1,721,000 research outputs found
Thermostability and Xylan-Hydrolyzing Property of Endoxylanase Expressed in Yeast Saccharomyces cerevisiae
No full textThe endoxylanase gene (xynB, GeneBank access code U51675), including its signal sequence, from Bacillus spp. was amplified and connected in frame downstream of yeast ADH1 promoter and then the resulting plasmid, pAEDX-1, was introduced into Saccharomyces cerevisiae. When the yeast transformants were grown on YPD medium, the majority of endoxylanase activity was detected in the extracellular culture medium, indicating that the signal peptide of Bacillus endoxylanase functioned well in yeast. In the batch cultivation of yeast transformants, the total expression level of endoxylanase and secretion efficiency were measured to be about 9.8 U/mL and 66.2%, respectively. The extracellular endoxylanase expressed in yeast showed an enhanced thermal stability due to the N-linked glycosylation. Through the hydrolysis of birchwood xylan with the endoxylanase, it was found that xylobiose and xylotriose were produced as major products with equimolar ratio.open
Enhancement of hydrolysis and biogas production of primary sludge by use of mixtures of protease and lipase
No full textThis study aims to improve the hydrolysis and degradation of primary sludge by using wild-type enzymes (protease and lipase) and establishing the optimal enzymatic cocktail ratio. Primary sludge from three wastewater treatment plants (WWTPs) in Korea (Ulsan, Pohang, and Busan) were subjected to enzymatic hydrolysis. Protease and lipase were isolated from enzyme-producing microorganisms cultured from secondary sludge collected at 8 different digester sites in Korea. Primary sludge degradation through enzymatic hydrolysis was monitored by measuring the reduction in the volatile suspended solids (VSS) content of the sludge and enzyme cocktail mix for 72 h at 40oC and pH 7.0. The enzymatic cocktail of Ulsan primary sludge treated with protease to lipase at a ratio of 1:3 was found to be optimal at 33.3% VSS reduction. Biochemical methane potential (BMP) tests were employed to the optimal enzyme cocktail to measure the potential of the hydrolyzed substrate for further degradation (VSS reduction) and bioconversion to biogas using 125 mL serum bottles as anaerobic reactors for 30 days. BMP tests showed that there was an increase in biogas production by 84.1%, methane production by 89.8%, and methane yield by 9.6%. Methane production rate was also increased. The significant VSS concentration reduction and higher biogas and methane yield of the enzyme-treated primary sludge correlate to the fact that the complex polymeric organic materials were degraded leading to efficient utilization by the microorganisms in the anaerobic digestion process. ⓒ 2020, The Korean Society for Biotechnology and Bioengineering and Springer.
Bacterial expression and purification of human papillomavirus type 18 L1
No full textThe human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.open
Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation
No full textTo investigate the effect of cellular fatty acids composition on ethanol tolerance in Escherichia coli, we overexpressed either des, encoding fatty acid desaturase from Bacillus subtilis, or fabA, encoding β-hydroxydecanoyl thio-ester dehydrase from E. coli, or both genes together, into E. coli. Recombinant E. coli harboring fabA had elevated tolerance against ethanol compared to wild type strain. In contrast, des decreased resistance to ethanol. Co-expression of both genes together complemented ethanol tolerance of E. coli. This result indicates how to engineer bacterial strains to be resistant to higher concentrations of ethanol.open
1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae
No full textCurrently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaBRA1 and dhaBRA2 encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain can produce 1,3-PD from glucose (2.4 g L-1) as well as glycerol (0.8 g L-1), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources.open
Isolation and Characterization of Xylanase-producing Paenibacillus sp. HY-8 from Moechotypa diphysis
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Characterization of a squalene synthase from the thraustochytrid microalga Aurantiochytrium sp. KRS101
No full textThe gene encoding squalene synthase (SQS) of the lipidproducing heterotrophic microalga Aurantiochytrium sp. KRS101 was cloned and characterized. The krsSQS gene is 1,551 bp in length and has two exons and one intron. The open reading frame of the gene is 1,164 bp in length, yielding a polypeptide of 387 predicted amino acid residues with a molecular mass of 42.7 kDa. The deduced krsSQS sequence shares at least four conserved regions known to be required for SQS enzymatic activity in other species. The protein, tagged with His6, was expressed into soluble form in Escherichia coli. The purified protein catalyzed the conversion of farnesyl diphosphate to squalene in the presence of NADPH and Mg2+. This is the first report on the characterization of an SQS from a Thraustochytrid microalgaopen
The role of aldehyde/alcohol dehydrogenase (AdhE) in ethanol production from glycerol by Klebsiella pneumoniae
No full textTranscriptome analysis of a K. pneumoniae GEM167 mutant strain derived by irradiation with gamma rays, which exhibited high-level production of ethanol from glycerol, showed that the mutant expressed AdhE at a high level. Ethanol production decreased significantly, from 8.8 to 0.5 g l-1, when an adhE-deficient derivative of that strain was grown on glycerol. Bacterial growth was also reduced under such conditions, showing that AdhE plays a critical role in maintenance of redox balance by catalyzing ethanol production. Overexpression of AdhE enhanced ethanol production, from pure or crude glycerol, to a maximal level of 31.9 g l-1 under fed-batch fermentation conditions; this is the highest level of ethanol production from glycerol reported to date.open
The production of 1,3-propanediol from mixtures of glycerol and glucose by a Klebsiella pneumoniae mutant deficient in carbon catabolite repression
No full textIn the present study, mutant strain of Klebsiella pneumoniae with deletion of the crr gene encoding EIIAGlc (a component of the glucose-specific phosphoenolpyruvate-dependent transferase system [PTS]) was prepared. This eliminated the ability of the strain to mediate carbon catabolite repression (CCR). Production of 1,3-propanediol (1,3-PD) from glycerol by the crr mutant strain was enhanced (compared to that of the parent) in the presence of glucose. Using molasses as a co-substrate of glycerol, the maximum yield of 1,3-PD was 60.4% greater (81.2g/l) than that obtained when glycerol was used alone, under optimum fermentation conditions.open
Synthesis of two new lipid mediators from docosahexaenoic acid by combinatorial catalysis involving enzymatic and chemical reaction
No full textOmega-3 polyunsaturated fatty acids (PUFAs) have been known to have beneficial effects in the prevention of various diseases. Recently, it was identified that the bioactivities of omega-3 are related to lipid mediators, called pro-resolving lipid mediators (SPMs), converted from PUFAs, so they have attracted much attention as potential pharmaceutical targets. Here, we aimed to build an efficient production system composed of enzymatic and chemical catalysis that converts docosahexaenoic acid (DHA) into lipid mediators. The cyanobacterial lipoxygenase, named Osc-LOX, was identified and characterized, and the binding poses of enzyme and substrates were predicted by ligand docking simulation. DHA was converted into three lipid mediators, a 17S-hydroxy-DHA, a 7S,17S-dihydroxy-DHA (RvD5), and a 7S,15R-dihydroxy-16S,17S-epoxy-DPA (new type), by an enzymatic reaction and deoxygenation. Also, two lipid mediators, 7S,15R,16S,17S-tetrahydroxy-DPA (new type) and 7S,16R,17S-trihydroxy-DHA (RvD2), were generated from 7S,15R-dihydroxy-16S,17S-epoxy-DPA by a chemical reaction. Our study suggests that discovering new enzymes that have not been functionally characterized would be a powerful strategy for producing various lipid mediators. Also, this combination catalysis approach including biological and chemical reactions could be an effective production system for the manufacturing lipid mediators.
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