Isolation and functional analysis of a 24-residue linear alpha-helical antimicrobial peptide from Korean blackish cicada, Cryptotympana dubia (Homoptera)

A new antimicrobial peptide, cryptonin, was isolated and characterized from the adult Korean blackish cicada, Cryptotympana dubia. It consists of 24 amino acid residues and has a molecular weight of 2,704 Da on mass spectroscopy. The predicted alpha-helical structure analysis and increased helix percent in 40% trifloroethanol of cryptonin suggests that it belongs to the typical linear alpha-helix forming peptide. Binding of the biotin-labeled cryptonin at the surface of E. coli cells and increased influx of propidium iodide in E. coli after cryptonin treatment indicates that it kills microbial cells by binding bacterial cell surfaces and disrupting the cell permeability. Cryptonin showed strong antibacterial (MIC 1.56-25 microg/ml) and antifungal (MIC 3.12-50 microg/ml) activities against tested bacteria and fungi including two antibiotic-resistant bacterial strains; methicilin-resistant S. aureus and vancomycin-resistant Enterococci (MIC 25 microg/ml, each).     

Characterization of an extracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle

Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60 degrees . A broad range of lipase substrates, from C4 to C18 rho-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was rho-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.     

Culture conditions for a new phytase-producing fungus

Extracellular phytase produced by Aspergillus sp. 5990 showed a 5-fold higher activity in liquid culture when compared with cultures of Aspergillus ficuum NRRL 3135. The optimum fermentation conditions were determined to be 35 °C, neutral pH, and 4 days incubation. The phytase had a higher optimum temperature for its activity than the commercial enzyme, Natuphos, from Aspergillus ficuum NRRL 3135.     

Halocynthiibacter namhaensis gen. nov., sp. nov., a novel alphaproteobacterium isolated from sea squirt Halocynthia roretzi

A Gram-negative, non-motile and rod-shaped bacterial strain, designated RA2-3^T, was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain RA2-3^T was observed to grow optimally at 25 °C, at pH 7.0–7.5 and in the presence of 2 % (w/v) NaCl. Strain RA2-3^T exhibited the highest 16S rRNA gene sequence similarity values to the type strains of Litoreibacter meonggei (95.7 %), Planktotalea frisia (95.6 %), Thalassobius gelatinovorus (95.5 %) and Pelagicola litoralis (95.4 %). A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain RA2-3^T clustered with the type strains of Planktotalea frisia, Pelagicola litoralis, Pacificibacter maritimus and Roseovarius marinus. Strain RA2-3^T was found to contain Q-10 as the predominant ubiquinone and C_18:1 ω7c as the major fatty acid. The major polar lipids detected in strain RA2-3^T were phosphatidylcholine, phosphatidylglycerol, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain RA2-3^T was 52.9 mol%. On the basis of the phylogenetic, chemotaxonomic and phenotypic properties, strain RA2-3^T is considered to represent a new genus and species within the family Rhodobacteraceae, for which the name Halocynthiibacter namhaensis gen. nov., sp. nov. is proposed. The type strain of H. namhaensis is RA2-3^T (=KCTC 32362^T=NBRC 109999^T).     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

Sunxiuqinia dokdonensis sp. nov., isolated from deep sub-seafloor sediment

A novel facultatively anaerobic strain DH1^T was isolated from deep sub-seafloor sediment at a depth of 900 m below the seafloor off Seo-do (the west part of Dokdo Island) in the East Sea of the Republic of Korea. The new strain was characterized using polyphasic approaches. The isolate was Gram-stain-negative, motile by gliding, non-spore-forming rods, oxidase-negative, and catalase-positive; and formed colonies of orange-red color. The NaCl range for growth was 0.5–7.0% (w/v) and no growth was observed in the absence of NaCl. The isolate grew optimally at 30°C, with 2% (w/v) NaCl and at pH 7. The cell-wall hydrolysates contained ribose as a major sugar. The DNA G+C content was 40.8 mol%. The closest related strains are Sunxiuqinia faeciviva JAM-BA0302^T and Sunxiuqinia elliptica DQHS-4^T (97.9 and 96.3% sequence similarity, respectively). The level of DNA-DNA relatedness between strain DH1^T and S. faeciviva JAM-BA0302^T was around 41% (but only 6% between DH1T and S. elliptica DQHS-4^T). The major cellular fatty acids of the isolate were contained iso-C_15:0 (25.9%), anteiso-C_15:0 (16.7%), and summed feature 9 (comprising C_16:0 3-OH and/or unknown fatty acid of dimethylacetal ECL 17.157; 13.2%). The predominant menaquinone was MK-7. On the basis of polyphasic evidence from this study, the isolate was considered to represent a novel species of the genus Sunxiuqinia, for which the name Sunxiuqinia dokdonensis sp. nov. is proposed; the type strain is DH1^T (=KCTC 32503^T =CGMCC 1.12676^T =JCM 19380^T).     

Isolation and characterization of a cellulase-free endo-β-1,4-xylanase produced by an invertebrate-symbiotic bacterium, Cellulosimicrobium sp. HY-13

A xylanolytic bacterium, Cellulosimicrobium sp. HY-13, was isolated from the digestive tract of an earthworm, Eisenia fetida. The purified cellulase-free endo-β-1,4-xylanase (XylK) produced by strain HY-13 was found to contain an N-terminal amino acid sequence of APSTLEAAAE and to have a relative molecular mass of 36 kDa. It was most active at pH 6.0 and 55 °C and had V_max and K_m values toward oat spelt xylan of 4067 IU/mg and 2.78 mg/ml, respectively. XylK primarily degraded xylan to a series of xylooligosaccharides composed of xylobiose to xylotetraose, but it could not further hydrolyze xylobiose to xylose. The results of the present study suggest that the relatively highly active XylK lacking exo-xylanolytic activity is a promising candidate for the efficient production of non-digestible xylooligosaccharides that may have beneficial effects to gastrointestinal health via promotion of the growth of probiotics.     

Catalytic properties of a GH10 endo-β-1,4-xylanase from Streptomyces thermocarboxydus HY-15 isolated from the gut of Eisenia fetida

A novel GH10 endo-β-1,4-xylanase (XylG) gene from Streptomyces thermocarboxydus HY-15, which was isolated from the gut of Eisenia fetida, was cloned, over-expressed, and characterized. The XylG gene (1182 bp) encoded a polypeptide of 393 amino acids with a deduced molecular mass of 43,962 Da and a calculated pI of 6.74. The primary structure of XylG was 69% similar to that of Thermobifida fusca YX endo-β-1,4-xylanase. It was most active at pH 6.0 and 55 °C. The susceptibilities of xylans to XylG were as follows: oat spelt xylan > birchwood xylan > beechwood xylan. The XylG also showed high activity (474 IU/mg) toward p-nitrophenylcellobioside. Moreover, at pH 6.0 and 50 °C, the V_max and K_m values of the XylG were 127 IU/mg and 2.51 mg/ml, respectively, for oat spelt xylan and 782 IU/mg and 5.26 mM, respectively, for p-nitrophenylcellobioside. A homology model indicated that XylG folded to form a (β/α)₈-barrel with two catalytic residues of an acid/base (Glu181) and a nucleophile (Glu289). The formation of a disulfide bond between Cys321 and Cys327 were predicted by homology modeling.