• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.251-259
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• 2013

An endoxylanase gene (PoxynA) that belongs to the glycoside hydrolase (GH) family 11 was cloned from a xylanolytic strain, Penicillium oxalicum B3-11(2). PoxynA was overexpressed in Trichoderma reesei QM9414 by using a constitutive strong promoter of the encoding pyruvate decarboxylase (pdc). The high extracellular xylanase activities in the fermentation liquid of the transformants were maintained 29~35-fold higher compared with the wild strain. The recombinant POXYNA was purified to homogeneity, and its characters were analyzed. Its optimal temperature and pH value were $50^{\circ}C$ and 5.0, respectively. The enzyme was stable at a pH range of 2.0 to 7.0. Using beechwood as the substrate, POXYNA had a high specific activity of $1,856{\pm}53.5$ IU/mg. In the presence of metal ions, such as $Cu^{2+}$, and $Mg^{2+}$, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of $Mn^{2+}$ and $Fe^{2+}$. The recombinant POXYNA hydrolyzed birchwood xylan, beechwood xylan, and oat spelt xylan to produce short-chain xylooligosaccharides, xylopentaose, xylotriose, and xylobiose as the main products. This is the first report on the expression properties of a recombinant endoxylanase gene from Penicillium oxalicum. The properties of this endoxylanase make it promising for applications in the food and feed industries.

• Journal of Life Science
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• v.19 no.7
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• pp.852-858
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• 2009

The homeostasis of the pyridine nucleotide pool [NAD(P)H and NAD(P)$^+$] is maintained in Rhodobacter sphaeroides mutant strains defective in the cytochrome bci complex or the cytochrome c oxidases in terms of its concentration and redox state. Aerobic derepression of the puf operon, which is under the control of the PrrBA two-component system, in the CBB3 mutant strain of R. sphaeroides was shown to be not the result of changes in the redox state of the pyridine nucleotides and the ubiquinone/ubiquinol pool. Using the bc$_1$ complex knock-out mutant strain of R. sphaeroides, we clearly demonstrated that the inhibitory effect of cbb$_3$, oxidase on spectral complex formation is not caused indirectly by the redox change of the ubiquinone/ubiquinol pool.

• Journal of Life Science
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• v.16 no.1
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• pp.76-81
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• 2006

A $\gamma-butyrolactone$ autoregulator receptor has a common activity as DNA-binding transcriptional repressors controlling secondary metabolism and/or morphological differentiation in Streptomyces. A gene (scaR) encoding it was cloned from Streptomyces cravuligerus, a clavulanic acid producer, and was in vitro characterized in a previous report. In this study to clarify the in vivo function of ScaR, a $\gamma-butyrolactone$ autoregulator receptor of Streptomyces clavuligerus, we constructed a scaR-deleted strain by means of homologous recombination. No difference in morphology was found between the wild-type strain and the scaR-disruptant, but the scaR-disruptant showed higher clavulanic acid production. This indicates that the ScaR in S. clavuligerus acts as a negative regulator of the biosynthesis of clavulanic acid, but plays no role in morphological differentiation.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.641-646
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• 2005

An approximately 10-fold higher level of adherence of Salmonella typhimurium strain TML to Int-407 cells was observed with organisms grown in Luria broth or in high-iron containing medium than those grown in low-iron containing medium. Iron specifically enhanced adherence, while other cations such as calcium, cobalt, copper, potassium, magnesium and manganese did not. It was suggested that iron did not act as a passive ligand - probably it stimulated production of bacterial factors necessary for adherence. A similar pattern of iron modulation of adhesiveness was also seen in Salmonella mutants with single or different combinations of multiple mutations in genes encoding the mannose sensitive hemagglutinin (type 1 fimbriae), mannose resistant hemagglutinin and flagellum. The adhesiveness of an isogenic fur mutant was modulated by iron in a manner similar to the wild-type strain, suggesting that iron modulation of adherence is independent of the fur gene product.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.631-636
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• 1999

The thermostable endo-chitosanase gene from the isolated strain Bacillus sp. KFB-C108 was identified on the basis of a phylogenetic analysis of the 16S rRNA gene sequence, and was cloned into plasmid pUCl8 using E. coli $DH5\alpha$ as the host strain. Positive clones carrying recombinant plasmids (pKCHO I and pKCHO II) containing chitosanase activity were selected using the direct activity staining method. Detailed physical maps showed the two plasmid inserts were identical except that the KCHO II insert (2.6 kb) was 1.8 kb smaller than that of the KCHO I. The recombinant plasmids were analyzed to determine the essential region for chitosanase activity, and a 1.3-kb fragment (KCHO-6) was subcloned into pTrc99A using the EcoRI and BamHI sites to construct pTrc99A/KCHO-6(pTrEB13). The resulting plasmid exerted high chitosanase activity upon transformation of E. coli $DH5{\alpha}cells$, overproducing about 20 times more in the cloned cells than in the wild-type cells. The cloned chitosanase protein exhibited the same molecular weight and catalytic activity similar to those of Bacillus sp. KFB-C108. The cloned enzyme was an endo-type that produced a chitosan tetramer as the major reaction product; however, it produced no monomers or dimers.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1644-1652
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• 2012

Iron plays a key role in host-pathogen interactions. Microbial pathogens require iron for survival and virulence, whereas mammalian hosts sequester and withhold iron as a means of nutritional immunity. We previously identified two paralogous genes, CFT1 and CFT2, which encode homologs of a fungal iron permease, Cft1 and Cft2, respectively, in the human fungal pathogen Cryptococcus neoformans. Cft1 was shown to play a role in the high-affinity reductive iron uptake system, and was required for transferrin utilization and full virulence in mammalian hosts. However, no role of Cft2 has been suggested yet. Here, we identified the third gene, CFT3, that produces an additional fungal iron permease homolog in C. neoformans, and we also generated the cft3 mutant for functional characterization. We aimed to reveal distinct functions of Cft1, Cft2 and Cft3 by analyzing phenotypes of the mutants lacking CFT1, CFT2 and CFT3, respectively. The endogenous promoter of CFT1, CFT2 and CFT3 was replaced with the inducible GAL7 promoter in the wild-type strain or in the cft1 mutant for gain-of-function analysis. Using these strains, we were able to find that CFT2 is required for growth in low-iron conditions in the absence of CFT1 and that overexpression of CFT2 compensates for deficiency of the cft1 mutant in iron uptake and various cellular stress conditions. However, unlike CFT2, no clear phenotypic characteristic of the cft3 mutant and the strain overexpressing CFT3 was observed. Overall, our data suggested a redundant role of Cft2 in the high-affinity iron uptake and stress responses in C. neoformans.

• Journal of Microbiology and Biotechnology
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• v.24 no.3
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• pp.337-345
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• 2014

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro $MAT{\alpha}$ sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

• Korean Journal of Microbiology
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• v.40 no.1
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• pp.7-11
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• 2004

eIF5B is a translation initiation factor that delivers Met-$tRNA^{Met}$ to AUG start codon and subsequently joins the small and large ribosomes. In order to study the function of eIF5B encoded by FUN12, we constructed FUN12 which lacked 5' end of its sequence. We found that this construct lacking almost all of its promoter in pRS plasmid partially complemented slow growth phenotype of fun12 deletion strain. Interestingly, this construct expressed N-terminally truncated eIF5B and its expression level was about 5% of that of wild type eIF5B. Low amount of the eIF5B expressed additionally in fun12 deletion strain played a direct role as a limiting factor for its growth. This limiting factor eIF5B in those strains also modulates activities of overall translation in vitro.

• Korean Journal of Food Science and Technology
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• v.32 no.2
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• pp.424-430
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• 2000

The purpose of the study was to investigate effect of addition of mutant strains of both Leuconostoc mesenteroides and Leu. paramesenteroides on extending shelf-life of Kimchi. The mutant strains have an increased adipic acid resistance in comparison with that of their paired wild types. Addition of both strains was more effective than that of one strain alone to extend shelf-life of Kimchi. The optimal amount of inoculation was determined as 0.005% of the two mixed mutant strains with a ratio of 1 : 10, Leu. mesenteroides : Leu. paramesenteroides based on the results of acidification and organoleptic tests.

• Mycobiology
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• v.47 no.3
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• pp.301-307
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• 2019

The $11{\alpha}$-hydroxylation of $16{\alpha}$, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The $11{\alpha}$-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the $11{\alpha}$-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The $H_2O_2$ levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the $11{\alpha}$-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.