• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.901-910
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• 2006

Poly(3-hydroxybutyrate) [P(3HB)] is a microbial polyester intracellularly accumulated as distinct granules in numerous microorganisms as an energy and carbon storage material. Recombinant Escherichia coli harboring the heterologous P(3HB) biosynthesis genes accumulates large amounts of P(3HB) granules, yet the granule-associated proteins have not been identified. Therefore, this study reports on an analysis of the P(3HB) granule-associated proteome in recombinant E. coli. Fiye proteins out of 7 spots identified were found to be involved in functions of translation, heat-stress responses, and P(3HB) biosynthesis. Two of the major granule-associated proteins, IbpA/B, which are already known to bind to recombinant proteins forming inclusion bodies in E. coli, were further analyzed. Immunoblotting and immunoelectron microscopic studies with IbpA/B antibodies clearly demonstrated the binding and localization of IbpA/B to P(3HB) granules. IbpA/B seemed to play an important role in recombinant E. coli producing P(3HB) by stabilizing the interface between the hydrophobic P(3HB) granules and the hydrophilic cytoplasm. Thus, IbpA/B were found to act like phasins in recombinant E. coli, as they are the major proteins bound to the P(3HB) granules, affect the morphology of the granules, and reduce the amount of cytosolic proteins bound to the P(3HB) granules.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.280-288
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• 1992

Molecular cloning of metalloprotease gene from Serratia marcescens ATCC 21074 into Escherichia coli JM109 was carried out. Chromosomal DNA of S. marcescens was completely digested with Hind111 and southern hybridization with a synthetic oligonucleotide probe revealed that a 50 KD metalloprotease gene was contained in 4.0 Kb chromosomal DNA fragment, 4.0 Kb chromosomal DNA fragments eluted from agarose gel were ligated with pUC19 and transformed into E. coli JM109. Nine positive clones were obtained from about $1\times 10^3$ transformants by colony hybridization. Their recombinant plasmids, pSPl and pSP2 have same chromosomal DNA fragments in pUC19 in opposite-orientations. When cloned metalloprotease gene was expressed in E. coli, about 52 KD precursor protein of metalloprotease was detected by western blot analysis from E. coli harboring a recombinant plasmid pSP2. Plasmid pSP2 showed no protease activities in E. coli but overproduced the active metalloprotease in S. rnarcescens ATCC 27117.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.206-215
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• 2005

Polyhydroxyalkanoates (PHAs) are homo or hetero polyesters of (R)-hydroxyalkanoates accumulated in various microorganisms under growth-limiting condition in the presence of excess carbon source. They have been suggested as biodegradable substitutes for chemically synthesized polymers. Recombinant Escherichia coli is one of the promising host strains for the economical production of PHAs, and has been extensively investigated for the process development. The heterologous PHA biosynthetic pathways have been established through the metabolic engineering and inherent metabolic pathways of E. coli have been redirected to supply PHA precursors. Fermentation strategies for cultivating these recombinant E. coli strains have also been developed for the efficient production of PHAs. Nowadays, short-chain-length (SCL) PHAs are being re-invited due to its improved mechanical properties and possible applications in the biomedical area. In this article, recent advances in the development of metabolically engineered E. coli strains for the enhanced production of SCL-PHAs are reviewed. Also, medical applications of SCL-PHAs are discussed.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.841-843
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• 2002

Pfu DNA polymerase from Pyrococcus furiosus was expressed in the E. coli periplasm, and the fully active polymerase was partially purified by applying osmotic shock, ammonium sulfate precipitation, and heat treatment. This method represents a new way of expressing and purifying functional Pfu DNA polymerase without the use of chromatography.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.607-613
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• 1994

The Bacillus stearothermophilus arfI gene encoding a-arabinofuranosidase was isolated from the genomic library, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101. The recombinant E. coli was selected from approximately 10,000 transformants screened by making use of its ability to produce a yellow pigment around the colony on the selective medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf), a chromogenic substrate. The functional clone was found to harbor a recombinant plasmid, pKMG11 with an insertion of about 5 kb derived from the B. stearothermophilus chromosomal DNA. Identity of the arfI gene on the insert DNA was confirmed by a zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate. The $\alpha$-arabinofuranosidase from the recombinant E. coli strain showed very high substrate specificity; the enzyme displayed high activity only with pNPAf among many other p- or $o$-nitrophenyl derivatives of several sugars, and acted only on arabinoxylan among various natural arabinose containing polysaccharides tested.

• Proceedings of the Microbiological Society of Korea Conference
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• 1991.04a
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• pp.237-242
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• 1991

In order to increase the production of glutathione by maximizing the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were cloned. A gshI gene was cloned onto pBR322 plasmid as 3.6Kb PstI DNA fragment from E. coli K-12 chromosomal DNA. Also gshII gene was cloned onto pUC13 plasmid as 2.2Kb PstI-BamHI DNA fragment. In order to improve the glutathione producing activity more efficiently, various recombinant plasmids containing tandem repeated gshI genes or both genes in various copy number onto the same vector were constructed. E. coli cells harboring pGH501 plasmid (pUC8-gshI$\cdot$I$\cdot$II) showed the highest glutathione synthesizing activity. The conditions for glutathione production with an ATP-generating system such as acetate kinase reaction of E. coli cells or glycolytic pathway of yeast cells were examined using the E. coli cells harboring the pGH501 plasmid. When the acetate kinase reaction of E. coli cells was used as an ATP generating system, 20mM of L-csteine was converted into glutathione with a yield of $100\%$.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.35-40
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• 1992

The influence of the nature of plasmids on fermentation parameters such as cell growth, cell viability, plasmid stability, and product formation has been investigated using E. coli M5248 and its recombinant derivatives M5248 [pBR322], M5248[pAS1], and M5248[pNKM21]. At a low temperature ($30^\circ{C}$), the cell growth, cell viability, and protein synthesis of the recombinants were nearly identical to those of the host cell. However, at high temperature ($42^\circ{C}$), in which transcription from the P_L$ promoter is derepressed, the recombinant cells showed decreased stability along with lower growth rates and cell viability. The ratio of total protein to cell mass was in the order of E. coli M5248>M5248[pBR322]>M5248[pAS1]>M5248[pNKM21]. It was found that transcription from the $P_L$ promoter adversely affect the plasmid maintenance and host cell metabolism even in the absence of the cloned-gene expression. Furthermore, profiles of ${\beta}$ activity were shown to vary with recombinant strains. E coli M5248[pBR322] showed highest ${\beta}-lactamase$ activity at $30^\circ{C}$, while at $42^\circ{C}\;{\beta}-lactamase$ activity was significantly reduced irrespective of the strains. The effect of the plasmid properties on plasmid-encoded gene expression has been further examined based on the relationship between $\{beta}-lactamase$ activity and plasmid-harboring cell numbers.

• Korean Journal of Microbiology
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• v.26 no.1
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• pp.6-12
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• 1988

Cellobiase ($\beta$-glucosidase) is an enzyme of the cellulase system in cellulolytic microor-ganisms. The chromosomal DNA fragment which include cellobiase gene of Cellulomonas biazotea was cloned in Eschericia coli via plasmid pBR 322 vector. Restriction enzyme Sal I was used to obtain adequate size of fragments from C. biazotea. chromosomal DNA. The transformant of E. coli HB101 with recombinant plasmid pBG101 showed cellobiase activity, which is not ordinary in E. coli HB101. The enzyme activity of the transformant was as of 20% lower than that of C. biazotea.

• Journal of Industrial Technology
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• v.36
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• pp.33-37
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• 2016

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli. Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

• Journal of Applied Biological Chemistry
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• v.57 no.2
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• pp.137-140
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• 2014

A gene encoding thermostable pectinase (TmPec) was isolated from hyperthermophilic microorganism, Thermotoga maritima. The open reading frame (ORF) of TmPec gene is 1,104 bp long and encodes 367 amino acid residues with a molecular weight of 40,605 Da. To analyze the enzymatic activity and biochemical properties, the ORF of TmPec gene excluding putative signal sequence of 27 amino acids was introduced into the E. coli expression vector, pRSET-B, and overexpressed in E. coli BL21. Protein concentration of purified recombinant TmPec was 1.1 mg/mL with specific activity of 56 U/mg protein on pectin. The recombinant TmPec showed the highest activity at around $85-95^{\circ}C$, and at around pH 6.5. It was stable at temperature below $85^{\circ}C$. In the presence of $Ca^{2+}$, the activity of recombinant TmPec was increased to 146.3% of normal level. In contrast, $Ba^{2+}$ and Mn2+ showed strong inhibition to the recombinant TmPec.