• Title/Summary/Keyword: biochemical characterization

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Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952

  • Rimal, Hemraj;Subedi, Pradeep;Kim, Ki -Hwa;Park, Hyun;Lee, Jun Hyuck;Oh, Tae-Jin
    • Journal of Microbiology and Biotechnology
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    • v.30 no.11
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    • pp.1750-1759
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    • 2020
  • The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated KD value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min-1 nmol-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

Biochemical Characterization and Genetic Diversity of Pongamia pinnata (L.) Pierre in Eastern India

  • Kumari, Kanchan;Sinha, Amrita;Singh, Sanjay;Divakara, B.N.
    • Journal of Forest and Environmental Science
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    • v.29 no.3
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    • pp.200-210
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    • 2013
  • Biochemical characteristics of 24 Pongamia pinnata genotypes (candidate plus trees) from three agroclimatic zones were estimated and molecular characterization through RAPD markers was done. Various biochemical characters viz. seed oil, total carbohydrates, protein, acid value and Iodine number recorded significant variation among different genotypes. The highest seed oil content was 41.87% while seeds of 14 genotypes recorded above average (32.11%) for the trait. Seed oil and protein content exhibited a significant positive correlation and moderate heritability. Out of the initially selected twenty-five random primers, twenty-two RAPD primers were found to be highly reproducible and produced a total of 183 loci of which 147 (80.32%) loci were polymorphic. Percentage of polymorphism varied from 44% to 100% with an average of 80.62%. High level of genetic variation was found among different genotypes of P. pinnata. Both molecular and oil content (biochemical) markers appeared useful in analyzing the extent of genetic diversity in Pongamia and the result of these analyses will help to better understand the genetic diversity and relationship among populations. Overall, the Pongamia genotypes included in the study showed a correlation with their geographical origins such that genotypes from the same region tend to have higher genetic similarity as compared to those from different regions. However, in UPGMA based Nei's analysis, some genotypes were found not to be grouped based on geographical origins possibly due to the exchange of germplasm over time between farmers across the regions. The results from oil content analyses showed that several genotypes in 'Central and Western Plateau' agroclimatic zone of Jharkhand displayed a good potential for high oil content. The study provides insight about P. pinnata populations in Jharkhand (India) and constitutes a set of useful background information that can be used as a basis for future breeding strategy and improvement of the species.

Purification and Characterization of a Recombinant Caulobacter crescentus Epoxide Hydrolase

  • Hwang, Seung-Ha;Hyun, Hye-Jin;Lee, Byoung-Ju;Park, Young-Seub;Lee, Eun-Yeol;Choi, Cha-Yong
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.11 no.4
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    • pp.282-287
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    • 2006
  • A Caulobacter crescentus epoxide hydrolase(CCEH) from a recombinant Escherichia coli was purified to homogeneity using a three-step procedure. The CCEH protein was purified 7.3-fold with a 22.9% yield in overalll activity. The optimal reaction temperature and pH were determined to be $37^{\circ}C$ and pH 8.0, respectively. The addition of 10%(v/v) dimethylsulfoxide as a cosolvent improved the enantioselectivity of CCEH for a batch kinetic resolution of racemic indene oxide.

Molecular and Morphological Characterization of a Taxol-Producing Endophytic Fungus, Gliocladium sp., from Taxus baccata

  • Sreekanth, D.;Sushim, G.K.;Syed, A.;Khan, B.M.;Ahmad, A.
    • Mycobiology
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    • v.39 no.3
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    • pp.151-157
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    • 2011
  • The endophytic fungal populations of different tissues of Taxus baccata grown at high altitudes in West Bengal, India were explored. These isolated fungal populations represented different genera, which were screened for taxol production using immunoassay technique. The culture AAT-TS-$4_1$ that produced taxol was identified as Gliocladium sp. based on its cultural, morphological characteristics, internal transcribed spacer, and 18S rRNA sequence analysis. Kinetics of taxol production as a function of culture growth were investigated.