• Title/Summary/Keyword: bioprocess

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Microcontact Printing of Biotin for Selective Immobilization of Streptavidin-fused Proteins and SPR Analysis

  • Lee, Sang-Yup;Park, Jong-Pil;Lee, Seok-Jae;Park, Tae-Jung;Lee, Kyung-Bok;Park, Insung S.;Kim, Min-Gon;Chung, Bong-Hyun
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.9 no.2
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    • pp.137-142
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    • 2004
  • In this study, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (CP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene of Streptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of $\mu$CP and cSA-fused proteins. is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications. one such being the use in protein-protein assays.

Expression and Characterization of a Novel Deoxyribose 5-Phosphate Aldolase from Paenibacillus sp. EA001

  • Kim, Yong-Mo;Choi, Nack-Shick;Kim, Yong-Ook;Son, Dong-Ho;Chang, Young-Hyo;Song, Jae-Jun;Kim, Joong-Su
    • Journal of Microbiology and Biotechnology
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    • v.20 no.6
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    • pp.995-1000
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    • 2010
  • A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding a protein of 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79% identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into an expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified using Ni-NTA affinity chromatography and then characterized. The optimum temperature and pH of the enzyme were $50^{\circ}C$ and 6.0, respectively. The specific activity for the substrate deoxyribose 5-phosphate (DR5P) was $62\;{\mu}mol/min/mg$. The $K_m$ value for DR5P was determined to be 145 mM with the $k_{cat}$ value of $3.2{\times}10^2/s$ from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).

Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource

  • Ko, Kyong-Cheol;Lee, Binna;Cheong, Dae-Eun;Han, Yunjon;Choi, Jong Hyun;Song, Jae Jun
    • Journal of Microbiology and Biotechnology
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    • v.25 no.11
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    • pp.1835-1841
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    • 2015
  • A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

A Dual Selection Marker Transformation System Using Agrobacterium tumefaciens for the Industrial Aspergillus oryzae 3.042

  • Sun, Yunlong;Niu, Yali;He, Bin;Ma, Long;Li, Ganghua;Tran, Van-Tuan;Zeng, Bin;Hu, Zhihong
    • Journal of Microbiology and Biotechnology
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    • v.29 no.2
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    • pp.230-234
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    • 2019
  • Currently, the genetic modification of Aspergillus oryzae is mainly dependent on protoplast-mediated transformation (PMT). In this study, we established a dual selection marker system in an industrial A. oryzae 3.042 strain by using Agrobacterium tumefaciens-mediated transformation (ATMT). We first constructed a uridine/uracil auxotrophic A. oryzae 3.042 strain and a pyrithiamine (PT)-resistance binary vector. Then, we established the ATMT system by using uridine/uracil auxotrophy and PT-resistance genes as selection markers. Finally, a dual selection marker ATMT system was developed. This study demonstrates a useful dual selection marker transformation system for genetic manipulations of A. oryzae 3.042.

Lipid Production by a $CO_2$-Tolerant Green Microalga, Chlorella sp. MRA-1

  • Zheng, Yanlin;Yuan, Cheng;Liu, Junhan;Hu, Guangrong;Li, Fuli
    • Journal of Microbiology and Biotechnology
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    • v.24 no.5
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    • pp.683-689
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    • 2014
  • Since $CO_2$ concentrations in industrial flue gases are usually 10%-20%, one of the prerequisites for efficient $CO_2$ removal by algae is the level of tolerance of microalgal species to exposure to high concentrations of $CO_2$. A newly isolated microalgal strain, Chlorella sp. MRA-1, could retain growth with high concentrations of $CO_2$ up to 15%. The highest lipid productivity for Chlorella sp. MRA-1 was 0.118 g/l/day with a 5% $CO_2$ concentration. Octadecenoic acid and hexadecanoic acid, the main components of biodiesel, accounted for 70% of the total fatty acids. A lipid content of 52% of dry cell weight was achieved with limited amounts of nitrogen. Chlorella sp. MRA-1 seems to be an ideal candidate for biodiesel production when cultured with high concentrations of $CO_2$.