• KSBB Journal
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• v.13 no.2
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• pp.133-140
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• 1998

Using rlFN-$\alpha$ and rhGH as the model proteins, the refolding performances of the published processes were evaluated and compared. Key engineering parameters such as the type of denaturant and this concentration, protein concentration in the refolding buffer, and pH and ionic strength of the buffer were experimentally investigated. Furthermore, the role of a co-solvent of surfactant type in aggregation reduction was also studied. Of the denaturants tested (8M urea, 6M guanidine HCI, 0.5% SDS), SDS at alkaline pH (9.5) and ambient temperature gave the highest recovery yield. The SDS process was effective in the refolding of observed where dissolution proceeded better under lower strength (10 mM) but aggregation was suppressed under higher strength (>50 mM.) When PEG-4000 and/or Tween were added as co-solvent or refolding-enhancing additive, 1.6-2 times higher yield was realized. The‘masking’of the hyrophobic patches located on the surface of the protein with the surfactant molecules was believed to be responsible for the considerable reduction in aggregation during refolding.

• Journal of Plant Biotechnology
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• v.39 no.2
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• pp.93-98
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• 2012

This study was conducted to evaluate effects of somatic embryos (SEs) induction with different kinds and concentrations of osmoticum, with gelrite and SEs germination with different abscisic acid (ABA) and embryogenic tissue lines (ETLs) in Japanese red pine (Pinus densiflora). In comparison of somatic embryos induction with different kinds and concentrations of osmoticum, the highest record (45/90 mg FW) was obtained from the treatment of 0.1 M maltose+3.75% PEG 4000. In addition, the higher one was also recorded from 0.2 M maltose (41 SEs), it turned out this treatment was also effective in induction of SEs with previous one. In effects of various gelrite concentrations for SEs production, no SEs were occurred in the treatment of 0.4 or 0.6% gelrite, however, the highest no. of SEs shown in 1.0% gellrite (41 SEs), and some comparable results were also marked with 0.8 (37.3 SEs) or 1.2% (39.7 SEs) gelrite. Therefore, SEs can be produced from above 0.8% concentration of gelrite. Finally, in comparison of SEs germination with different concentrations of ABA, ETLs, and AC, the best germination rates (45%) were obtained from the SEs derived from both concentrations of 150 and $200{\mu}M$ ABA in 06-6 ETL and when cultured on germination supplemented with 0.2% AC. When SEs were cultured on germination medium without AC, best germination rate (28.9%) came from the SEs which cultured on $250{\mu}M$ ABA in 06-6 ETL.

• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.460-465
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• 1990

Regeneration of protoplast was effective by preincubating spore suspension containing 30$\mu g$/ml of 2-DG for 4 hours, and CBE medium containing casamino acid, bovine serum albumin, ergosterol and myoinositol was found to be more efficient than any other regeneration medium tested in this experiment. The regeneration frequency was about 30%. Optimal conditions for conidial protoplast fusion were obtained by treatment of protoplasts with 10 mM $CaCl_2$ and 30% polyethylene glycol 4000 (pH 7.5) as fusogenic agent at $37^{\circ}C$ for 10 minutes. The fusion frequency was $8.2\times 10^{-4}$. The higher productivity of enzyme of fusant FWN-56 was achived: 2.3-fold for CMCase, 1.5-fold for avicelase, 1.8-fold for $\beta$-glucosidase and 2.5-fold for xylanase compared to that obtained in two parental strains. The genetic stability of fusant after maintenance on minimal medium for more than 4 weeks was high because segregant rate was below 1%. The conidial DNA content of fusant was 1.4-1.6 times higher than that of the parental strains, The nucleus size of fusants were also higher than that of each parental strains.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.6
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• pp.964-969
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• 1995

For the effective utilization of cellulosic biomass, conidial protoplast fusion between Aspergillus niger MAN-831(${\beta}-glucosidase$) and A. wentii MAW-538(CMCase and avicelase), which produced potently cellulolytic enzymes was carried out. Optimal conditions for formation and regeneration of protoplast were conidiospore age-5 dyuas. $2-DG-30\mu\textrm{g}/ml$, preincubation time-4 hours, osmotic stabilizer-0.7M KCl, novozyme(7mg/ml)＋driselase(2.5mg/ml) and reaction time of enzyme-5 hours. Optimal conditions for protoplast fusion were obtained by treatment of protoplasts with 15mM CaCl2 and 25% polyethylene glycol 4000(pH 6~7) as fusogenic agent at $36^{\circ}C$ for 25~30 minutes. The frequency was then $7.94{\times}10^{-4}$. CMCase, avicelase and ${\beta}-glucosidase$ activity of fusant F-208 strain was 1.5, 1.3, 1.2 times higher than those of parental strains, respectively.

• Korean Journal of Microbiology
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• v.30 no.3
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• pp.207-212
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• 1992

To develope the new strains of microorganisms having the degradative ability for various aromatic hydrocarbons. spheroplast cell fusions were performed with Arthrobacter spp. degrading phthalate ester and Pseudomonas putida degrading alkylbenzen sulfonate(ABS) and the characteristics of the fusants were investigated. The spheroplasts of P. putia KUD15 and Arthrobacter sp. were formed effectively by lysozyme-EDTA treatment and by Ampicillin-lysozyme-EDTA treatment. respectively. The Spheroplast formation frequency and the regeneration frequency of the strains were 98-99% and 5-8%, respectively. For cell fusion. 40% PEG6000 was used as a fusogenic agent and the formation frequencies of fusion product were $1.8{\times}10^{4}-$2.9{\times}10^{4}$ Most of the fusants, which were selected in complemented antibiotics media showed the degradative ability in minimal selective medium added phthalate ester or ABS as sole carbon source. ABS degradation by fusant strain was increased about 20% with compared with the parental strain, while the degradative ability of phthalate ester was simillilar to that of parental strain.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.356-361
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• 1991

- To investigate the possibility of genetic development for a multi-purpose strain of Bacillus subtilis YBL-7 against Fusat-iurn solani causing root rot of many impotant corps, the plasmid pGU66 inserting urease gene of Bacillus pasteurii had been introduced into Bacillus subtilis YBL-7 by PEG-induced protoplast (PIP) transformation system. Protoplasts of B. subtilis YBL-7 were prepared by treating the cells with lysozyme (200 $\mu g$/ml) in hypertonic buffer (SMMP). The highest transformation frequency was achieved when cells of the strain with lysozyme at $42^{\circ}C$ for 90 minutes. Optimal transformation was obtained using polyethylene glycol (MW 4000) at final concentration of 30% (V/V). The transformation frequency was increased proportionally to 1.2 $\mu g$ of plasmid DNA. At best condition, the transformation frequency (transformants/ regenerants/$\mu g$ of DNA) for pGU66 was appoximately $4 \times 10^{-3}$. Also, the urease gene was strongly expressed in the transformants of B. subtilis YBL-7 and maintained steadily. The antifungal ability of transformant was very similar to that of B. ssubtilis YBL-7.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.

• The Korean Journal of Mycology
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• v.21 no.4
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• pp.323-331
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• 1993

The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.283-286
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• 2008

The methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFDC) from the thermoacidophilic archaeon Thermoplasma acidophilum is a 30.6kDa molecular-mass enzyme that sequentially catalyzes the conversion of formyltetrahydrofollate to methylenetetrahydrofolate, with a preference for NADP as a cofactor, rather than NAD. In order to elucidate the functional and structural features of MTHFDC from archaeons at a molecular level, it was overexpressed in Escherichia coli and crystallized in the presence of its cofactor, NADP, at 295K using polyethylene glycol (PEG) 4000 as a precipitant. The crystal is a member of the monoclinic space group $P2_1$, with the following unit cell parameters: $a=66.333{\AA},\;b=52.868{\AA},\;c=86.099{\AA},\;and\;{\beta}=97.570^{\circ}$, and diffracts to a resolution of at least $2.40{\AA}$ at the synchrotron. Assuming a dimer in the crystallographic asymmetric unit, the calculated Matthews parameter $(V_M)\;was\;2.44{\AA}^3/Da$ and the solvent content was 49.7%.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.295-298
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• 2008

Creatine kinase (CK; E.C. 2.7.3.2) is an important enzyme that catalyzes the reversible transfer of a phosphoryl group from ATP to creatine in energy homeostasis. The brain-type cytosolic isoform of creatine kinase (BB-CK), which is found mainly in the brain and retina, is a key enzyme in brain energy metabolism, because high-energy phosphates are transfered through the creatine kinase/phosphocreatine shuttle system. The recombinant human BB-CK protein was overexpressed as a soluble form in Escherichia coli and crystallized at $22^{\circ}C$ using PEG 4000 as a precipitant. Native X-ray diffraction data were collected to $2.2{\AA}$ resolution using synchrotron radiation. The crystals belonged to the tetragonal space group $P4_32_12$, with cell parameters of a=b=97.963, $c=164.312{\AA},\;and\;{\alpha}={\beta}={\gamma}=90^{\circ}$. The asymmetric unit contained two molecules of CK, giving a crystal volume per protein mass $(V_m)$ of $1.80{\AA}^3\;Da^{-1}$ and a solvent content of 31.6%.