-
[show abstract]
[hide abstract]
ABSTRACT: We have demonstrated that 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI could enhance the levels of adiponectin and its receptors in differentiated 3T3-L1 adipocytes, which has been shown to be effective in lowering blood glucose levels and enhancing insulin sensitivity. DNJ was not toxic to differentiated 3T3-L1 adipocytes for up to a concentration of 5 microM. In terms of expression levels of adiponectin and its receptors (AdipoR1 and AdipoR2), DNJ in concentrations as low as 0.5 microM elevated both mRNA and protein levels of adiponectin and transcript levels of AdipoR1 and AdipoR2. In addition, DNJ increased phosphorylation of 5' adenosine monophosphateactivated protein kinase (AMPK) in a statistically significant manner. Finally, treatment with DNJ resulted in increased mRNA expression of glucose transporter 4 (GLUT4), which encodes for a glucose transporter, along with a significant increase in glucose uptake into the adipocytes based on results of a 2-deoxy-D-[3H] glucose uptake assay. Our findings indicate that DNJ may greatly facilitate glucose uptake into adipose tissues by increasing the action of adiponectin via its up-regulated expression as well as its receptor genes. In addition, the glucose-lowering effects of DNJ may be achieved by an increased abundance of GLUT4 protein in the plasma membrane, as a consequence of the increased transcript levels of the GLUT4 gene and the activation of AMPK.
Journal of Microbiology and Biotechnology 05/2013; 23(5):637-43. · 1.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase delta of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'-->5' exonuclease activity. The resulting HpPOL3* gene encoding the error-prone proofreading-deficient DNA polymerase delta was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a URA3- mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of HpPOL3*. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.
Journal of Microbiology and Biotechnology 03/2013; 23(3):304-12. · 1.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The thermotolerant methylotrophic yeast Hansenula polymorpha is able to grow at elevated temperature up to 48 °C as one of a few yeast strains which are naturally capable of alcoholic fermentation of xylose, a pentose sugar abundant in lignocellulosic biomass. However, the current level of ethanol production from xylose by H. polymorpha is still very low compared to those of other xylose-fermenting strains. Therefore, it is necessary to analyze and remodel the xylose metabolism in H. polymorpha at the whole genome level to identify and overcome these limits. In the present study, the transcriptomes of H. polymorpha grown on xylose were compared with those of glucose-grown cells under both aerobic and microaerobic conditions. Approximately, two percent of H. polymorpha genes were either up- or down-regulated by more than two-fold during the growth on xylose. The majority of the up-regulated genes were involved in metabolism. Some genes involved in xylose metabolism, such as XYL1, XYL2, and TAL1 were also up-regulated, despite the fact that the differences in their induction level were only about three-fold. On the other hand, the majority of the down-regulated genes were involved in metabolism and cellular transport. Interestingly, some genes involved in glycolysis and ethanol fermentation were also repressed during growth on xylose, suggesting that these genes are good targets for engineering H. polymorpha to improve xylose fermentation.
Bioprocess and Biosystems Engineering 02/2013; · 1.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1 - 6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2 - 3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2 - 6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.
Glycoconjugate Journal 10/2012; · 2.12 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this study, the putative target genes of the Arc two-component system of the rumen bacterium Mannheimia succiniciproducens were determined by analyzing the transcriptome of the ArcA overexpression strain and by the in silico scanning of the entire genome sequence with the position weight matrix of the ArcA binding sequence developed for Escherichia coli. The majority of 79 repressed genes were involved in energy metabolism and carbohydrate transport and metabolism, while the majority of 82 induced genes were involved in hypothetical or unknown functions. Our results suggest that the Arc system in M. succiniciproducens has a specific function that differs from that in E. coli.
The Journal of Microbiology 08/2012; 50(4):665-72. · 1.10 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The influences of impeller types on morphology and protein expression were investigated in a submerged culture ofAspergillus oryzae. The impeller types strongly affected mycelial morphology and protein production in batch and fed-batch fermentations. Cells
that were cultured by propeller agitation grew in the form of a pellet, whereas cells that were cultured by turbine agitation
grew in a freely dispersed-hyphal manner and in a clumped form. Pellet-grown cells showed high levels of protein production
for both the intracellular heterologous protein (β-glucuronidase) and the extracellularly homologous protein (α-amylase).
The feeding mode of the carbon source also influenced the morphological distribution and protein expression in fed-batch fermentation
ofA. oryzae. Pulsed-feeding mainly showed high protein expression and homogeneous distribution of pellet whereas continuous feeding resulted
in less protein expression and heterogeneous distribution with pellet and dispersed-hyphae. The pellet growth with propeller
agitation paralleling with the pulsed-feeding of carbon source showed a high level of protein production in the submerged
fed-batch fermentation of recombinantA. oryzae.
Biotechnology and Bioprocess Engineering 04/2012; 9(3):184-190. · 1.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The encapsulated fungal pathogen Cryptococcus neoformans causes cryptococcosis in immunocompromised individuals. Although cell surface mannoproteins have been implicated in C. neoformans pathogenicity, the structure of N-linked glycans assembled on mannoproteins has not yet been elucidated. By analyzing oligosaccharide profiles combined with exoglycosidase treatment, we report here that C. neoformans has serotype-specific high mannose-type N-glycans with or without a β1,2-xylose residue, which is attached to the trimannosyl core of N-glycans. Interestingly, the neutral N-glycans of serotypes A and D were shown to contain a xylose residue, whereas those of serotype B appeared to be much shorter and devoid of a xylose residue. Moreover, analysis of the C. neoformans uxs1Δ mutant demonstrated that UDP-xylose is utilized as a donor sugar in N-glycan biosynthesis. We also constructed and analyzed a set of C. neoformans mutant strains lacking genes putatively assigned to the reconstructed N-glycan biosynthesis pathway. It was shown that the outer chain of N-glycan is initiated by CnOch1p with addition of an α1,6-mannose residue and then subsequently extended by CnMnn2p with multiple additions of α1,2-mannose residues. Finally, comparative analysis of acidic N-glycans from wild-type, Cnoch1Δ, Cnmnn2Δ, and Cnuxs1Δ strains strongly indicated the presence of xylose phosphate attached to mannose residues in the core and outer region of N-glycans. Our data present the first report on the unique structure and biosynthesis pathway of N-glycans in C. neoformans.
Journal of Biological Chemistry 04/2012; 287(23):19501-15. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: As a step forward to achieve the generation of human complex-type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc(1)Man(5)GlcNAc(2) and GlcNAc(1)Man(3)GlcNAc(2), respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.
The Journal of Microbiology 04/2012; 50(2):341-8. · 1.10 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha. However, unlike the cytosolic HpADH1, HpADH3 appears to be a mitochondrial enzyme, as a mitochondrial targeting extension exists at its N terminus. The recombinant HpADH3 overexpressed in Escherichia coli showed similar catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The HpADH3 displayed substrate specificities with clear preferences for medium chain length primary alcohols and acetaldehyde for an oxidation reaction and a reduction reaction, respectively. Although the H. polymorpha ADH3 gene was induced by ethanol in the culture medium, both an ADH isozyme pattern analysis and an ADH activity assay indicated that HpADH3 is not the major ADH in H. polymorpha DL-1. Moreover, HpADH3 deletion did not affect the cell growth on different carbon sources. However, when the HpADH3 mutant was complemented by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium. In contrast, in a xylose medium, the ethanol production was dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain. Taken together, our results suggest that the expression of HpADH3 would be an ideal engineering target to develop H. polymorpha as a substrate specific bioethanol production strain.
Applied Microbiology and Biotechnology 01/2012; 96(3):697-709. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mannheimia succiniciproducens, a rumen bacterium belonging to the family Pasteurellaceae, has two putative β-galactosidase genes, bgaA and bgaB, encoding polypeptides whose deduced amino acid sequences share 56% identity with each other and show approximately 30% identity to the Escherichia coli gene for LacZ. The M. succiniciproducens bgaA (MsbgaA) gene-deletion mutant was not able to grow on lactose as the sole carbon source, suggesting its essential role in lactose metabolism, whereas the MsbgaB gene-deletion mutant did not show any growth defect on a lactose medium. Furthermore, the expression of the MsbgaA gene was induced by the addition of lactose in the growth medium, whereas the MsbgaB gene was constitutively expressed independently of a carbon source. Biochemical characterization of the recombinant proteins revealed that MsBgaA is more efficient than MsBgaB in hydrolyzing o-nitrophenyl-β-d-galactopyranoside and p-nitrophenyl-β-d-galactopyranoside. MsBgaA was highly specific for the hydrolysis of lactose, with a catalytic efficiency of 46.9 s(-1) mM(-1). However, MsBgaB was more efficient for the hydrolysis of lactulose than lactose, and the catalytic efficiency was 10.0 s(-1) mM(-1). Taken together, our results suggest that the β-galactosidase paralogues of M. succiniciproducens BgaA and BgaB play a critical role in lactose metabolism and in an unknown but likely specific function for rumen bacteria, respectively.
Journal of bacteriology 11/2011; 194(2):426-36. · 3.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the present study, we functionally analysed two yapsin genes of the thermotolerant methylotrophic yeast Hansenula polymorpha, HpYPS1 and HpYPS7, for their roles in maintaining cell wall integrity and proteolytic processing. Both HpYPS1 and HpYPS7 proteins were shown to largely localize on the cell wall via glycosylphosphatidylinositol anchor. Heterologous expression of HpYPS1 completely restored all of the growth defects of the Saccharomyces cerevisiae yps1-deletion strains, while HpYPS7 expression exhibited a limited complementation effect on the S. cerevisiae yps7-deletion strain. However, different from S. cerevisiae, deletion of the HpYPS genes generated only minor influence on the sensitivity to cell wall stress. Likewise, HpYPS1 expression was significantly induced only by a subset of stressor agents, such as sodium dodecyl sulphate and tunicamycin. HpYps1p was shown to consist of two subunits, whereas HpYps7p comprises a single long polypeptide chain. Biochemical analysis revealed that HpYps1p has much stronger proteolytic cleavage activity at basic amino acids, compared to HpYps7p. Consistent with the much higher proteolytic activity and expression level of HpYps1p compared to HpYps7p, the sole disruption of HpYPS1 was sufficient in eliminating the aberrant proteolytic cleavage of recombinant proteins secreted by H. polymorpha. The results indicate that, although their roles in the maintenance of cell wall integrity are not critical, HpYps1p and HpYps7p are functional aspartic proteases at the cell surface of H. polymorpha. Furthermore, our data present the high biotechnological potential of H. polymorpha yps1-mutant strains as hosts useful for the production of secretory recombinant proteins.
Yeast 10/2011; 29(1):1-16. · 1.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The α(1,6)-fucose attached to the core N-glycan (core fucose) of glycoproteins has been known to play essential roles in various pathophysiological events, including oncogenesis and metastasis. Aspergillus oryzae lectin (AOL) encoded by the fleA gene has been reported to bind to N-glycans containing core fucose. The fleA gene encoding AOL was cloned into an Escherichia coli expression vector and then fused with genes of fluorescent proteins for production of fusion proteins. The resulting FleA-fluorescent fusion proteins were expressed well in E. coli and shown to detect glycoproteins containing N-glycans with core fucose by lectin blot assay. It was also shown to bind to the surface of cancer cells highly expressing the fucosyltransferase VIII for attachment of core fucose. Surprisingly, we found that FleA-fluorescent fusion proteins could be internalized into the intracellular compartment, early endosome, when applied to live cells. This internalization was shown to occur through a clathrin-mediated pathway by endocytosis inhibitor assay. Taken together, these results suggest that FleA-fluorescent fusion proteins can be employed as a valuable fluorescent probe for the detection of fucosylated glycans and/or a useful vehicle for delivery of substances to the inside of cells.
Applied Microbiology and Biotechnology 09/2011; 93(1):251-60. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Sialidases, or neuraminidases (EC 3.2.1.18), belong to a class of glycosyl hydrolases that release terminal N-acylneuraminate residues from the glycans of glycoproteins, glycolipids, and polysaccharides. In bacteria, sialidases can be used to scavenge sialic acids as a nutrient from various sialylated substrates or to recognize sialic acids exposed on the surface of the host cell. Despite the fact that bacterial sialidases share many structural features, their biochemical properties, especially their linkage and substrate specificities, vary widely. Bacterial sialidases can catalyze the hydrolysis of terminal sialic acids linked by the α(2,3)-, α(2,6)-, or α(2,8)-linkage to a diverse range of substrates. In addition, some of these enzymes can catalyze the transfer of sialic acids from sialoglycans to asialoglycoconjugates via a transglycosylation reaction mechanism. Thus, some bacterial sialidases have been applied to synthesize complex sialyloligosaccharides through chemoenzymatic approaches and to analyze the glycan structure. In this review article, the biochemical features of bacterial sialidases and their potential applications in regioselective hydrolysis reactions as well as sialylation by transglycosylation for the synthesis of sialylated complex glycans are discussed.
Applied Microbiology and Biotechnology 07/2011; 91(1):1-15. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation, xhe genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative Operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this Operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.
The Journal of Microbiology 06/2011; 49(3):431-40. · 1.10 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mannosylphosphorylation of N- and O-glycans, which confers negative charges on the surfaces of cells, requires the functions of both MNN4 and MNN6 in Saccharomyces cerevisiae. To identify genes relevant to mannosylphosphorylation in the dimorphic yeast Yarrowia lipolytica, the molecular functions of five Y. lipolytica genes showing significant sequence homology with S. cerevisiae MNN4 and MNN6 were investigated. A set of mutant strains in which Y. lipolytica MNN4 and MNN6 homologues were deleted underwent glycan structure analysis. In contrast to S. cerevisiae MNN4 (ScMNN4), the Y. lipolytica MNN4 homologue, MPO1 (YlMPO1), encodes a protein that lacks the long KKKKEEEE repeat domain at its C terminus. Moreover, just a single disruption of YlMPO1 resulted in complete disappearance of the acidic sugar moiety in both the N- and O-linked glycan profiles. In contrast, even quadruple disruption of all ScMNN6 homologues, designated YlKTR1, YlKTR2, YlKTR3, and YlKTR4, resulted in no apparent reduction in acidic sugar moieties. These findings strongly indicate that YlMpo1p performs a significant role in mannosylphosphorylation in Y. lipolytica with no involvement of the Mnn6p homologues. Mutant strains harboring the YlMPO1 gene disruption may serve as useful platforms for engineering Y. lipolytica glycosylation pathways for humanized glycans without any yeast-specific acidic modifications.
Applied and environmental microbiology 02/2011; 77(4):1187-95. · 3.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: L-amino acid deaminases catalyze the deamination of natural L-amino acids. Two types of L-amino acid deaminase have been identified in Proteus species. One exhibits high levels of activity toward a wide range of aliphatic and aromatic L-amino acids, typically L-phenylalanine, whereas the other acts on a relatively narrow range of basic L-amino acids, typically L-histidine. In this study, we cloned, expressed, and characterized a second amino acid deaminase, termed Pm1, from P. mirabilis KCTC 2566. Homology alignment of the deduced amino acid sequence of Pm1 demonstrated that the greatest similarity (96%) was with the L-amino acid deaminase (LAD) of P. vulgaris, and that homology with Pma was relatively low (72%). Also, similar to LAD, Pm1 was most active on L-histidine, indicating that Pm1 belongs to the second type of amino acid deaminase. In agreement with this conclusion, the V(max) and K(m) values of Pm1 were 119.7 (μg phenylpyruvic acid/mg/min) and 31.55 mM phenylalanine, respectively, values lower than those of Pma. The Pml deaminase will be very useful industrially in the preparation of commercially valuable materials including urocanic acid and α-oxoglutarate.
Journal of Basic Microbiology 02/2011; 51(2):129-35. · 1.27 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mesenchymal stem cells (MSCs) undergo cellular senescence during in vitro expansion culture, which accompanies the loss of migration and homing abilities. In this study, we analyzed expression levels of several surface markers of human MSCs at different passages of expansion culture. It has been shown that expression of vascular cell adhesion molecule-1 (VCAM-1) was most markedly decreased among the tested markers in the senescent MSCs. Interestingly the reduced VCAM-1 expression could be restored by applying hyaluronan, a major glycosaminoglycan ligand of CD44, to the culture. It was found that the hyaluronan level in extracellular and pericellular matrices was greatly reduced in the senescent MSCs, mainly due to the decreased expression of hyaluronan synthases, suggesting a correlation between the reduced VCAM-1 expression and hyaluronan synthesis. In fact, when hyaluronan synthases were knock-downed by siRNA transfection, the VCAM-1 expression was also reduced. Our results indicate that VCAM-1 expression in the senescent MSCs was down-regulated because of the reduced synthesis of hyaluronan. Thus, we suggest that hyaluronan supplementation in expansion culture of MSCs would compensate adverse effects induced by its decreased synthesis and subsequently enhance cell adhesion and migration abilities.
Biochemical and Biophysical Research Communications 01/2011; 404(1):463-9. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p-Aga2p platform to immobilize the enzyme on the surface of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage. The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling.
Applied Microbiology and Biotechnology 10/2010; 88(4):893-903. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 degrees C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l(-1). Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.
Applied Microbiology and Biotechnology 09/2010; 88(2):497-507. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Corynebacterium diphtheriae, a pathogenic Gram-positive bacterium, contains sialic acids on its cell surface, but no genes related to sialic acid decoration or metabolism have been reported in C. diphtheriae. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. Interestingly, the recombinant NanH protein was secreted as a catalytically active sialidase into the periplasmic space in Escherichia coli, while the short region at its C-terminus was truncated by proteolysis. We reconstructed a truncated NanH protein (His(6)-NanH(DeltaN)) devoid of its signal sequence as a mature enzyme fused with the 6xHis tag at the N-terminal region. The purified His(6)-NanH(DeltaN) can cleave alpha-2,3- and alpha-2,6-linked sialic acid from sialic acid-containing substrates. In addition, even though the efficiency was low, the recombinant His(6)-NanH(DeltaN) was able to catalyse the transfer of sialic acid using several sialoconjugates as donor, suggesting that the reversible nature of C. diphtheriae NanH can be used for the synthesis of sialyl oligosaccharides via transglycosylation reaction.
Journal of biochemistry 12/2009; 147(4):523-33. · 1.95 Impact Factor
