• Microbiology and Biotechnology Letters
• /
• v.23 no.4
• /
• pp.454-460
• /
• 1995

Acetylxylan esterase II was produced by Escherichia coli HB101 harboring the recombinant plasmid pKMG7 which contained the estII gene of Bacillus stearothermophilus. Optimal medium for the production of the acetylxylan esterase by E. coli HB101/pKMG7 was determined to contain 0.5% galactose, 1% yeast extract and 1% NaCl. The enzyme produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The temperature and pH optimum of the esterase were 45$\circ$C and pH 6, respectively. The essential amino acids for the esterase activity were found to be methionine, serine, and cysteine. Molecular weight of the esterase was determined to be 28 kDa by SDS-polyacrylamide gel electrophoresis, and 120 kDa by gel filtration. This suggests that the functional enzyme is a homomeric tetramer. The esterase had an isoelectric point of pH 3.4. The N-terminal amino acid sequence of the enzyme was Ala-Leu-Phe-Glu-Ser-Arg-Phe-Phe-Ser-Glu-Val-Leu-Gly-Leu.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.5
• /
• pp.680-686
• /
• 2003

This study was carried out to characterize the antilisterial substances produced by Leuconostoc sp. W65 and to evaluate the effects of pH, temperature, and time on inhibitory activity using response surface methodology. Leucocin W65, an antilisterial substance produced by Leuconostoc sp. W65, markedly inhibited the growth of Listeria monocytogenes, L. innocua, and L. ivanovii, whereas other pathogens including Gram-negative bacteria were not susceptible. The pH was the most effective factor with regard to bacteriocin activity, while temperature and time of heat treatment had no significant effect. Fifty percent of inhibitory activity remained after 22.8 min at pH 4.2 and $121^{\circ}C$. Leucocin W65 was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and tricine-SDS-PAGE. Compositional analysis originally estimated the peptide to be 56 amino acids in length without asparagine, glutamine, and tryptophane. The sequence of partial N-terminal amino acid residues of purified bacteriocin was identified as follows: $NH_{2}-XGXAGVXPXGGQQPXVPLXYP$.

• BMB Reports
• /
• v.36 no.6
• /
• pp.572-579
• /
• 2003

An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts >500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of $\alpha$-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.

• Microbiology and Biotechnology Letters
• /
• v.44 no.4
• /
• pp.470-478
• /
• 2016

A bacterium producing a large amount of chitinolytic enzyme was isolated from the intestinal tract of earthworm. The isolate was identified as Bacillus licheniformis by 16S ribosomal RNA analysis and designated as B. licheniformis GA9. The enzyme was purified by 40-60% ammonium sulfate precipitation, diethyl-aminoethyl groups exchange chromatography, and gel filtration chromatography. The molecular weight was estimated to be 52.1 kDa and the N-terminal amino acid sequence was D-S-G-K-N-G-K-I-I-R-Y-YP-I-R. The optimum activity of the purified chitinolytic enzyme was shown at pH 5.0 and $40^{\circ}C$, and the enzyme was stable in the ranges of $20-50^{\circ}C$ and pH 5.0-6.0. Enzyme activity was increased by $Co^{2+}$, while it was inhibited by $Cu^{2+}$ and $Fe^{2+}$. But it was recovered by chelating metals with ethylenediaminetetraacetic acid. The $K_m$ and $V_{max}$ values of the purified enzyme were 4.02 mg/ml and 0.52 mg/min, respectively. The chitinolytic enzyme characterized in this study has potential applications in areas such as biotechnology, biomedicine, agriculture, and nutrition.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.1
• /
• pp.160-166
• /
• 2008

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoding 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with $endo-{\beta}-1,4-D-mannanase$ from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by $Ni^{2+}$ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa ${\beta}$-mannanase having a specific activity of 481.55U/mg. The optimal activity of the purified protein, MANB48, was at $58^{\circ}C$ and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.114.1-114
• /
• 2003

A new isolate of rod-shaped virus was identified from grafted cactus, Gymnocalycium mihanovichii grafted onto Hylocereus trigonus, in Korea. The virus proved to be a new Tobamovirus and called previously as Tobamovirus-Ca for which we suggest the name Cactus mild mottle virus(CMMoV), because it produced systemic mild mosaic symptoms on its original host. CMMoV is distantly related to known species of the genus Tobamovirus on the basis of host range, serological and sequence analyses. Western blot analysis showed that CMMoV is serologically unrelated to Summons' Opuntia virus which is the only known species of the genus found in cactus plants. The 3'-terminal 2,910 nucleotides have been sequenced for the virus. The coat protein (CP) and movement protein (MP) genes encode 161 and 306 amino acids residues, respectively. The nucleotide and amino acid sequences of the CP were 39.6 ％ to 49.2 ％ and 26.4 ％ to 40.3 ％ identical to other tobamoviruses, respectively. The MP and 3' noncoding region shared 16.3 ％ to 23.3 ％ and 44.6 ％ to 63.4 ％ identities, respectively, with the members of the genus. Phylogenetic tree analysis of the CP gene revealed that CMMoV clusters with members of subgroup I of Tobamovirus. CMMoV particles contained genomic RNA along with two subgenomic RNAs, and this characteristics is common in the members of the subgroup II. This is the first information of sequence and comparative analysis of a Tobamovirus that infects cactus.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.2
• /
• pp.206-212
• /
• 1999

The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28 $\mu$M and 295 nmol $min^{-1} (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of this enzyme.$NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.2
• /
• pp.337-343
• /
• 2004

A bacterial strain concurrently producing extracellular chitosanase and chitinase was isolated from soil and identified as a member of the $\beta$-subgroup of Proteobacteria through its 16S rRNA analysis and some biochemical analyses. The newly discovered strain, named as KNU3, had 99% homology of its 16S rRNA sequence with chitinolytic $\beta$-Proteobacterium CTE108. Strain KNU3 produced 34 kDa of chitosanase in addition to two chitinases of 68 kDa and 30 kDa, respectively. The purified chitosanase protein (ChoK) showed activity toward soluble, colloidal, and glycol chitosan, but did not exhibit any activity toward colloidal chitin. The optimum pH and temperature of ChoK were 6.0 and $70^{\circ}C$, respectively. The chitosanase was stable in the pH 4.0 to 8.0 range at $70^{\circ}C$, while enzyme activity was relatively stable at below $45^{\circ}C$. MALDI-TOF MS and N-terminal amino acid sequence analyses indicated that ChoK protein is related to chitosanases from Matsuebacter sp. and Sphingobacterium multivorum. HPLC analysis of chitosan lysates revealed that glucosamine tetramers and hexamers were the major products of hydrolysis.

• Journal of Applied Biological Chemistry
• /
• v.43 no.3
• /
• pp.125-130
• /
• 2000

An antiviral protein (CAP30) with ribosome-inactivating activity was purified from the leaves of Chenopodium album L. through ammonium sulfate precipitation and column chromatography using S-Sepharose, Blue-Sepharose, FPLC Suprose12 HR, and FPLC Mono-S. The molecular wight of CAP30 was estimated to be 30kD. CAP30 was thermostable, maintaing its activity even after incubation at $70^{\circ}C$ for 30 min, and was stable in the pH range of 6 to 9. In a cell-free in vitro translation system using rabbit reticulocyte lysate, protein synthesis was inhibited by the addition of CAP30 with an $IC_{50}$ of 2.26pM. The comparison of N-terminal amino acid sequences of this protein with known ribosome-inactivating proteins (RIPs) revealed that it had some sequence homology with PAP-S and PAP-R from pokeweed (Phytolacca americana)and dodecandrin from P. dodecandra, but had no sequence homology with RIPs from other plants belonging to different orders. The mosaic symptoms on tobacco leaves caused by cucumber mosaic virus infection was completely inhibited by 100 ng/ml of the pure CAP30 protein.

• 한국생물공학회:학술대회논문집
• /
• 2003.10a
• /
• pp.434-437
• /
• 2003

A new bacterial strain, was isolated from activated sludge, identified and named P. aeruginosa EMS1. The new strain produced surface-active rhamnolipids by batch cultivation in mineral salts medium with waste flying oils. The mutant strain KH7, designated P. aeruginosa EMS1, derived by random mutagenesis with N-methyl-N-nitro-N-nitrosogoanidine treatment producing high levels of the biosurfactants was selected by an ion-pair plate assay. The mutant strain KH7 showed 4-5 times more hydrocarbon emulsification as compared to the parent when grown on waste frying oils and various hydrocarbons. Furthermore, P. aeruginosa EMS1 and mutant strain KH7 was also able to use whey as a co-substrate for growth and biosurfactant production. As results of this study, mutant strain KH7 is a very efficient biosurfactant producer, and its culture conditions are relatively inexpensive and economical. Rhamnolipid is synthesized by the rhlAB-encoded rhamnosyltransferase. To be convinced of these genes, we performed PCR based on P. aeruginosa PAO1 whole-genome database. rhl gene cluster nucleotide and amino acid sequences were compared for both parent and mutant. Comparison of nucleotide sequence of rhlAB, there were usually terminal's codons exchange.