecanoate-1,3-13C via fatty acid de novo biosynthesis pathway and then further processed into (R)-3-((R)-3-hydroxyalkanoyloxy)alkanoic acids (HAAs) via RhlA. The ratio of mono- to dirhamnolipids in the product depends on the type of carbon sources.

Tautomycetin (TMC), a secondary metabolite produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412 [1]. Within the TMC biosynthetic gene cluster, we found two putative pathway-specific genes, tmcN and tmcT. Both tmcN and tmcT were identified as positive regulators by targeted gene disruption as well as in silico sequence comparison [2]. Here, we isolated, identified, and disrupted a wblA ortholog gene (named wblA-tmc) from Streptomyces sp. CK4412, which was previously reported as a strong global down-regulator of antibiotic biosynthesis in various Streptomycetes speices. Moreover, we added additional tmcN or tmcT into ΔtmcT or Δ tmcN mutants and also disrupted wblA-tmc gene from each of ΔtmcN or ΔtmcT to understand how these regulatory genes be related in TMC biosynthetic gene cluster of Streptomyces sp. CK4412. The detailed results will be further discussed.

Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD+ and NADP+ as cofactors. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37°C. The Km and Vmax values for propionaldehyde in the presence of NAD+ were 1.18 mM and 0.35 U mg-1, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild type strain (from 0.18 g L-1 to 0.72 g L-1) under shake-flask culture conditions and the highest titer (2.51 g L-1 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.

Bacilli are widely used as functional probiotics, feed and food additives. In this work, Bacillus sp. LS 1-2, was isolated from Korean transitional soybean paste foods and had similar 16S rRNA of Bacillus sp. of about 98%. LS 1-2 was cultured in citrus-processing-waste containing 5% solid as a culture medium and produced an antimicrobial agent against both of the gram-positives and negatives at adequate culture conditions (pH, Temperature, Culture period and etc). An antimicrobial agent produced by LS 1-2 was purified by alcohol precipitation, C-18 cartridge, YM-10, silica gel open column, prep TLC and prep HPLC. Antimicrobial activity was detected in the fermentation supernatant of LS 1-2. The active substance had more activity through the each purification step. Molecular weight of the antimicrobial agent was lower 10,000Da and it was a hydrophilic compound. The active substance was not sensitive to lipase, proteinase K, amylase, catalase but was affected by pronase E. So this antimicrobial agent was expected as a bacteriocin-like-substance. In order to evaluate its commercial value, the minimum inhibitory concentration of purified antimicrobial agent from Bacillus sp. LS 1-2 was compared with commercialized antimicrobial agents (Colistin, Chlortetracycline, Ampicillin) against Staphylococcus aureus and Escherichia coli O-157.

In the present study, a novel oleaginous Thraustochytrid containing high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain was identified as an Aurantiochytrium sp. by 18S rRNA sequencing and was named KRS101. The strain utilized various carbon and nitrogen sources, indicating the versatility of this novel isolate. Optimal medium condition for maximal cell growth and lipid and DHA yield was determined as glucose (60 g L-1) as carbon source, corn steep solid (10g L-1) as nitrogen source, and sea salt (15 g L-1). The highest biomass, lipid, and DHA yield of KRS101 with fed-batch fermentation using medium composition was 52.6 g L-1 , 21.9 g L-1, and 8.8g L-1, respectively, in 63.5 h, which is comparable to the highest level reported in the species, indicating the strain is valuable as a single cell oil producer.

In this study, a process of removing a half volume of the culture broth and replacing it with the same volume of substituted solution after 48 h of cultivation was developed to enhance the production of heteropolysaccharide-7 (PS-7) by Beijerinckia indica HS-2001. The optimal substitution time and volume of the substituted solution were found to be 48 h after cultivation and a half of the initial volume of the culture broth. The optimal concentration of glucose in the substituted solution and inoculum size were 20.0 g/L and 5.0% (v/v). Optimal composition for the substituted solution was found to be 20.0 g/L glucose, 10.0 g/L soybean pomace, 0.1 g/L MgSO4 .7H2O, 0.9 g/L NH4NO3, and 5.0 g/L potassium phosphate, which was the same composition as the medium developed in previous study for the production of PS-7 by B. indica HS-2001. Total amount and productivity of PS-7 by B. indica HS-2001 with a substitution under optimized conditions in a 7 L bioreactor for 96 were 49.28 g and 0.51 g/h, which were 1.76 and 1.31 times higher than those without a substitution for 72 h.

Glycerol dehydratase (DhaB) and its reactivase (GdrAB) are essential for the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA), which is a key intermediate for commercially valuable chemicals, 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Both DhaB and GdrAB are multicomponent proteins, whose expression levels often limit the production of 3-HP and 1,3-PDO. In order to improve the optimal expression of multisubunits, a synthetic operon was constructed with high affinity ribosomal binding sites (RBS) and “spacer sequence” at the upstream of each subunit of dhaB and gdrAB genes. The synthetic operon was successfully expressed in Escherichia coli BL21 for the production of 3-hydroxypropionic acid from glycerol.

Protease (EC 3.4.21) catalyzes the cleavage of the peptide bonds in proteins, and it is found in a wide variety of microorganisms. The application of alkaline protease has increased in foods, leathers, pharmaceuticals, bioremediations, and textiles.1) Bacterial protease has become one of the largest classes of industrial enzymes, accounting for 60% of the total global enzyme sales.2) Microbial proteases represent a good source of enzymes due to their broad biochemical diversity, the rapid growth of microorganisms, the limited space for cultivation, and the easy manipulation of cells for the production of novel enzymes.This study aims to conduct purification and characterization of protease from Bacillus subtilis RKY3. The protease produced by B. subtilis RKY3 was purified in two steps including ammonium sulfate precipitation and anion exchange chromatography. Molecular weight of the purified protease was approximately 38 kDa. The purified protease was stable over a broad pH range from 5.0 to 11.0, and it exhibited a relatively high activity at a broad range of pH between 7.0 and 9.0. The optimum temperature for enzyme activity was 60℃, but the activity was sharply deactivated beyond 60℃. The purified protease was found to be serine protease because the enzyme activity was almost inhibited by 1 mM of PMSF. It was stable with oxidants (H2O2), reducing agents (SDS), and organic solvents (bezene, hexane, and toluene). The protease purified in this study has a possibility to be used in detergent and bleaching industries.

3-Hydroxypropionic acid (3-HP) is a potential feedstock for producing many industrially important chemicals. Previously, we reported the production of 3-HP from glycerol by recombinant E. coli SH-BGK1 with a highest titer of 2.8 g/L in shake-flask experiments and 38.7 g /L in bioreactor experiments. However, the 3-HP yield on glycerol was too low (35%). To improve the 3-HP yield on glycerol the gene encoding glycerol kinase (glpK), which converts glycerol to glycerol-3-phosphate, was disrupted. When the resting cells of resulting recombinant strain, harboring glycerol dehydrate and aldehyde dehydrogenase, was subjected to shake flask cultivation the theoretical yield of 3-HP on glycerol was improved to >95%. This study demonstrates the highest yield of 3-HP on glycerol.

Chitosan oligosaccharides are depolymerization products of chitosan, which have a wide range of functional properties such as antimicrobial and antitumor effects.1) Chitooligosaccharides can be produced by chemical and enzymatic methods. The enzymatic production of chitooligosaccharides has become popular because chemical synthesis such as acid hydrolysis typically results in a large quantity of short-chain oligosaccharides and low yields.2,3) The enzymatic degradation of chitosan is generally conducted with chitosanase (EC 3.2.1.132), which catalyzes the endohydrolysis of β-1,4-linkages between D-glucosamine residues in a partially deacetylated chitosan.This study aims to optimize the production of chitosanase from Bacillus subtilis RKY3 through statistical experimental tools. The parameters significantly influencing both chitosanase production and cell growth were identified through Plackett-Burman design, by which maltose, beef extract, MgSO4, and incubation time were selected as the most significant variables. The optimum levels of the screened variables and their mutual interactions were determined through the steepest ascent method and Box-Behnken design. Under the optimized conditions, 62.30 U/mL of chitosanase activity was predicted, which was in good accordance with the validation experiments, The optimized conditions resulted in a maximum chitosanase activity of 63.53±1.22 U/mL. This result is 11.3-fold increased chitosanase activity compared to that before optimization.

The SGNH superfamily is characterized by four highly conserved sequence blocks containing serine (S), glycine (G), asparagine (N), and histidine (H) residues. High specificities of SGNH hydrolases could make them appropriate for a wide variety of applications such as preparations of chiral compounds and enantiomeric resolutions. Here, acyl-CoA thioesterase1 precursor from L. innocus CLIP 11262 was used as a model enzyme to investigate its industrial applications of SGNH hydrolase family. Especially, cross-linked enzyme aggregates (CLEAs) from this enzyme were investigated for the immobilization process. Efficient cross-linking was observed when using 80%-saturation ammonium sulfate precipitations followed by crosslinking with 0.5%(v/v) glutaraldehyde for 12h-14h. The CLEA product was much more resistant to denaturation than the free soluble enzyme. For example, while soluble enzyme retained only 20% of its initial activity after 1 h incubation at 80 °C, 75% of activity in CLEA product was observed after 2h of incubation. In addition, after 15 day of storage at 4℃, the LI22-CLEA retained more than 85% of its initial activity. We are currently investigating biochemical and biophysical properties of LI22-CLEA using several biochemical and biophysical approaches including scanning electron microscopy (SEM).