• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1472-1482
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• 2017

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity ($K_d$ value) to 4-androstenedione was $387{\pm}37{\mu}M$. Moreover, the crystal structure of BaCYP106A2 was determined at $2.7{\AA}$ resolution. Structural analysis suggested that the ${\alpha}8-{\alpha}9$ loop region of BaCYP106A2 is intrinsically mobile and might be important for initial ligand binding. The hydroxyl activity of BaCYP106A2 was identified using in vitro enzyme assays. Its activity was confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using chromatography and mass spectrometry methods. The main products were mono-hydroxylated compounds with high conversion yields. This is the second study on the structure of CYP106A steroid hydroxylases, and should contribute new insight into the interactions of bacterial CYP106A with steroid substrates, providing baseline data for studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.244-255
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• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.60-61
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• 2004

Metabolic engineering is now a well established discipline, used extensively to determine and execute rational strategies of strain development to improve the performance of micro-organisms employed in industrial fermentations. The basic principle of this approach is that performance of the microbial catalyst should be adequately characterised metabolically so as to clearlyidentify the metabolic network constraints, thereby identifying the most probable targets for genetic engineering and the extent to which improvements can be realistically achieved. In order to harness correctly this potential, it is clear that the physiological analysis of each strain studied needs to be undertaken under conditions as close as possible to the physico-chemical environment in which the strain evolves within the full-scale process. Furthermore, this analysis needs to be undertaken throughoutthe entire fermentation so as to take into account the changing environment in an essentially dynamic situation in which metabolic stress is accentuated by the microbial activity itself, leading to increasingly important stress response at a metabolic level. All too often these industrial fermentation constraints are overlooked, leading to identification of targets whose validity within the industrial context is at best limited. Thus the conceptual error is linked to experimental design rather than inadequate methodology. New tools are becoming available which open up new possibilities in metabolic engineering and the characterisation of complex metabolic networks. Traditionally metabolic analysis was targeted towards pre-identified genes and their corresponding enzymatic activities within pre-selected metabolic pathways. Those pathways not included at the onset were intrinsically removed from the network giving a fundamentally localised vision of pathway functionality. New tools from genome research extend this reductive approach so as to include the global characteristics of a given biological model which can now be seen as an integrated functional unit rather than a specific sub-group of biochemical reactions, thereby facilitating the resolution of complexnetworks whose exact composition cannot be estimated at the onset. This global overview of whole cell physiology enables new targets to be identified which would classically not have been suspected previously. Of course, as with all powerful analytical tools, post-genomic technology must be used carefully so as to avoid expensive errors. This is not always the case and the data obtained need to be examined carefully to avoid embarking on the study of artefacts due to poor understanding of cell biology. These basic developments and the underlying concepts will be illustrated with examples from the author's laboratory concerning the industrial production of commodity chemicals using a number of industrially important bacteria. The different levels of possibleinvestigation and the extent to which the data can be extrapolated will be highlighted together with the extent to which realistic yield targets can be attained. Genetic engineering strategies and the performance of the resulting strains will be examined within the context of the prevailing experimental conditions encountered in the industrial fermentor. Examples used will include the production of amino acids, vitamins and polysaccharides. In each case metabolic constraints can be identified and the extent to which performance can be enhanced predicted