• Journal of Microbiology and Biotechnology
• /
• v.12 no.4
• /
• pp.603-609
• /
• 2002

DFA IV is di-D-fructose-2,6':6,2'-dianhydride, consisting of two fructose residues. It can be enzymatically synthesized from levan by levan fructotransferase, and can be used for mineral absorption. Understanding of the structure and composition of levan is important to obtain high-level production of DFA IV. A bacterial strain, Pseudomonas aurantiaca 5-4380, was identified to produce low-branched levan, and the levansucrase gene (lsch) from this bacterium was found to be composed of 1,275 Up coding for a protein of 424 amino acids, with an estimated molecular weight of 47 kDa. The bacterial levansucrase gene was expressed in Escherichia coli DH5${\alpha}$ by its own promoter and lac promoter. The recombinant levansucrase was produced in soluble form with 170U of levansucrase activity from 1-ml E. coii culture broth. The expressed enzyme from the clone showed similar biochemical properties, such as size of active levansucrase, degree of branching, and optimum temperature, with P.aurantiaca 5-4380 levansucrase.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.9
• /
• pp.937-946
• /
• 2011

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on $LC_{50}$ values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.4
• /
• pp.483-488
• /
• 2014

In order to improve the expression of heat-resistant xylanase XYNB from Aspergillus niger SCTCC 400264, XynB has been cloned into Pichia pastoris secretary vector pPIC9K. The XynB production of recombinant P. pastoris was four times that of E. coli, and the $V_{max}$ and specific activity of XynB reached $2,547.7{\mu}mol/mg$ and 4,757 U/mg, respectively. XynB still had 74% residual enzyme activity after 30 min of heat treatment at $80^{\circ}C$. From the van der Waals force analysis of XYNB (ACN89393 and AAS67299), there is one more oxygen radical in AAS67299 in their catalytic site, indicating that the local cavity is much more free, and it is more optimal for substrate binding, affinity reaction, and proton transfer, etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB indicated that there are two more H-bonds in the 33rd Ser of XYNB (AAS67299) than in the 33rd Ala(ACN89393 ), and two H-bonds between Ser70 and Asp67.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.3
• /
• pp.304-312
• /
• 2013

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'${\rightarrow}$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
• /
• v.27 no.7
• /
• pp.1216-1222
• /
• 2017

To develop starters for the production of functional foods or materials, lactic acid bacteria producing ${\gamma}-aminobutyric$ acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium $\text\tiny{L}$-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced $18.47{\pm}1.26mg/ml$ GABA when incubated for 48 h at $37^{\circ}C$ in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and $55^{\circ}C$and the activity was dependent on pyridoxal 5'-phosphate. The $K_m$ and $V_{max}$ values of GAD were $3.26{\pm}0.21mM$ and $0.0120{\pm}0.0001mM/min$, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.

• Korean Chemical Engineering Research
• /
• v.55 no.2
• /
• pp.237-241
• /
• 2017

Acetoin is variously applicable platform chemical in chemical and food industry. In this study, Klebsiella pneumoniae was engineered for acetoin production using metabolic engineering. From the recombinant Klebsiella pneumoniae (KMK-05) producing 2,3-butanediol, budC and dhaD genes encoding two 2,3-butanediol dehydrogenases were deleted to reduce 2,3-butanediol production. Furthermore, a transcriptional regulator, AcoK, was deleted to reduce the expression levels of acetoin degrading enzyme. Lastly, NADH oxidase was overexpressed for adjusting intracellular redox balance. The resulting strain (KJW-03-nox) produced considerable amount of acetoin, with concentration reaching 51 g/L with 2.6 g/L/h maximum productivity in 36 h fed-batch fermentation.

• Biomedical Science Letters
• /
• v.15 no.1
• /
• pp.37-45
• /
• 2009

Insect cell cultures have become important tools in the production of biological substances for use in a variety of research, human and veterinary medicine, and pest control applications. These applications often require the introduction of foreign DNA into the cells and have generally used methods originally developed for use with human and other mammalian cell cultures. While these methods can be successfully employed, they are often less efficient with insect cells and frequently involve complex procedures or require specialized equipment. Even when they do work, they may require substantial modification because of differences in the culture medium or growth patterns of insect cells. In this study, We have optimized transfection conditions of Sf9 cell line using insect expression vector pIZT/V5-His which expresses green fluorescent protein effectively. Human stem cell factor (hSCF) is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a key role in mast cell development, gametogenesis, and melanogenesis. It can exist in membrane-bound form and in proteolytically released soluble form. As determined by an enzyme-linked immunosorbent assay performed, hSCF level in supernatant averaged 995ng/ml. The human hSCF was partially purified by immunoaffinity chromatography and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the hSCF has N-linked carbohydrate and corresponds to the soluble form, at or about 223 amino acids in length. The findings suggest functional importance for soluble hSCF in cells.

• BMB Reports
• /
• v.47 no.7
• /
• pp.399-404
• /
• 2014

B7-H4 is a member of B7 family of co-inhibitory molecules and B7-H4 protein is found to be overexpressed in many human cancers and which is usually associated with poor survival. In this study, we developed a therapeutic vaccine made from a fusion protein composed of a tetanus toxoid (TT) T-helper cell epitope and human B7-H4IgV domain (TT-rhB7-H4IgV). We investigated the anti-tumor effect of the TT-rhB7-H4IgV vaccine in BALB/c mice and SP2/0 myeloma growth was significantly suppressed in mice. The TT-rhB7-H4IgV vaccine induced high-titer specific antibodies in mice. Further, the antibodies induced by TT-rhB7-H4IgV vaccine were capable of depleting SP2/0 cells through complement-dependent cytotoxicity (CDC) in vitro. On the other hand, the poor cellular immune response was irrelevant to the therapeutic efficacy. These results indicate that the recombinant TT-rhB7-H4IgV vaccine might be a useful candidate of immunotherapy for the treatment of some tumors associated with abnormal expression of B7-H4.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.23 no.2
• /
• pp.223-229
• /
• 2011

Cyclophilins are originally identified as cytosolic binding protein of the immunosuppressive drug cyclosporine A. They have an activity of peptidyl prolyl cis/trans-isomerases (PPIase), which may play important roles in protein folding, trafficking, assembly and cell signaling. In this study, we report the cloning and characterization of a Bombyx mori cyclophilin A (bCypA) cDNA. The full-length cDNA of bCypA consist of 947 nucleotides with a polyadenylation signal sequence AATAAA and contain an open reading frame of 498 nucleotides encoding a polypeptide of 166 amino acids. The deduced amino acid sequence of bCypA shares a central peptidyl prolyl cis/trans-isomerase and a cyclosporin-A-binding domain with other cyclophilin sequences. Relative quantification real-time (RT) PCR analysis shows that mRNA transcripts of bCypA are detected in all the investigated tissues and highest expression level in the skin of 3-day-old 5 instar larva. Also, bCypA had PPIase activity on the proline-containing peptides. Accordingly, we suggest that bCypA is a new member of the cyclophilin A (CyPA) family and will be useful for quality control of bioactivity recombinant proteins with proline-containing peptides.

• Food Science and Biotechnology
• /
• v.18 no.3
• /
• pp.776-781
• /
• 2009

A putative cyclomaltodextrinase (BHCD) gene was found from the genome of Bacillus halodurans C-125, which encodes 578 amino acids with a predicted molecular mass of 67,279 Da. It shares 42-59% of amino acid sequence identity with common cyclomaltodextrinase (CDase)-family enzymes. The corresponding gene was cloned by polymerase chain reaction (PCR) and the dimeric enzyme with C-terminal 6-histidines was successfully overproduced and purified from recombinant Escherichia coli. BHCD showed the highest activity against ${\beta}-CD$ at pH 7.0 and $50^{\circ}C$. Due to its versatile hydrolysis and transglycosylation activities, BHCD has been confirmed as a member of CDases. However, BHCD can be distinguished from other typical CDases on the basis of its novel multisubstrate specificity. While typical CDases have over 10 times higher activity on ${\beta}-CD$ than starch or pullulan, the CD-hydrolyzing activity of BHCD is only 2.3 times higher than pullulan. In particular, it showed significantly higher activity ratio of maltotriose to acarbose than other common CDase-family enzymes.