• Microbiology and Biotechnology Letters
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• v.15 no.6
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• pp.441-445
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• 1987

The gene coding for alkaline amylase of alkalophilic Bacillus sp. AL-8 was cloned and expressed in Escherichia coli which was lack of amylase activity. For the cloning of the alkaline amylase gene, the chromosomal DNA and plasmid vector pBR322 were cleaved at the site of EcoRI and the gene was cloned. The selection of the transformants carrying the amylase gene was based on the their antibiotics resistance and amylase activity of the transformants. The recombinant plasmids pJW8 and pJW200 containing 5.8Kb and 3.0Kb EcoRI inserts respectively were proved to can the alkaline amylase gene. Alkaline amylase expressed in E. coli was characterized. The enzyme was proved to be stable at the range of alkaline pH.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.436-439
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• 1989

The chromosomal DNA fragments of thermophilic alkalophilic Bacillus sp, K-17, a potent xylanhydrolyzing bacterium, were ligated to a vector plasmid pBR322 and transformed into Escherichia coli HB101. The plasmid pAX278, isolated from a transformant forming yellow color on the LB agar plate containing 1 mM p-nitrophenyl- $\beta$-xylopyranoside, was found to enable the transformants to produce p-xylosidase. The 5.0 kilobase insert of pAX278 had single sites for EcoRI, PstI, XbaI, and PvuII, and 2 sites for BglII. Biotinylated pAX218 was hybridized to 0.9 kb as well as 5.0 kb fragment from Bacillus sp. K-17 DNA on nitrocellulose filter. pGX718 was constructed by inserting the 5.0 kb HindIII fragment of pGX278 at the HindIII site of pGR71, E. coli and B. subtilis shuttle vector. The enzymatic properties of $\beta$-xylosidase from E. coli HB101 carrying recombinant plasmid were the same those of $\beta$-xylosidase from Bacillus sp. K-17.

• Microbiology and Biotechnology Letters
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• v.30 no.3
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• pp.230-234
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• 2002

To describe characteristics of a hemolysin in mosquitocidal Bacillus thuringiensis subsp. gyangiensis strain 21-2, Escherichia coli HB101 was transformed with a gene encoding hemolysin in the strain 21-2. Transformant 47 con-tained 2.5 kb DNA was selected by ELISA, immunoblot and DNA electrophoresis. Transformant 47-5 was recon-structed after digestion of the 2.5 kb DNA with Hind m. Transformant 47-5 contained 1.8 kb DNA and expressed 23 kDa Protein which had mosquitocidal activity to Aedes aegypti. The 23 kDa Protein itself in vitro didn't show hemolytic activity on human erythrocytes, but the protein had the activity after proteinase K treatment.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.369-373
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• 1988

The gene for thermostable alpha-amylase from the thermostable bacterium Bacillus licheniformis has been cloned and expressed in Escherichia coli. The Alpha-amylase producing E. coli cells contained a 7.4 kb chimeric plasmid (pTA 322) which was composed of the vector pBR322 and a 3.1 kb EcoRI fragment of B. licheniformis DNA. The alpha-amylase from cloned fragement was shown to be indistlnguishable from that of B. licheniformis in the optimum temperature of 9$0^{\circ}C$, heat stability and the pH stability. The foreign gene was expressed efficiently in E. coli and stably maintained.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.349-354
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• 1985

A 4.7 kb Hind III fragment containing $\alpha$-amylase gene of Bacillus stearothermophilus IAM 11062 was cloned in Escherichia coil HB101, using plasmid pBR322 and runaway plasmid pSY343 as a vector. The cloned gene was stably maintained and expressed In E.coli. The constructed strain of E. coli have at least 3 times higher amylase activity than the donor strain, of B. stearothermophilus. About 75％ of the $\alpha$-amylase produced by the constructed strain of E. coli was localized in the periplasm and it was found that the enzymes can be released by an osmotic shock using EDTA. The enzymatic properties of L-amylase produced in E. coli were very similar to those produced by B. stearothermophilus in terms of optimum temperature, heat stability and molecular weight.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.87-92
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• 2005

Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.224-231
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• 1993

The nucleotide sequence of the xylanase gene (xynY) from alkalophilic Bacillus sp. YC-335 was determined and analyzed. An open reading frame of 1, 062 base pairs for xynY gene was observed and encoded for a protein of 354 amino acids with a molecular weight of 38, 915. S1 nuclease mapping showed that the transcription initiation sites of the xynY gene were different in Bacillus sp. YC-335 and Escherichia coli HB101 (pYS55). S1 mapping also showed that -10 region of the xynY gene recognized by RNA polymerases of E. coli and Bacillus sp. YC-335 were TACAGT and TATGAT , respectively. A ribosome binding site sequence with the free energy of -17.0 Kcal/mol was observed 9 base pairs upstream from the unusual initiation codon, TTG. The proposed signal sequence consisted of 27 amino acids, 2 of which were basic amino acid residues and 21 were hydrophobic amino acid residues. When the amino acid sequences of xylanases were compared, Bacillus sp. YC-335 xylanase showed more than 50% homology with xylanases from B. pumilus, B. subtilis, and B. circulans.

• Korean Journal of Veterinary Research
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• v.24 no.1
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• pp.40-49
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• 1984

To investigate the factors influencing the artifical transformation in Escherichia coli, E. coli C600 was transformed by pBR322 DNA with tetracycline and ampicillin resistant gene purified by CsCl-Etbr equilibrium density gradient centrifugation from E.coli HB 101. The influencing factors in the transformation such as concentration of calcium chloride, time of ice incubation, temperature and time of heat shock, time of gene expression, effects of plasmid DNA concentration and adding time were examined in these experiments. The results obtained were as follows; 1. The highest transformation frequency was observed in the treatments of 100 mM $CaCl_2$ before heat shock and the treatment of $CaCl_2$ was essential step in the process of E. coli transformation. 2. The highest transformation frequency was observed in the treatment of heat shock at $42^{\circ}C$ for 4 min. or $37^{\circ}C$ for 6 min., but the prolonged heat shock resulted a decreased transformation frequency. 3. Treatments of ice incubation at $0^{\circ}C$ for 45 min. before heat stocks or at $0^{\circ}C$ for 30min. after heat shock resulted an increased transformation frequency. 4. There was a linear relationship between DNA concentration and transformation frequency at the concentration of $8{\times}10^3$ recipient cells. The highest transformation frequency reached in carte of 7 mcg of donor DNA, but above 1 mcg of DNA concentration, transformation frequency was not remarkably increased. Addition of donor DNA just after the treatment of $CaCl_2$ was the best. 5. The best condition of gene expression at $37^{\circ}C$ were 40min. for TC-resistant gene and 100min. for AP-resistant gene. TC-resistant gene was higher in the transformation frequency and faster in the gene expression time than AP-resistant gene. In these results, the best conditions for the transformation of E. coli C 600 with pBR322 DNA were: treatment with 100mM $CaCl_2$, ice incubation at $0^{\circ}C$ for 45 min, heat shock at $42^{\circ}C$ for 4 min., 30 min. of ice incubation and incubation at $37^{\circ}C$ for 100min. for gene expression in that order.

• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.136-142
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• 1992

Bacillus stearothemophilus isolated from soil was identified to express multiple extracellular xylanases. Two HindIII restriction fragments of 5.4 and 6.4 kb from B. stearothermophilus genomic DNA were cloned into pBR322 to obtain recombinant plasmids pMG0l and pMG02, respectively, which enabled E. coli HBlOl cells to produce $\beta$-D-xylosidase activity. By subcloning into pUC18 and Southern blotting, the loci of the $\beta$-D-xyiosidase genes were elucidated to be on non-homologous DNA fragments of 2.2 kb from pMGOl(pMG1) and 1.0 kb from pMG02(pMG2), respectively. The two enzymes produced in E. coli cleaved xylobiose, xylotriose, xylotetrose and xylotetrose to produce xylose as a major end product. The gene on pMG1, distinct from that on pMG2 was observed to encode a bifunctional protein that displayed both P-D-xylosidase (EC.3.2.1.37) and a-L-arabinofuranosidase activities (EC.3.2.1.55).

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.414-419
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• 1996

The xylnX gene encoding a xylanase from Clostridium thermocellum ATCC27405 was cloned in the plasmid pJH27, an E. coli-Bacillus shuttle vector and the resultant recombinant plasmid, pJX18 was transformed into E. coli HB101. The overexpressed xylanase was found to be secreted into the periplasmic space of the recombinant E. coli cells. The crude enzyme was obtained by treating the E. coli cells with lysozyme, and purified by DEAE-Sepharose column chromatography. Molecular wieght of the xylanase was estimated to be 53 kDa by gel filtration. The pI value was determined to be pH 8.8. The N-terminal sequence of the enzyme protein was Asp-Asp-Asn-Asn-Ala-Asn-Leu-Val-Ser-Asn which was considered to be the sequence of that of the mature form protein. The Km value of the enzyme for oat spelt xylan was calculated to be 2.63 mg/ml and the Vmax value was $0.47 {\mu}mole/min$. The xylanase had a pH optimum for its activity at pH 5.4 and a temperature optimum at $60^{\circ}C$. The enzyme hydrolyzed xylan into xylooligosaccharides which were composed mainly of xylobiose (40%) and xyloltriose (12%) after 5 hour reaction. This result indicates that the xylanase from C. thermocellum ATCC27405 is an endo-acting enzyme.