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Hyeoncheol Francis Son,Kyung-Jin Kim 한국구조생물학회 2017 Biodesign Vol.5 No.2
meso-Diaminopimelic acid decarboxylase from Corynebacterium glutamicum (CgDAPDC) is the key enzyme for the production of L -lysine and it catalyzes meso-DAP to produce the final product, L -lysine. The CgDAPDC was overexpressed and purified to homogeneity by Ni-NTA affinity and size-exclusion chromatography. The CgDAPDC protein was crystallized using sitting-drop vapor-diffusion method in the presence of 0.8 M sodium citrate tribasic and 0.1 M sodium cacodylate, pH 6.5 at 293 K. X-ray diffraction data were collected to a maximum resolution of 2.4 Å. The crystal belonged to space group P2 1 2 1 2, with unit cell parameters a = 114.54 Å, b = 91.702 Å, c = 95.161, α = β = γ = 90°. With one molecules per asymmetric unit, the crystal volume per unit protein mass was 2.64 Å 3 Da -1 , which correspond to a solvent content of approximately 53.37%.
Crystal Structure and Biochemical Characterization of Xylose Isomerase from Piromyces sp. E2
( Hyeoncheol Francis Son ),( Sun-mi Lee ),( Kyung-jin Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.4
Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a D-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize D-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino- acid sequence comparison with other reported XIs and relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XIs. Moreover kinetic analysis of PsXI was also performed using Mn<sup>2+</sup>, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance D-xylose utilization for biofuel production.
( Hyeoncheol Francis Son ),( Kyung-jin Kim ) 한국미생물 · 생명공학회 2022 Journal of microbiology and biotechnology Vol.32 No.2
3-Hydroxypropionic acid (3HP) is a platform chemical and can be converted into other valuable C3- based chemicals. Because a large amount of glycerol is produced as a by-product in the biodiesel industry, glycerol is an attractive carbon source in the biological production of 3HP. Although eight 3HP-producing aldehyde dehydrogenases (ALDHs) have been reported so far, the low conversion rate from 3-hydroxypropionaldehyde (3HPA) to 3HP using these enzymes is still a bottleneck for the production of 3HP. In this study, we elucidated the substrate binding modes of the eight 3HPproducing ALDHs through bioinformatic and structural analysis of these enzymes and selected protein engineering targets for developing enzymes with enhanced enzymatic activity against 3HPA. Among ten AbKGSADH variants we tested, three variants with replacement at the Arg281 site of AbKGSADH showed enhanced enzymatic activities. In particular, the AbKGSADHR281Y variant exhibited improved catalytic efficiency by 2.5-fold compared with the wild type.
Seo, Hogyun,Kim, Seongmin,Son, Hyeoncheol Francis,Sagong, Hye-Young,Joo, Seongjoon,Kim, Kyung-Jin Elsevier 2019 Biochemical and biophysical research communication Vol.508 No.1
<P><B>Abstract</B></P> <P>Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from <I>Ideonella sakaiensis</I> 201-F6 (<I>Is</I>PETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level <I>Is</I>PETase expression and purification procedures in industry. To overcome this difficulty, the expression of <I>Is</I>PETase can be improved by using a secretion system. This is the first report on the production of an extracellular <I>Is</I>PETase, active against PET film, using Sec-dependent translocation signal peptides from <I>E. coli</I>. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from <I>E. coli</I> secretory proteins into <I>Is</I>PETase, and successfully produced the extracellular enzyme using pET22b-SP<SUB>MalE</SUB>:<I>IsPETase</I> and pET22b-SP<SUB>LamB</SUB>:<I>IsPETase</I> expression systems. We also confirmed that the secreted <I>Is</I>PETase has PET-degradation activity. The work will be used for development of a new <I>E. coli</I> strain capable of degrading and assimilating PET in its culture medium.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PETase from <I>Ideonella sakaiensis</I> (<I>Is</I>PETase) was successfully produced using the protein secretory expression system. </LI> <LI> Extracellular production of <I>Is</I>PETase is achieved by sec-dependent secretion system. </LI> <LI> The extracellularly produced <I>Is</I>PETase shows a PET degradation activity. </LI> </UL> </P>