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Math Renukaradhya K.,Nagakumar Bharatham,Palaksha K. Javaregowda,윤한대 한국현미경학회 2021 Applied microscopy Vol.51 No.1
Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H ( Bl Cel5H) and Paenibacillus polymyxa Cel5A ( Pp Cel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in Bl Cel5H but not in Pp Cel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69 ± 19 pN for wild-type, 58 ± 19 pN for M2, 53 ± 19 pN for M3, and 49 ± 19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.
Cho, Kye-Man,Lee, Sun-Mi,Math, Renukaradhya K.,Islam, Shah Md. Asraful,Kambiranda, Devaiah M.,Kim, Jong-Min,Yun, Myoung-Geun,Cho, Ji-Joong,Kim, Jong-Ok,Lee, Young-Han,Kim, Hoon,Yun, Han-Dae The Korean Society for Microbiology and Biotechnol 2008 Journal of microbiology and biotechnology Vol.18 No.12
Bacterial diversity and the composition of individual communities during the composting process of swine and mushroom cultural wastes in a field-scale composter (Hazaka system) were examined using a PCR-based approach. The composting process was divided into six stages based on recorded temperature changes. Phylogenetic analysis of eighty 16S rRNA sequences from uncultured composting bacterial groups revealed the presence of representatives from three divisions, including plant pathogenic bacteria, high-molecule-degrading bacteria and spore-forming bacteria. The plant pathogen A. tumefaciens gradually decreased in abundance during the composting process and eventually disappeared during the thermophilic and cooling stage. A bacterium homologous to Bacillus humi first appeared at the early thermophilic stage and was established at the intermediate thermophilic, post-thermophilic, and cooling stages. It was not possible to isolate the B. humi during any of the stages using general culture techniques.
Expression of esterase gene in yeast for organophosphates biodegradation
Kambiranda, Devaiah M.,Asraful-Islam, Shah Md.,Cho, Kye Man,Math, Renukaradhya K.,Lee, Young Han,Kim, Hoon,Yun, Han Dae Elsevier 2009 Pesticide biochemistry and physiology Vol.94 No.1
<P><B>Abstract</B></P><P>Organophosphates are esters of phosphoric acid and can be hydrolyzed and detoxified by carboxylesterase and phosphotriesterase. In this work esterase enzyme (Est5S) was expressed in yeast to demonstrate the organophosphorus hydrolytic activity from a metagenomic library of cow rumen bacteria. The esterase gene (<I>est</I>5S) is 1098bp in length, encoding a protein of 366 amino acid residues with a molecular weight of 40kDa. Est5S enzyme was successfully produced by <I>Pichia pastoris</I> at a high expression level of approximately 4.0g L<SUP>−1</SUP>. With <I>p</I>-nitrophenol butyrate as the substrate, the optimal temperature and pH for enzyme activity were determined to be 40°C and pH 7.0, respectively. The esterase enzyme was tested for degradation of chlorpyrifos (CP). TLC results obtained inferred that CP could be degraded by esterase enzyme (Est5S) and HPLC results revealed that CP could be efficiently degraded up to 100ppm. Cadusafos (CS), coumaphos (CM), diazinon (DZ) dyfonate (DF), ethoprophos (EP), fenamiphos (FM), methylparathion (MPT), and parathion (PT) were also degraded up to 68, 60, 80, 40, 45, 60, 95, and 100%, respectively, when used as a substrate with Est5S protein. The results highlight the potential use of this enzyme in the cleanup of contaminated insecticides.</P>
Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells
Islam, Shah Md. Asraful,Cho, Kye Man,Hong, Sun Joo,Math, Renukaradhya K.,Kim, Jong Min,Yun, Myoung Geun,Cho, Ji Joong,Heo, Jae Young,Lee, Young Han,Kim, Hoon,Yun, Han Dae Springer-Verlag 2010 Applied microbiology and biotechnology Vol.86 No.1
Kye Man Cho,Devaiah M Kambiranda,Seong Weon Kim,Renukaradhya K Math,Woo Jin Lim,Su Young Hong,Han Dae Yun 한국식품과학회 2008 Food Science and Biotechnology Vol.17 No.6
Kimbab is the most popular ready-to-eat (RTE) food in Korea. A rapid detection method based on multiplex PCR technique was developed for detection of major food-borne pathogens like Salmonella spp., Shigella spp., Bacillus cereus, Listeria monocytongenes, and Staphylococcus aureus. Specific bands were obtained as 108 bp (Sau, S. aureus), 284 bp (Sal, S. enterica, S. enteritids, and S. typhmurium), 404 bp (Lmo, L. monocytogenes), 475 bp (Bce, B. cereus), and 600 bp (Shi, S. flexineri and S. sonnei). Visible cell numbers varied from 4.14-5.03, 3.61-4.47, and 4.10-5.11 log CFU/g in randomly collected June, July, and August samples, respectively. Among the 30 kimbab samples obtained 83.3% samples were contaminated and 16.7% samples were free from contamination. The highest rate of contamination was with S. aureus (56.7%) followed by B. cereus (43.3%), Salmonella spp. (36.7%), Shigella spp. (13.3%), and L. monocytogenes (6.7%). The identification of the pathogenic species could be faster using one polymerase chain reaction (PCR) and the ability to test for food-borne pathogenic species in kimbab will save time and increase the ability to assure its quality.
N. Abhilash,S. K. Ajay,K. V. Ramesh,Renukaradhya K. Math 경희대학교 융합한의과학연구소 2023 Oriental Pharmacy and Experimental Medicine Vol.23 No.2
Complex synergistic interplay of the composite mixture of compounds present in plants may be the reason of their beneficial effects. In this perspective an important yet not well studied ethnomedicinal plant Lasiosiphon glaucus was subjected to invitro anticancer analysis. The flowcytometry study revealed that the plant has cytotoxic effect against breast cancer cells (MCF-7) acting upon multiple pathways, inhibiting cell proliferation (Downregulation of Bcl2 and Akt) and inducing apoptosis (Mitochondrial membrane depolarisation, Upregulation of p53, caspase 8, caspases 3 and DNA fragmentation). It is interesting that the plant activated both intrinsic and extrinsic pathways while inhibiting cancer cell proliferation. The study, a first report, highlights the use of L. glaucus in ethnomedicine for treating cancer.
Cloning and Characterization of a Novel Carboxylesterase Gene from Cow Rumen Metagenomic Library
Shah Md. Asraful Islam(아스라풀 이스람),Min Keun Kim(김민근),Renukaradhya K. Math(아라디아),Srinivasa Reddy R.N.(스리니 래디),Eun Jin Kim(김은진),Jungho Kim(김정호),Hoon Kim(김훈),Han Dae Yun(윤한대) 한국생명과학회 2010 생명과학회지 Vol.20 No.9
한우의 반추위에서 게놈 DNA를 분리하여 메타게놈 은행을 구축한 다음 carboxylesterase를 암호화하는 유전자를 클로닝 및 유전자를 선별하였다. 선별된 유전자의 DNA 염기서열 및 아미노산 서열을 분석하고 유전산물의 생화학적인 특성을 조사하였다. est1R 유전자는 2,465 bp로 366개의 아미노산 잔기를 가진 단백질을 암호화하였으며 이 단백질의 이론적인 분자량은 61,166 Da이었다. Est1R단백질은 PNB carboxylesterase (P37967), acetylcholinesterase(1EEAA) 및 chain A (1H23A)와 각각 5.9%, 6.1%, 6.1% 상동성을 가지고 있었다. 이러한 검색 결과 기존의 알려진 lipase 및esterase와의 상동성이 낮은 것으로 보아 새로운 그룹의 효소로 추정된다. Est1R효소의 최적 pH는 7.0 근방이었으며 최적 온도는 40℃ 부근이었다. 한편 10% 유기용매를 함유한 기질의 효소활성측정에서 대조구에 비해 methanol은 95%의 상대적인 활성을 가진 반면에 hexane 용액에서는 그 활성이 반으로 감소하였다. 따라서 유기용매 농도의 작용성에 따라 이 효소의 산업적 이용성도 가능하리라 추정된다. The gene encoding esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est1R) was 2,465 bp in length, encoding a protein of 366 amino acid residues, and the molecular weight of the enzyme was 61,166 Da. Est1R of rumen cosmid library shared 5.9% amino acid identity with Est1R (P37967) of PNB carboxylesterase, 6.1% with Est1R (1EEAA) of acetylcholinesterase and 6.1% with Est1R (1H23A) of chain A. BlastP in NCBI database analysis of Est1R revealed that it was not homologous to previous known lipases and esterases. Est1R showed optimum activity at pH 7.0 and 40℃. On the other hand, the enzyme was found to be most active without organic solvent, followed by 95% activity with methanol, and the enzyme activity was highly affected by hexane (lost 51% activity). Therefore, the novel esterase gene est1R is likely obtainable from cow rumen metagenome and may be utilized for industrial purposes.
조계만,김은주,레누카라디아마스,샤모허마드아스라풀,홍선주,김종옥,신기재,이영한,김훈,윤한대,Cho, Kye-Man,Kim, Eun-Ju,Math, Renukaradhya K.,Asraful Islam, Shah Md.,Hong, Sun-Joo,Kim, Jong-Ok,Shin, Ki-Jae,Lee, Young-Han,Kim, Hoon,Yun, Han-Dae Korean Society of Life Science 2007 생명과학회지 Vol.17 No.9
연부균인 Pectobacterium carotovorum subsp. carotovorum LY34로부터 이소아밀라제 유전자 (glgX)를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 ${\alpha}-1$,6-글루코시드 결합을 가수분해하였으나 ${\alpha}-1$,4-글루코시드 결합은 가수분해 하지 못하였다. 유전자는 658개의 아미노산을 암호화하는 1,977개의 DNA 염기서열로 이루어져 있었고 이 유전자에 의해 암호화되는 아미노산 서열을 다른 아밀라제 효소들과 비교한 결과 이소아밀라제 유전자와 유사하였으며 4개의 보존 지역을 확인하였다. SDS-PAGE에 의해 확인된 단백질의 크기는 약 74 kDa 이었다. 효소 활성은 pH 7.0, $40^{\circ}C$에서 가장 높은 활성을 나타났으며 $Ca^{2+}$ 첨가로 활성이 증가되었다. 이 효소의 보존되어 있는 아미노산 중에 글루탐산 370번, 아스파르트산 335번 및 442번 잔기를 알라닌으로 치환시킨 결과 활성이 약해졌다. 이 결과로부터 이들 잔기들이 효소활성에 중요한 역할을 하는 것으로 추정된다. The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze ${\alpha}-1,4-glycosidic$ linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and $40^{\circ}C$. GlgX was $Ca^{2+}-dependent$. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.