• The Korean Journal of Mycology
• /
• v.21 no.4
• /
• pp.279-284
• /
• 1993

The metE gene coding for $N^{5}-methyl-H_{4}-folate;$ homocysteine methyltransferase from the basidiomycete Ganoderma lucidum was cloned by complementation of methionine-requiring mutants of E. coli. The size of a inserted DNA was about 1.54 kb and had 5 restriction enzyme sites. A physical map was constructed. Southern blot analysis confirmed the presence of a transforming DNA in the genome of Ganoderma lucidum. indicating the presence of a single copy.

• Korean Journal of Microbiology
• /
• v.40 no.2
• /
• pp.73-80
• /
• 2004

Coenzyme Q is a quinone derivative that acts as a lipid electron carrier in the respiratory chain located at mito-chondrial inner membrane in eucaryotes or plasma membrane in procaryotes and also functions as antioxidant. A putative Neurospora crassa coq-4 gene was cloned and functionally expressed in Saccharomyces cerevisiae coq4 mutant. Complemented S. cerevisaie mutant strain was able to produce coenzyme $Q_{6}$ and showed a normal growth rate. They also showed less sensitivities to polyunsaturated fatty acids such as linoleic acid or linolenic acid. The predicted sequence of N. crassa COQ4 is consisted of 347 amino acids with a molecular mass of 39.7 kDa and showed 35% identity and 52% similarity with that of S. cerevisiae.

• Microbiology and Biotechnology Letters
• /
• v.16 no.4
• /
• pp.316-319
• /
• 1988

Pectate Lyase (PL) was cloned from alkali-tolerant Bacillus sp. YA-14 into Escherichia coli MB1000 by inserting HindIII-generated DNA fragment into the HindIII site of pBR322 and then screening recombinant transformant for the ability to hydrolyze sodium polypectate on agar plate, The recombinant plasmid, called pYPC29, was isolated, and the size of the cloned HindIII fragment was found to be 1.6 kb. The PL gene was stablely maintained and expressed efficiently in Escherichia coli. The Pt accumulated largely in the periplasmic space of Escherichia coli clones.

• Korean Journal of Microbiology
• /
• v.35 no.1
• /
• pp.18-24
• /
• 1999

Kinesin has been discovered in Saccharomyces cerevisiae, Aspergillus nidulans, and Drosophila melanogaster and it has major roles in the movemenl of chromosomes and separation of spindle poles. In this study, a gene encoding kinesin heavy chain in Schizosaccharo~n)~ces pombe was cloned by using the polymerase chain reaction with degenerated primcrs corresponding to highly conserved regions of the kinesin heavy chain motor domain. The kinesin gene in S pombe contains an open reading frame of 2496 base pairs and encodes a kinesin prolein of 832 amino acids with a molecular weight of 96 kd. From thc comparison of the predictcd amino acids of the newly cloned kinesin, the kinesin in S. pornbe belongs to the C-terminal motor subfamily of kincsin-related protein.

• Korean Journal of Microbiology
• /
• v.39 no.3
• /
• pp.201-205
• /
• 2003

Laccase, lignin- and manganese peroxidase are implicated in the lignin degradation. The nucleotide sequences of four copper-binding domains in fungal laccases, and heme-binding domains of lignin- and manganese peroxidases are well conserved, and therefore these short fragments can be used for the PCR for the gene amplification. We synthesized several PCR primers according to their sequences, and run PCR to amplifiy the lignin degrading genes of Trametes versicolor isolated in Korea. PCR products were cloned with pGEM-T vector in order to determine their nucleotide sequences. A laccase fragment (1.3 kb) showed 65-97％ homologies, lignin peroxidase fragment (185 bp) showed 80-95％ homologies, and manganese peroxidase fragment (443 bp) showed 61-83％ homologies when compared with other white-rot fungal enzymes.

• Korean Journal of Microbiology
• /
• v.26 no.4
• /
• pp.291-297
• /
• 1988

Three alleles of rbsB gene, rbsB, rbsB103, and rbsB106 from the wild type, the mutant and the revertant strain, respectively, were cloned for overproduction of proteins under the control of lambda $P_{L}$ promoter. Five different species of precursor and mature ribose-binding proteins were purified to homogeneity using DEAE-Sephadex column chromatography, osmotic shock pocedure, CM-Sephadex column chromatography, and Chromatofocusing column chromaography. pI of the precursor proteins and mature proteins were determined and found to be pH 8.0 and 7.5, respectively. The purified proteins were subjected to amino acid sequencing. The results confirmed the amino acid changes deduced from the DNA sequencing.

• The Korean Journal of Food And Nutrition
• /
• v.9 no.4
• /
• pp.459-465
• /
• 1996

Bacillus thuringiensis serovar. darmstadiensis produced bipyramidal endo-toxin. The toxin protein was purified by Renografin-76 step gradient centrifugation and investigated by electron microscope. Analysis of total plasmid DNA patterns showed that four different size of plasmids existed in wild type B. thuringiensis serovar. darmstadiensis. Total plasmids DNA was isolated and transformed into pst I site of pBR322 cloning vector. Ten clones containing crystal toxin gene were forst screened colony hybridization by using PUYBT 9044 probe ontained B. thuringiensis kurskaki HD 1 toxin gene. Cloned-DNA was digested with EcoR1 and HindIII and transformed to pIBI30 sequencing vector. Finally, 2.6kb and 3.6kb size fragments contatined toxin-gene were cloned with restriction analysis.

• Microbiology and Biotechnology Letters
• /
• v.17 no.4
• /
• pp.334-342
• /
• 1989

The Bacillus stearothermophilus cdd gene encoding cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5) was isolated through shot gun cloning by oomplementation of an E. coli cdd mutation. Primarily 3.0 kbp of the exogenote was cloned into the Pstl site of pBR322 (pJSC101). By subsequent deletion and subcloning from the insert of pJSC101 with cdd$^+$ and tetracycline resistancy, about 1.35 kbp of the EcoRI$_1$/PstI$_2$ fragment containing the cdd gene was isolated as pJSC201. The minicell experiment revealed a molecular mass of 33,000 dalton for polypeptide from the cloned DNA fragment complementing the cdd gene. From the lacZ fusion of 550 bp fragment of the EcoRI$_1$/AuaI as a putative promoter region, the transcription direction of the cdd gene on pJSC201 is from EcoRI towards the PstI sites, When the cdd gene was expressed in B. subtilis ED4O (cdd$^-$, pyr$^-$) by transformation with the E. coli-B. subtilis shuttle vector, the gene expression occured more efficiently than in E. coli and the gene appears to be stably maintained in B. subtitis as well as in E. coli.

• Korean Journal of Microbiology
• /
• v.52 no.3
• /
• pp.336-343
• /
• 2016

A mannanase gene was cloned into Escherichia coli from Cellulosimicrobium sp. YB-43, which had been found to produce two kinds of mannanase, and sequenced completely. This mannanase gene, designated manB, consisted of 1,284 nucleotides encoding a polypeptide of 427 amino acid residues. Based on the deduced amino acid sequence, the ManB was identified to be a modular enzyme including two carbohydrate binding domains besides the catalytic domain, which was highly homologous to mannanases belonging to the glycosyl hydrolase family 5. The N-terminal amino acid sequence of ManB, purified from a cell-free extract of the recombinant E. coli carrying a Cellulosimicrobium sp. YB-43 manB gene, has been determined as QGASAASDG, which was correctly corresponding to signal peptide predicted by SignalP4.1 server for Gram-negative bacteria. The purified ManB had a pH optimum for its activity at pH 6.5~7.0 and a temperature optimum at $55^{\circ}C$. The enzyme was active on locust bean gum (LBG), konjac and guar gum, while it did not exhibit activity towards carboxymethylcellulose, xylan, starch, and para-nitrophenyl-${\beta}$-mannopyranoside. The activity of enzyme was inhibited very slightly by $Mg^{2+}$, $K^+$, and $Na^+$, and significantly inhibited by $Cu^{2+}$, $Zn^{2+}$, $Mn^{2+}$, and SDS. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose, which was the most predominant product resulting from the ManB hydrolysis for mannooligosaccharides and LBG.

• Journal of Life Science
• /
• v.27 no.9
• /
• pp.1003-1010
• /
• 2017

An open reading frame coding for mannanase predicted from the partial genomic sequence of Paenibacillus woosongensis was cloned into Escherichia coli by polymerase chain reaction amplification, and completely sequenced. This mannanase gene, designated man26AT, consisted of 3,162 nucleotides encoding a polypeptide of 1,053 amino acid residues. Based on the deduced amino acid sequence, Man26AT was identified as a modular enzyme, which included a catalytic domain belonging to the glycosyl hydrolase family 26 and two carbohydrate-binding modules, CBM27 and CBM11. The amino acid sequence of Man26AT was homologous to that of several putative mannanases, with identity of 81% for P. ihumii and identity of less than 57% for other strains of Paenibacillus. A cell-free extract of recombinant E. coli carrying the man26AT gene showed maximal mannanase activity at $55^{\circ}C$ and pH 5.5. The enzyme retained above 80% of maximal activity after preincubation for 1 h at $50^{\circ}C$. Man26AT was comparably active on locust bean gum (LBG), galactomanan, and kojac glucomannan, whereas it did not exhibit activity on carboxymethylcellulose, xylan, or para-nitrophenyl-${\beta}$-mannopyranoside. The common end products liberated from mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, or LBG by Man26AT were mannose, mannobiose, and mannotriose. Mannooligosacchrides larger than mannotriose were found in enzymatic hydrolyzates of LBG and guar gum, respectively. However, Man26AT was unable to hydrolyze mannobiose. Man26AT was intracellularly degraded into at least three active proteins with different molecular masses by zymogram.