• Journal of Applied Biological Chemistry
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• 제42권3호
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• pp.107-112
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• 1999

A carboxymethyl-cellulase (CMCase) was purified from the culture supernatant of Bacillus sp. KD1014 by ultrafiltration, ammonium sulfate precipitation, and a series of chromatography on QAE-Sephadex A-50, hydroxylapatite and Sephadex G-75. The purified CMCase was a single protein of 32 kDa, showed an optimum activity at $60^{\circ}C$ and pH 6.0, and had a half-life of 23 min at $70^{\circ}C$. The enzyme activity was not influenced by metal ions such as $Mg^{2+},\;Fe^{3+},\;K^+,\;Zn^{2+}$, and $Cu^{2+}$ at a concentration of 1.0 mM, partially inhibited by $Mn^{2+}$ and $Ag^+$, and significantly inhibited by pentachlorophenol (PCP). The purified enzyme showed a 3.9-times higher activity on lichenan than on CMC, but hardly cleaved xylan, starch, avicel, laminarin, filter paper and levan. The results of activity staining of the purified enzyme separated by native and denaturing gel electrophoresis suggested that the CMCase might exist in dimeric, oligomeric or aggregated form as well as in monomeric form. The enzymatic cleavage products from cellotetraose indicated that the CMCase possessed transglycosylation activity.

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• BMB Reports
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• 제38권1호
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• pp.17-23
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• 2005

During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.759-765
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• 2013

The gene encoding squalene synthase (SQS) of the lipid-producing heterotrophic microalga Aurantiochytrium sp. KRS101 was cloned and characterized. The krsSQS gene is 1,551 bp in length and has two exons and one intron. The open reading frame of the gene is 1,164 bp in length, yielding a polypeptide of 387 predicted amino acid residues with a molecular mass of 42.7 kDa. The deduced krsSQS sequence shares at least four conserved regions known to be required for SQS enzymatic activity in other species. The protein, tagged with $His_6$, was expressed into soluble form in Escherichia coli. The purified protein catalyzed the conversion of farnesyl diphosphate to squalene in the presence of NADPH and $Mg^{2+}$. This is the first report on the characterization of an SQS from a Thraustochytrid microalga.

• Journal of Microbiology
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• 제38권1호
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• pp.18-23
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• 2000

Streptomyces coelicolor produces three kinds of catalases to cope with oxidative stress and to allow normal differentiation. Catalase A is the major vegetative catalase which functions in removing hydrogen peroxide generated during the process of aerobic metabolism. To understand the regulatory mechanism of response against oxidative stress, hydrogen peroxide-resistant mutant (HR4O) was isolated from S. coelicolor J1501 following UV mutagenesis. The mutant overproduced catalase A more than 50-fo1d compared with the wild type. The mutation locus catRI was mapped closed to the mthB2 locus by genetic crossings. An ordered cosmid library of S. coelicolor encompassing the mthB2 locus was used to isolate the regulator gene (catR) which represses catalase overproduction when introduced into HR4O. A candidate catR gene was found to encode a Fur-like protein of 138 amino acids (15319 Da).

• 한국잠사곤충학회지
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• 제53권2호
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• pp.92-96
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• 2015

본 연구는 장려 품종 누에고치로부터 실크 세리신과 실크 피브로인으로 분리하여 이들 단백질을 세포 첨가제로 개발하기 위하여 수행되었다. 장려품종 누에고치를 고온 고압기를 통하여 실크 세리신을 먼저 추출하였으며, 남겨진 실크 피브로인을 이용하여 시간별로 용해하여 품종별 실크 피브로인을 얻었다. 이들 실크 세리신과 실크 피브로인을 이용하여 세포 독성, 세포 증식 및 분열 관련 유전자 발현 분석을 확인하였다. 먼저, 누에고치의 특성을 분석하기 위하여 공극률을 측정하였다. 공극률은 금옥잠 누에고치의 경우 84.71%, 대성잠 81.58%, 백옥잠 73.23%의 공극률 값이 나타났다. 분자량 차이에 있어서 실크 세리신은 품종의 영향이 크지 않았으나 실크 피브로인의 경우, 금옥잠은 5시간의 장시간의 용해에도 불구하고 100 kDa 이상의 큰 분자량을 나타내었다. 장려품종 누에를 사용하여 제조된 실크 세리신과 실크 피브로인을 이용하여 세포 증식 실험을 하였으며 백옥잠으로부터 얻은 실크 세리신의 경우 농도에 따라 유의적으로 세포 증식 효과가 나타났으며, 금옥잠으로부터 얻은 실크 피브로인의 경우 5시간 용해 시 세포 증식에 있어서 가장 우수한 결과를 보였다. 또한, 백옥잠 실크 세리신 처리 결과 세포 증식 관련 유전자의 발현수준이 증가됨을 확인할 수 있었다.

• BMB Reports
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• 제34권3호
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• pp.194-200
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• 2001

Secreted arginase from Evernia prunastri thallus has been purified 616-fold from the incubation medium. Purified arginase was resolved as only one peak in a capillary electrophoresis with a pI value of 5.35. The protein contained high amounts of acidic amino acids, such as Asx and Glx, and a relatively high quantity of Ser and Gly. The molecular mass of native, purified arginase was estimated as about 26 kDa by SE-HPLC. Substrate saturated kinetic showed a typical Michaelis-Menten relationship with a K_m value of 3.3 mM L-arginine. Atranorin behaved as a mixed activator of the enzyme (apparent $K_m$ = 0.96 mM); whereas evernic and usnic acid were revealed as non competitive inhibitors (apparent $K_m$ values were 3.16 mM and 3.05 mM, respectively). Kinetics of atranorin binding indicated that saturation was reached from 0.18 ${\mu}mol$ of the total atranorin and the occurrence of multiple sites for the ligand. This agrees with a possible aggregation of several enzyme subunits during the interaction process. A value of binding sites of about 12 was obtained. The binding of evernic acid was saturated from 23 nmol of total phenol. The number of binding sites was about 5. The loss of the binding ability of evernic acid could be interpreted as a single negative cooperatively. Usnic acid behaves in a similar way to evernic acid, although the binding saturation occurs at $0.14\;{\mu}moles$ of the ligand. This binding appears to be unspecific, and has 28 usnic acid binding sites to the protein.

• Journal of Microbiology and Biotechnology
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• 제21권7호
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• pp.734-738
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• 2011

Streptococcus pneumoniae is a major cause of invasive infection in young infants and older adults. There are currently 90 capsular serotypes identified and 23 serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F) are responsible for about 90% of invasive disease. Among the more than 90 different S. pneumoniae serotypes, serotype 19A is globally very prevalent. A simplified purification procedure including adjustment of cell lysate pH to 4.5, fractionation with 50. 80% ethanol, and dialysis rendered capsular polysaccharide (CPS) in a yield of $31.32{\pm}3.11$ mg from 1 l culture (75% recovery after lyses). The product contained only 69.6 ${\mu}g$ of protein (99.78% purity) and 0.8mg (sum of the precipitants from 50~60%, 60~70%, and 70~80%) of nucleic acid (97.45% purity). The purified CPS was conjugated with bovine serum albumin; the product size ranged from 100 to 180 kDa.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10079-10083
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• 2015

Background: Endothelin-1 and Endostar are both significant for the progression, proliferation, metastasis and invasion of cancer. In this paper, we studied the effect of ET-1 RNAi and Endostar in PC-3 prostatic cancer cells. Materials and Methods: The lentiviral vector was used in the establishment of ET-1 knockdown PC-3 cells. Progression and apoptosis were assessed by CKK-8 and flow cytometry, respectively. Transwell assay was used to estimate invasion and signaling pathways were studied by Western blotting. Results: ET-1 mRNA and protein in ET-1 knockdown PC-3 cells were reduced to 26.4% and 22.4% compared with control group, respectively. ET-1 RNAi and Endostar both were effective for the suppression of progression and invasion of PC-3 cells. From Western blotting results, the effects of ET-1 regulation and Endostar on PC-3 cells were at least related to some signaling pathways involving PI3K/Akt/Caspase-3, Erk1/2/Bcl-2/Caspase-3 and MMPs (MMP-2 and MMP-9). Furthermore, combined treatment of ET-1RNAi and Endostar was found to be more effective than single treatment. Conclusions: Both ET-1 RNAi and Endostar can inhibit the progression and invasion of PC-3 cells, but combined treatment might be a better therapeutic schedule.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2006년도 International Meeting of the Microbiological Society of Korea
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• pp.105-107
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• 2006

Two kinds of nucleoside hydrolases (NHs) encoded by rih1 and rih2 were cloned from Corynebacterium ammoniagenes using deoD- and gsk-defective Escherichia coli. Sequence analysis revealed that NH 1 was a protein of 337 aa with a deduced molecular mass of 35,892 Da, whereas NH 2 consisted of 308 aa with a calculated molecular mass of 32,310 Da. Experiments with crude extracts of IPTG-induced E. coli CGSC 6885(pTNU23) and 6885(pTNI12) indicated that the Rihl enzyme could catalyse the hydrolysis of uridine and cytidine and showed pyrimidine-specific ribonucleoside hydrolase activity. Rih2 was able to hydrolyse both purine and pyrimidine ribonucleosides with the following order of activity-inosine>adenosine>uridine>guanosine>xanthosine>cytidine-and was classified in the non-specific NHs family. rih1 and rih2 deletion mutants displayed a decrease in cell growth on minimal medium supplemented with pyrimidine and purine/pyrimidine nucleosides, respectively, compared with the wild-type strain. Growth of each mutant was substantially complemented by introducing rih1 and rih2, respectively. Furthermore, disruption of both rih1 and rih2 led to the inability of the mutant to utilize purine and pyrimidine nucleosides as sole carbon source on minimal medium. These results indicated that rih1 and rih2 play major roles in the salvage pathways of nucleosides in this micro-organism.