[Show abstract][Hide abstract] ABSTRACT: The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9,
yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS
4 locus (38 of Mut+ clone) or into theAOX1 locus (45 of Muts clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into
thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity,
290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared
with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of
the recombinantS. cerevisiae.
Biotechnology and Bioprocess Engineering 04/2012; 5(1):7-12. · 1.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields--dental, ophthalmic, and drug delivery--to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.
Applied Microbiology and Biotechnology 08/2009; 84(4):597-606. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A filamentous fungus Aspergillus terreus produces itaconic acid, which is predicted to be derived from cis-aconitic acid via catalysis by cis-aconitic acid decarboxylase (CAD) in the carbon metabolism of the fungus. To clarify the enzyme's function and a pathway for itaconic acid biosynthesis, we cloned a novel gene encoding the enzyme. The open reading frame of this gene (CAD1) consists of 1,529 bp encoding 490 amino acids and is interrupted by a single intron. Among the identified proteins in the database, the primary structure of the protein encoded by CAD1 shared high identity with the MmgE/PrpD family of proteins, including a number of 2-methylcitrate dehydratases of bacteria. The cloned gene excluding an intron was introduced into the expression plasmid pAUR-CAD1 controlled by the ADH1 promoter. The CAD activity in Saccharomyces cerevisiae was confirmed by directly detecting itaconic acid as a product from cis-aconitic acid as a substrate. This result reveals for the first time that this gene encodes CAD, which is essential for itaconic acid production in A. terreus.
Applied Microbiology and Biotechnology 07/2008; 80(2):223-9. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.
[Show abstract][Hide abstract] ABSTRACT: In order to study the feasibility of commercial-scale L-lactic acid production by Rhizopus sp. MK-96-1196 using large scale airlift bioreactors (ALBs), a scale-up study from 0.003 m3 to 5 m3 ALB was carried out using oxygen transfer rate (OTR) as the scale-up criterion. Enhanced L-lactic acid production was achieved at OTRs higher than 0.28 (g-O2/l/h) irrespective of the scale of the bioreactor in question: in the range of 0.003 m3 to 5 m3, more than 90 g/lL-lactic acid was produced with a yield of approximately 80%, based on the initial glucose concentration. In future research, we plan to design an ALB greater than 3000 m3 (working volume: 2000 m3) for further studies on the production of L-lactic acid in large quantities.
Journal of Bioscience and Bioengineering 02/2006; 101(1):9-12. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose
concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture,
1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h−1. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam,
and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively,
compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production
Biotechnology and Bioprocess Engineering 01/2005; 10(6):522-527. · 1.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The optimum temperature, initial pH, amount of added enzyme and substrate (corncob) for the hydrolysis of corncob by Acremonium cellulase were 35 degrees C, 4.5, 10 u/g-corncob and 100 g/l, respectively. Under the optimum conditions, more than 55 g/l of reducing sugars were hydrolyzed from 100 g/l of corncob to 34 g/l of glucose and 12 g/l of xylose based on dried corncob. More than 25 g/l of L-lactic acid was produced from this enzymatic hydrolyzate and less than 5 g/l of xylose remained in the 3-l airlift bioreactor. The production of L-lactic acid by simultaneous saccharification and fermentation (SSF) was also carried out in the 3-l airlift bioreactor using Acremonium thermophilus (cellulose-producer) and Rhizopus sp. MK-96-1196 (lactic acid-producer). More than 24 g/l of L-lactic acid was produced from 100 g/l of untreated raw corncob.
Journal of Bioscience and Bioengineering 02/2004; 97(3):153-7. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: By a monospore isolation technique, Rhizopus sp. MK-96-1 was selected from colonies of Rhizopus sp. MK-96, which was isolated from the soil sample collected in Fujieda, Japan, and used as a parent strain. By the ammonia-concentration-gradient agar plate technique after mutation using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) method, a mutant strain designated Rhizopus sp. MK-96-1196 producing more than 90 g/l L-lactic acid under pH control using liquid ammonia in an airlift bioreactor was successfully isolated. Compared with the parent strain, this mutant strain produced about twofold the amount of L-lactic acid in half fermentation time under the same culture conditions. Ammonium L-lactate was recovered and purified as free L-lactic acid via n-butyl L-lactate. The ammonia used for pH control in the fermentation broth was recovered as liquid ammonia during the recovery and purification process and subsequently reused for the next fermentation. Thus, we have developed a new highly purified L-lactic acid production process without producing recalcitrant wastes, e.g., CaSO4 (gypsum).
Journal of Bioscience and Bioengineering 02/2004; 97(1):19-23. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We determined the optimum culture conditions such as inoculum size, initial starch concentration, pH during the fermentation and aeration rate for L-lactic acid production by Rhizopus sp. MK-96-1196 in a 3-l airlift bioreactor. More than 90 g/l of L-lactic acid was produced from only partially enzymatically hydrolyzed corn starch with a production rate of 2.6 g/l/h and a product yield of 87% based on the starch consumed under the optimum conditions in the 3-l airlift bioreactor. Scale-up from the 3-l to a 100-l airlift bioreactor for L-lactic acid fermentation was carried out using V(s)(cm/s) as a scale-up criterion. The production rates and yields of L-lactic acid in both bioreactors appeared to be fairly well correlated with k(L)a (1/h).
Journal of Bioscience and Bioengineering 02/2003; 96(1):65-9. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper deals with studies on epsilon-poly-L-lysine (epsilon-PL) production in an airlift bioreactor (ABR) using Streptomyces albulus S410 (S410) to minimize the production cost including the downstream processing of epsilon-PL. In a 5-l ABR, 30 g/l of epsilon-PL was produced with a power consumption of 0.3 kW/m3, the production level being similar to that in a 5-l jar fermentor with a power consumption of 8.0 kW/m3. Furthermore, the leakage of intracellular nucleic acid (INA)-related substances into the culture broth in the ABR was less than that in the jar fermentor. Due to the high-level power consumption (8.0 kW/m3) in the jar fermentor, the morphology of the cells changed from the pellet to filament form due to the extensive shear stress arising from continuous agitation, thereby increasing the leakage of the INA-related substances into the culture broth. This suggested that ABR would have an advantage in the low-cost production of epsilon-PL over stirred tank type reactors (STR).
Journal of Bioscience and Bioengineering 02/2002; 93(3):274-80. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: cis-Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45 degrees C. The K(m) value for cis-aconitic acid was determined as 2.45 mM (pH 6.2, 37 degrees C). The enzyme was completely inactivated by Hg+, Cu2+, Zn2+, p-chloromercuribenzoate, and 5,5'-dithio-bis(2-nitrobenzoate).
Journal of Bioscience and Bioengineering 02/2002; 94(1):29-33. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper deals with studies on ε-poly-l-lysine (ε-PL) production in an airlift bioreactor (ABR) using Streptomyces albulus S410 (S410) to minimize the production cost including the downstream processing of ε-PL. In a 5-l ABR, 30 g/l of ε-PL was produced with a power consumption of 0.3 kW/m3, the production level being similar to that in a 5-l jar fermentor with a power consumption of 8.0 kW/m3. Furthermore, the leakage of intracellular nucleic acid (INA)-related substances into the culture broth in the ABR was less than that in the jar fermentor. Due to the high-level power consumption (8.0 kW/m3) in the jar fermentor, the morphology of the cells changed from the pellet to filament form due to the extensive shear stress arising from continuous agitation, thereby increasing the leakage of the INA-related substances into the culture broth. This suggested that ABR would have an advantage in the low-cost production of ε-PL over stirred tank type reactors (STR).
Journal of Bioscience and Bioengineering. 01/2002;
[Show abstract][Hide abstract] ABSTRACT: An optimal feed rate profile of a substrate (tylosin) for a novel antibiotic, acetyl-isovaleryl tylosin (AIV) production process was investigated. In the first step of optimization, a kinetic model for production of AIV from tylosin by Streptomyces thermotolerans was established properly using the least square method, followed by the confirmation that the proposed model could be used to predict the production process of AIV from tylosin. An objective function, state equations and an inequality constraint with respect to the tylosin feeding rate profile were applied to maximize the amount of AIV produced from tylosin in a fed-batch culture. The optimized tylosin feeding rate profile was determined using a direct iterative search algorithm based on the modified complex method. The simulation of AIV production at the optimal tylosin feeding profile indicates that the final amount of AIV is expected to be about 30% higher than that at the conventional constant tylosin feeding rate, which was also confirmed experimentally using a 30-l jar fermentor.
Journal of Bioscience and Bioengineering 02/2001; · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enhancement of epsilon-poly-l-lysine (epsilon-PL) production by Streptomyces albulus strain no. 410 (S410) by means of a pH control strategy was investigated. S140 cells produce epsilon-PL at a high concentration if the culture pH remains at about 4.0; however, if it shifts to higher than 4.0, the accumulated epsilon-PL is depolymerized. We therefore suggest a pH control strategy for cell growth and epsilon-PL production aimed at increasing the amount of epsilon-PL produced. The cultivation was divided into two control phases. In phase I, cell growth was accelerated by maintaining the pH at higher than 5.0; in phase II, epsilon-PL production was increased by maintaining the pH at about 4.0. To avoid an increase in the pH during phase II as a result of glucose depletion, the glucose concentration was kept at around 10 g/l by glucose feeding. This control strategy enhanced the production of epsilon-PL to 48.3 g/l from 5.7 g/l in the case of batch culture.
Journal of Bioscience and Bioengineering 02/2001; 91(2):190-4. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A strain designated MK107-39, producing kojic acid with a high yield, was obtained by a new screening method using a 96-well microtiter plate after NTG treatment of Aspergillus oryze ATCC 22788. The amount of kojic acid produced by strain MK107-39 in a shaking flask was 28 g/l from 100 g/l of glucose, which was 7.7-times higher than that produced by parent strain. The kojic acid yields per cell and the amount of glucose consumed were 9.8 and 6.0-times higher than those of the parent strain. Based on differences in the use of carbohydrates and organic acids, it seems that strain MK107-39 has some mutation regarding carbohydrate metabolism. By customizing the medium and culture conditions such as glucose concentration, dissolved oxygen concentration and pH of the fermentation broth, more than 110 g/l of kojic acid was produced in a 3-l jar fermentor. Upon scale up to a 600-l pilot fermentor, enhanced production of kojic acid was successfully achieved. The kojic acid yield from glucose consumed, Y(P/S), was 0.43 (g/g) in this pilot plant-scale fermentation.
Journal of Bioscience and Bioengineering 02/2001; 91(3):272-6. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a culture of Aspergillus oryzae MK-107-39 in a 3-l airlift bioreactor, kojic acid was not produced when glucose/wheat germ medium (GM1) was used. However, when a jar fermentor was used, the kojic acid yield was high. A suitable medium for culture in an airlift bioreactor consisting of partially hydrolyzed corn starch and a small amount of corn steep liquor (CSL) (SM1) was selected. In the cultivation in the airlift bioreactor using SM1, nearly 40 g/l of kojic acid was produced, which was the same as the amount produced in the jar fermentor containing GM1. The optimum aeration rate for the airlift bioreactor was 2.0 vvm (0.66 cm/s of superficial linear velocity (Vs)). The cost of SM1 using the airlift bioreactor was reduced to 40% that of GM1 using the jar fermentor. Furthermore, the energy cost of kojic acid production using SM1 in the airlift bioreactor was less than one-fourth of that for the jar fermentor using GM1.
Journal of Bioscience and Bioengineering 02/2001; 92(4):360-5. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa.s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa.s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 x 10(4) microm(2). In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 x 10(4) microm(2) comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.
[Show abstract][Hide abstract] ABSTRACT: Expression of the recombinant protein beta-galactosidase in the Spodoptera frugiperda Sf-9 insect cell line infected by the Autographa californica nuclear polyhedrosis virus expressing beta-galactosidase (AcNPV-betagal) was visualized using confocal scanning laser microscopy with fluorescent staining of both the recombinant protein and the cell nucleus. The average size of the insect cells and the intracellular DNA concentration both increased markedly, respectively reading 3.8- and 2.3-fold the values before infection. The average beta-galactosidase activity began to increase at 20-24 h post infection and finally reached 1.9 x 10(4) units/ml. As the post infection time increased, the stained nucleus images expanded and spread broadly. Beta-galactosidase was first identified by fluorescent staining at 12 h post-infection, filled the cell at 27 h, began to be released at 36 h, and finally spread out of the cell. The locations of the nucleus and expressed beta-galactosidase were identified from computerized tomograms and 3-dimensional images.
Journal of Bioscience and Bioengineering 02/1999; 87(6):756-61. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of nitrogen source on arachidonic acid (AA) production and morphological changes during the culture of Mortierella alpina were investigated using an image analysis system. When yeast extract, gluten meal, or corn steep liquor was used, a circular pellet morphology was obtained. However, when Pharmamedia, fish meal, or soybean meal was used, M. alpina formed radial filamentous mycelia. The radial filamentous area in the case of soybean meal was 75% of the total mycelial area. In a jar fermentor culture, M. alpina morphology varied with the cultivation period: (i) at 0-6 h culture, the inoculated pellet-like mycelia were adapted to the new environment, (ii) at 6 h-1 d culture, filamentous mycelia grew exponentially which yielded a feather-like morphology, (iii) at 1-2 d culture, the filamentous mycelia became disentangled as a result of the mechanical agitation; consequently, the proportion of filamentous mycelia was increased, (iv) at 2-4 d culture, mycelia showed stationary growth, but the AA concentration increased rapidly, and (v) at 4-6 d culture, hyphae grew thick radially with the AA concentration continuing to increase gradually. In the case of the cultures with feather-like morphology obtained using soybean meal, the AA yield was 0.14 g/g dry cell weight, which was two times higher than that in cultures grown using yeast extract. These results suggest that the feather-like morphology of culture of M. alpina is suitable for AA production.
Journal of Bioscience and Bioengineering 02/1999; 88(1):61-7. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A simple kinetics of soybean oil consumption and cephamycin C production in Streptomyces sp. culture using a mineral support is proposed in this study. The mineral support was used for both suspending the soybean oil as fine oil droplets and immobilizing mycelia. The optimum concentrations of oil and mineral support for obtaining the maximum cephamycin C production were determined to be 50 and 15 g/l, respectively, by the proposed kinetics. At the optimal concentrations, the concentration of cephamycin C estimated from the proposed model and from the experimental data was 2.82 and 2.80 g/l, respectively. The results of the simulation coincided well with the experimental data for various concentrations of the soybean oil and the support. This demonstrates that our model can explain the kinetics of a culture using vegetable oil as the carbon source and mineral support for both oil suspension and mycelial immobilization.
Journal of Bioscience and Bioengineering 02/1999; 87(3):390-3. · 1.74 Impact Factor