• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.1882-1893
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• 2019

β-Glucosidases and β-xylosidases are two categories of enzymes that could cleave out non-reducing, terminal β-D-glucosyl and β-D-xylosyl residues with release of D-glucose and D-xylose, respectively. In this paper, two functional β-glucosidase Dth3 and β-xylosidase Xln-DT from Dictyoglomus thermophilum were heterologously expressed in E.coli BL21 (DE3). Dth3 and Xln-DT were relatively stable at 75℃ and were tolerant or even stimulated by glucose and xylose. Dth3 was highly tolerant to glucose with a K_i value of approximately 3 M. Meanwhile, it was not affected by xylose in high concentration. The activity of Xln-DT was stimulated 2.13-fold by 1 M glucose and 1.29-fold by 0.3 M xylose, respectively. Furthermore, the βglucosidase Dth3 and β-xylosidase Xln-DT showed excellent selectivity to cleave the outer C-6 and C-3 sugar moieties of ASI, which established an effective and green method to produce the more pharmacologically active CAG, an exclusive telomerase activator. We measured temperature, pH and dosage of enzyme using a single-factor experiment in ASI biotransformation. After optimization, the optimal reaction conditions were as follows: 75℃, pH 5.5, 1 U of Dth3 and 0.2 U of Xln-DT, respectively. Under the optimized conditions, 1 g/l ASI was transformed into 0.63 g/l CAG with a corresponding molar conversion of 94.5% within 3 h. This is the first report to use the purified thermostable and sugar-tolerant enzymes from Dictyoglomus thermophilum to hydrolyze ASI synergistically, which provides a specific, environment-friendly and cost-effective way to produce CAG.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1650-1655
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• 2009

The $\beta$-glucuronidase (GUS) gene from Lactobacillus brevis RO1 was cloned and expressed in Escherichia coli GMS407. The GUS gene was composed of 1,812 bp, encoding a 603-amino-acid protein belonging to glycosyl hydrolase family 2 with three conserved domains. The amino acid similarity was higher than 70% with the $\beta$-glucuronidases of various microorganisms, yet less than 58% with the $\beta$-glucuronidase of L. gasseri ADH. Overexpression and purification of the GUS was performed in $\beta$-glucuronidase-deficient E. coli GMS407. The purified GUS protein was 71 kDa and showed 1,284 U/mg of specific activity at optimum conditions of pH 5.0 and $37^{\circ}C$. At $37^{\circ}C$, the GUS remained stable for 80 min at pH values ranging from 5.0 to 8.0. The purified enzyme exhibited a half-life of 1 h at $60^{\circ}C$ and more than 2 h at $50^{\circ}C$. When the purified GUS was applied to transform baicalin and wogonoside into their corresponding aglycones, $150\;{\mu}M$ of baicalin and $125\;{\mu}M$ of wogonoside were completely transformed into baicalein and wogonin, respectively, within 3 h.

• Applied Biological Chemistry
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• 제43권4호
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• pp.231-235
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• 2000

담배 배양 세포의 성장과정 중의 칼모듈린 농도변화 및 칼모듈린 결합 단백질의 종류에 대하여 조사하고 이들 단백질들 중 글루탐산 탈탄산효소를 immunodetection과 활성측정으로 확인하였다. 담배세포는 유도기(초기 $1{\sim}2$일간), 대수증식기($3{\sim}5$일), 정지기 등의 전형적인 성장 패턴을 보였다. 칼모듈린의 농도는 비록 대수증식기에 약간 감소하는 경향을 보이다 정지기에 이르면서 유도기의 수준을 회복하는 것으로 나타났지만 전체적으로는 성장단계에 관계없이 유사한 수준을 유지하는 것으로 나타났다. 주요 칼슘-의존형 칼모듈린 결합단백질은 56, 46, 36, 32-kDa의 4종류인 것으로 조사되었고, 모노클로날 항체를 이용하여 immunodetection을 실시해 본 결과 56-kDa 단백질이 담배 글루탐산 탈탄산효소로 확인되었다. 56-kDa의 글루탐산 탈탄산효소는 대수증식기에 수확한 세포에서 가장 많이 검출되었고, 이와같은 패턴은 효소활성 측정에서도 확인되었다. 이러한 결과들은 담배세포의 성장과정 중에 칼슘/칼모듈린-의존형 글루탐산 탈탄산효소 농도가 조절되고 있음을 제안해 주는 것이다.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1751-1757
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• 2007

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli $DH5{\alpha}$ at $37^{\circ}C$ for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at $20^{\circ}C$ in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature ($T_t$) of levansucrase-ELP[V-40] was $17^{\circ}C$ with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

• BMB Reports
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• 제33권3호
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• pp.242-248
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• 2000

Phage display of proteins is a powerful tool for protein engineering since a vast library of sequences can be rapidly screened for a specific property. In this study, we develop da new phage display vector that was derived from a pET-25b(+) vector. The pET-25b(+) was modified in order that the expressed protein would have a T7-tag at the amino terminus and GpS (a major coat protein of M13 phage) at the carboxyl terminus. Another vector without the gp8 gene was also constructed. The newly developed phagemid vectors have several advantageous features. First, it is easy to examine whether or not the target proteins are functional and faithfully transported into the periplasmic space. This feature is due to the fact that recombinant proteins are produced abundantly in the pET system. Second, the T7-tag makes it possible to detect any target proteins that are displayed on the surface of filamentous bacteriophage. To verify the utility of the vector, the clones containing the glutathione S-transferase (GST) gene as a target were examined. The result showed that the GST produced from the recombinant vector was successfully transported into the periplasmic space and had the anticipated enzyme activity. Western blot analysis using a T7-tag antibody also showed the presence of the target protein displayed on the surface of the phage. The phages prepared from the recombinant clones were able to bind to glutathione-Sepharose and then eluted with glutathione. These results showed that the new vectors developed in this study are useful for the phage display of proteins.

• Parasites, Hosts and Diseases
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• 제44권3호
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• pp.187-196
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• 2006

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2007년도 춘계학술발표회
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• pp.65-73
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• 2007

The objective of this study is to understand how regulatory mechanisms respond to sugar status for more efficient carbon utilization and source-sink regulation in plants. So, we need to identify and characterize many components of sugar-response pathways for a better understanding of sugar responses. For this end, genes responding change of sugar status were screened using Arabidpsis cDNA arrays, and confirmed thirty-six genes to be regulated by sucrose supply in detached leaves by RNA blot analysis. Eleven of them encoding proteins for amino acid metabolism and carbohydrate metabolism were repressed by sugars. The remaining genes induced by sugar supply were for protein synthesis including ribosomal proteins and elongation factors. Among them, I focused on three hydrolase genes encoding putative $\beta$-galactosidase, $\beta$-xylosidase, and $\beta$-glucosidase that were transcriptionally induced in sugar starvation. Homology search indicated that these enzymes were involved in hydrolysis of cell wall polysaccharides. In addition to my results, recent transcriptome analysis suggested multiple genes for cell wall degradation were induced by sugar starvation. Thus, I hypothesized that enzyme for cell wall degradation were synthesized and secreted to hydrolyze cell wall polysaccharides producing carbon source under sugar-starved conditions. In fact, the enzymatic activities of these three enzymes increased in culture medium of Arabidopsis suspension cells under sugar starvation. The $\beta$-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved condition with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. Further, contents of cell wall polysaccharides especially pectin and hemicellulose were markedly decreased associating with sugar starvation in detached leaves. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These results supported my idea that cell wall has one of function to supply carbon source in addition to determination of cell shape and physical support of plant bodies.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1363-1370
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• 2016

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector $pPICZ{\alpha}A$, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.231-236
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• 2004

The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2005년도 2005 Annual Meeting & International Symposium
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• pp.245-247
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• 2005

A cellulolytic fungus isolated from Agave plantation in northeastern Thailand was identified as Acrophialophora nainiana. The fungus was capable of growing at pH between 3 - 7 and 25 - 45 $^{\circ}C$, with the optimum conditions at pH 5.0 and 40 $^{\circ}C$. The wild isolate produced cellulases, comprising of exoglucanase (0.019 U/mg protein), endoglucanase (0.366 U/mg protein), and ${\beta}$-glucosidase (0.001 U/mg protein). Mutations with UV and NTG produced the UV 10-2 mutant with cellulases activities including exoglucanase (0.093 U/mg protein), endoglucanase (0.585 U/mg protein), and ${\beta}$-glucosidase (0.013 U/mg protein). Purification of the enzymes with ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography yielded the maximal cellulase specific activities of 2.736 U/mg protein (exoglucanase), 0.235 U/mg protein (endoglucanase), and 0.008 U/mg protein (${\beta}$-glucosidase). The mutant's cellulases were the most active at pH 5.0 and 60 $^{\circ}C$. Ion-exchange chromatography revealed that A. nainiana UV 10-2 cellulases were comprised of two peaks with one peak showing the single endoglucanase activity while the other peak showed a mixture of the three enzyme activities. Production of A. nainiana UV 10-2 cellulases using banana leaf stalk as the sole carbon source gave comparable yields to that of the pure ${\alpha}$-cellulose. The enzymes were used in the simultaneous saccharification and fermentation (SSF) of plant residue (Coix aquatica) along with Kluveromyces marxianus to produce ethanol. Moreover, when the enzymes were used in the bioscouring process of fabric, the desiravle traits of textile processing including immediate water absorbency, increased in whiteness and reduction of yellowness of the treated fabric were observed.