• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.550-558
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• 2021

Lactobacillus kefiranofaciens contains two types of β-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36n-lacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-β-galactopyranoside (ONPG) showed that the β-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two β-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, the maximal growth rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentation assay for practical purpose. The results displayed that 37℃ and 20-40 U/ml nisin were the optimal fermentation conditions for the growth of recombinant β-galactosidase strains. Altogether the food-grade Expression system of recombinant β-galactosidase was feasible for applications in the food and dairy industry.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.191-194
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• 2003

The phospholipase C (PLC) hydrolyzes the polar head groups such as phosphocholine or phosphoethanolamine residues esterified at the sn-3 position of phospholipids. Pichia pastoris can utilize methanol as a carbon source and produce recombinant proteins under the control of the strong, tightly-regulated alcohol oxidase (AOX) promoter. In this study, we developed recombinant P. pastoris system for PLC expression and analyzed PLC activity.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1688-1694
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• 2007

A gene encoding the mannanase of Bacillus subtilis WL-3, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and the nucleotide sequence of a 2.7-kb DNA fragment containing the mannanase gene was subsequently determined. The mannanase gene, designated manA, consisted of 1,080 nucleotides encoding a polypeptide of 360 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to glycosyl hydrolase family 26. The manA gene was strongly expressed in B. subtilis 168 by cloning the gene downstream of a strong B. subtilis promoter of plasmid $pJ27{\Delta}88U$. In flask cultures, the production of mannanase by recombinant B. subtilis 168 reached maximum levels of 300 units/ml and 450 units/ml in LB medium and LB medium containing 0.3% locust bean gum, respectively. Based on the zymogram ofthe mannanase, it was found that the mannanase produced by recombinant B. subtilis could be maintained stably without proteolytic degradation during the culture time.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.405-407
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• 2009

A Bacillus subtilis mannanase gene was subcloned into an Escherichia coli- Corynebacterium lactofermentum shuttle vector pHE83, and the resultant plasmid pHE83M was transferred into an endogenous plasmid-free strain of C. lactofermentum. Mannanase produced by the recombinant C. lactofermentum (pHE83M) was secreted extracellulary at the level of 86%, and the remaining activity of mannanase was detected in the cell-free extract. The maximum mannanase productivity of the recombinant strain reached 8.1 unit/mL in the culture filtrate of LB medium. According to the zymogram of mannanase on SDS-PAGE, it was found that the mannanase produced by the recombinant C. lactofermentum could be maintained stably with a migration identical to the mannanase produced by the parental strain, B. subtilis WL-3.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.916-920
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• 2002

This report describes a high-level expression of human alpha-2a interferon ($IFN{\alpha}-2a$) in Escherichia coli and its pilot scale purification by using a monoclonal antibody-independent chromatographic procedure that is based on anion-exchange, cation-exchange, hydrophobic interaction, and gel filtration. The recombinant E. coli produced much more $IFN{\alpha}-2a$ in a soluble form, when cultivated at low temperatures than at high-temperature fermentation. However, if the bacterial growth was taken into consideration, fermentation at $30^{\circ}C$ seemed optimal for the interferon production. By using our new protocol, we recovered approximately 160 mg of $IFN{\alpha}-2a$ with a specific activity of $3.59{\times}10^8$ IU/mg from 201 of the broth. The gel permeation chromatographic and SDS-PAGE indicated that the interferon preparation was purified to homogeneity and was of the correctly folded fast-migrating monomer.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1709-1713
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• 2015

Sepiapterin is a precursor for the synthesis of tetrahydrobiopterin (BH4), which is a wellknown cofactor for aromatic amino acid hydroxylation and nitric oxide synthesis in higher mammals. In this study, a recombinant Escherichia coli BL21(DE3) strain harboring cyanobacterial guanosine 5’-triphosphate cyclohydrolase 1 (GCH1) and human 6-pyruvoyltetrahydropterin synthase (PTPS) genes was constructed to produce sepiapterin. The optimum conditions for T₇ promoter–driven expression of GCH1 and PTPS were 30℃ and 0.1 mM isopropyl-β-D-thioglucopyranoside (IPTG). The maximum sepiapterin concentration of 88.1 ± 2.4 mg/l was obtained in a batch cultivation of the recombinant E. coli, corresponding to an 18-fold increase in sepiapterin production compared with the control condition (37℃ and 1 mM IPTG).

• Journal of Industrial Technology
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• v.37 no.1
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• pp.16-20
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• 2017

A new putative laccase gene (soncotA) which show 78% homology with that from Bacillus licheniformis (liccotA) was isolated from draft genome sequence of Bacillus sonorensis KCTC 13918. A 1,545 bp of PCR product corresponding 514 amino acids was cloned into NdeI-NotI site of pET21c and expressed as soluble form in E. coli. About 59 kDa size of recombinant laccase was purified into homogenity by Ni-NTA column and laccase activity was confirmed by zymography. The enzymatic properties of recombinant laccase were characterized. The specific activity of B. sonorensis laccase was 0.033 fold lower than that of Bacillus licheniformis laccase. The finding of new laccase gene broadened the enzymatic diversity of Bacillus species laccases.

• Bulletin of the Korean Chemical Society
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• v.32 no.12
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• pp.4337-4340
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• 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.174-182
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• 1992

The hemagglutinin-esterase glycoprotein (HE) gene of bovine coronavirus, coupled with a simian virus 40 early promoter and polyadenylation signal, was inserted into a human adenovirus transfer vector. The transfer vector was used to co-transfect 293 cells along with adenovirus genomic DNA. The hemagglutinin-esterase transcription unit was rescued into the adenovirus genome by homologous in vivo DNA recombination between the vector plasmid DNA and the adenovirus genomic DNA, and a recombinant adenovirus was isolated by several rounds of plaque assays. Thus the recombinant adenovirus carries the hemagglutinin-esterase gene in the early transcription region 3 (E3) of the adenovirus genome in the parallel orientation to the E3 transcription. The recombinant adenovirus synthesized the HE polypeptide in HeLa cells as demonstrated by immunoprecipitation with anti-coronavirus rabbit antisera. The recombinant HE polypeptide could be labelled by $[^3H]$glucosamine, demonstrating that the recombinant HE was glycosylated. Cells expressing the HE polypeptide exhibited hemadsorption activity when incubated with mouse erythrocytes. The HE was transported to the plasma membrane as shown by the cell surface immunofluorescence, indicating that the recombinant HE polypeptide retained its biological activities. Potential for the use of infectious recombinant adenovirus as a live virus-vectored vaccine candidate for bovine coronavirus disease is discussed.

• The Microorganisms and Industry
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• v.19 no.4
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• pp.14-26
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• 1993

Optimizing protein production in recombinant E. coli strains involves manipulation of genetic and environmental factors. In designing a production system, attention must be paid to gene expression efficiency, culture conditions and bioreactor configuration. Although not much emphasis was given to the physiology of host strains in this review, an understanding of the relationship between the physiology of host cell growth and the overproduction of a cloned gene protein is of primary importance to the improvement of the recombinant fermentation processes. Sometimes it is desirable to make use of gene fusion systems, e.g. protein A, polypeptide, gutathione-S-transferase, or pneumococcal murein hydrolase fusion, to facilitate protein purification.