• Journal of Microbiology
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• v.33 no.2
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• pp.165-170
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• 1995

The 356 amino acid long lambda integrase protein of bacteriophage .lambda. constains two autonomous DNA binding domains with distinct sequence specificities. The amino terminal domain of integrase is implicated to bind to the arm-type sequences and the carboxyl domain interacts with the coretype sequencess. As a first step to understand the molecular mechanism of the integrase-DNA interaction at the arm-type site, the int(am)94 gene carrying an amber mutation at the 94th codon of the int was cloned under the control of the P$\_$tac/ promoter and the lacI$\_$q/ gene. The Int(am)94 mutant protein of amino terminal 93 amino acid residues can be produced at high level from a suppressor free strain harboring the plasmid pInt(am)94. The arm-type binding activity of Int(am)94 were measured in vivo and in vitro. A comparison of the arm-type binding properties of the wild-type integrase and the truncated Int(am)94 mutant indicated that the truncated fragment containing 93 amino acid residues carry all the determinants for DNA binding at the arm-type sites.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.625-630
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• 1998

Using a modified yeast secretory expression vector, $\alpha$-amylase of Schwanniomyces occidentalis was produced from Saccharomyces cerevisiae. The expression vector contains the a-amylase gene (AMY) harboring its own promoter without the regulatory region and the adenine base at the -3 position from the ATG start codon, its own signal sequence, CYC1 transcription terminator, and SV40 enhancer. The expressed $\alpha$-amylase activity from cells carrying the plasmid was approximately 26 times higher than that from the cells harboring an unmodified plasmid. When Saccharomyces diastaticus was transformed with this modified vector, a 2.5 times higher level of amylolytic activity than that from Sch. occidentalis was observed.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.223-228
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• 1997

A DNA fragment containing the gene for cell wall hydrolase of alkalophilic Bacillus subtilis BL-29 was cloned into E. coli JM109 using pUC18 as a vector. A recombinant plasmid, designated pCWL45B, was contained in the fragment originating from the alkalophilic B. subtilis BL-29 chromosomal DNA by Southern hybridization analysis. The nucleotide sequence of a 1.6-kb HindIII fragment containing a cell wall hydrolase-encoding gene was determined. The nucleotide sequence revealed an open reading frame (ORF) of 900 bp with a concensus ribosome-binding site located 6 nucleotide upstream from the ATG start codon. The primary amino acid sequence deduced from the nucleotide sequence revealed a putative protein of 299 amino acid residues with an M.W. of 33, 206. Based on comparison of the amino acid sequence of the ORF with amino acid sequences in the GenBank data, it showed significant homology to the sequence of cell wall amidase of the PBSX bacteriophage of B. subtilis.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.45-49
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• 1991

The complete nucleotide sequence of alkalophilic Bacillus sp. K-17 $\beta$-xylosidase gene and its flanking regions were established. A 1263-bp of an open reading frame for $\beta$-xylosidase was observed. The molecular weight (50, 521 dalton), deduced from the nucleotide sequence of $\beta$-xylosidase gene, agreed with the result obtained by SDS-polyacrylamide gel electrophoresis of the purified enzyme (51, 000 dalton). The Shine-Dalgarno sequence, 5'-GAGGAGG-3', was found 8 bp upstream of the initiation codon ATG. The -10 sequence (TAAAAT) in the promoter region for $\beta$-xylosidase gene was similar to the consensus sequence for Bacillus subtilis RNA polymerase, whereas the -35 sequence (TCGATCA) different from all the known -35 regions in the promoter for Bacillus subtilis RNA polymerase.

• Korean Journal of Environmental Biology
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• v.21 no.1
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• pp.56-59
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• 2003

The Salmonella typhimurium cdd gene encoding cytidine deaminase (cyti-dine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5.) was isolated through shotgun clon-ing by complementation of the E. coli odd mutation. By subsequent deletion and sub-cloning from the original 3.7 Kb of EcoRI insert (pSAMI), the precise region of the cdd structural gene is located around the BglII site in the middle part of 1.7 Kb of NruI/PvuI segment. The 1.7 Kb containing odd gene wag subcloned to the pUC18 vector and the nucleotide sequence of the cdd gene was determined. When the putative ribosorne-binding site (Shine-Dalgarno sequence) and initiation codon were predicted to be GAGG at the position 459 and ATG at the position 470, respectively, there was an open reading frame of 885 nucleotides, encoding an 294 amino acid protein. The cdd gene expression in E. coli JF611/pSAMI was amplified about 50 fold compared to that of the wild type. The cdd gene expression was maintained in the stationary phase after rea-ching the peak in the late logarithmic phase.

• Journal of Microbiology and Biotechnology
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• v.13 no.3
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• pp.344-347
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• 2003

Unnatural amino acid S-(2-nitrobenzyl)cysteine was incorporated into human erythropoietin by using a programmed suppression of nonsense codon in a cell-free protein synthesis system. Several controlling factors affecting the operational efficiency of the suppression were investigated and optimized. The amount of suppressor tRNA and the concentration of $Mg^2+$ were crucial not only for the efficiency but also for the control of the exact suppression. In addition, some general optimization factor are reported in order to improve the efficiency in an unnatural amino acid mutagenesis.

• Journal of Applied Biological Chemistry
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• v.48 no.4
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• pp.218-221
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• 2005

Nucleotide sequence of Brevibacterium flavum ptsG gene capable of complementing Escherichia coli ZSC113 mutations defective to glucose permease activity of phosphotransferase system was completely determined, and the gene product was compared with other glucose-specific enzyme II ($EII^{Glc}$). A ptsG gene of B. flavum consisted of open reading frame of 2,025 nucleotides putatively encoding polypeptide of 675 amino acid residues and TAA stop codon. Deduced amino acid sequence of B. flavum ($EII^{Glc}$) had high homology with ($EIIs^{Glc}$) of Corynebacterium glutamicum, C. efficiens, and B. lactofermentum. Arrangement of structural domains, IIBCA, of B. flanum ($EII^{Glc}$) protein was identical to that of EIIs belonging to glucose-phosphotransferase system.

• Microbiology and Biotechnology Letters
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• v.30 no.2
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• pp.105-110
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• 2002

The sequence of 3,221 nucleotides immediately adjacent to rpsA gene encoding 30S ribosomal protein S1 of Brevibacterium ammoniagenes was determined. A putative open reading frame (ORF) of 2,670 nucleotides for a polypeptide of 889 amino acid residues and a TAG stop codon was found, which is located at a distance of 723 nucleotides upstream from rpsA gene with same translational direction. The deduced amino acid sequence of the ORF was found to be highly homologous to the DNA polymerase I of Streptomyces griseus (75.48％), Rhodococcus sp. ATCC 15963 (56.69％), Mycobacterium tuberculosis (55.46％) and Mycobacterium leprae (53.99％). It was suggested that the predicted product of the ORF is a DNA polymerase I with three functional domains. Two domains of 5 → 3 exonuclease and DNA polymerase are highly conserved with other DNA polymerase I, but 3 → 5 exonuclease domain is less conserved.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.138-144
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• 1995

The cDNA, encoding human lysozyme (HLY) which was isolated from a human placenta cDNA library, has been well characterized (Yoshimura et al., 1988). Based on the communication, we have prepared an artificial HLY gene from chemically synthesized 38-oligomer with high codon usage in Saccharomyces cerevisiae. For directing the synthesis and secretion of HLY in S. cerevisiae, an expression vector, pHKl was constructed by inserting the HLY gene, containing a synthetic HLY secretion signal sequence, between the yeast GAP promoter and PH05 terminator. From a lysoplate assay, we have confirmed an yeast transformant harboring a pHK1 which makes a clearing zone on the overlayed Micrococcus luteus. This result means a chemically synthesized HLY gene which was normally expressed and secreted in yeast.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1202-1207
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• 2012

A gene, ClCDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.