Cloning, sequencing and expression of the amylase isozyme gene from Pseudomonas sp. KFCC 10818

Summary A gene coding for amylase was cloned and sequenced from an alkalophilic Pseudomonas sp. KFCC 10818. The coding region for the amylase precursor contained 1,692 nucleotides. The presumed Shine-Dalgarno sequence, AAGG, was located at 8 nucleotides upstream from the ATG initiation codon. The precursor protein had a putative signal peptide of 25 amino acid residues at its amino terminus. The Pseudomonas amylase had four highly conserved regions as other -amylases. The amylase expressed from E. coli harboring the Pseudomonas gene produced maltose and maltotriose from soluble starch.The nucleotide sequence reported in this paper has been submitted in the GenBank/EMBL database with accession number(s) U40056.     

Cloning, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp.

A new gene encoding an -amylase has been cloned, sequenced and expressed in E. coli from an alkaliphilic Pseudomonas sp. KFCC10818. The structural gene is 1356 base pairs long and encodes a protein of 452 amino acids. The recombinant -amylase has been purified and biochemically characterized. Molecular mass of the protein deduced from SDS-PAGE was 50 kDa. The enzyme showed an activity optimum at pH 8 and at 40 °C with complete stability at pH 13 for 3 h. The enzyme released maltose and maltotriose on hydrolysis of soluble starch. Amylose was hydrolysed over 5 times faster than amylopectin by the enzyme while the hydrolysis of cyclodextrin or pullulan was negligible.     

Effects of sub-sonic vibration on the proliferation and maturation of 3T3-L1 cells

AimsAlthough low and high intensity sub-sonic vibrations (SSV) have been shown to facilitate wound healing, very few studies have investigated the effects of SSV on 3T3-L1 preadipocytes. Therefore, the present study was undertaken to investigate the influence of SSV on the proliferation and maturation of 3T3-L1 preadipocytes.Main methodsTo evaluate the effect of SSV on 3T3-L1 cell proliferation, the cells were maintained in an apparatus that administered SSV (0.5 V) for 3 days at a frequency of 10, 20, 30, or 40 Hz. In addition, to study the effect of SSV on 3T3-L1 cell maturation, the cells were stimulated with SSV for 6 days at a frequency of 10, 20, 30, or 45 Hz.Key findingsSub-sonic vibrations inhibited the proliferation of 3T3-L1 preadipocytes at frequencies of 20 and 30 Hz. Triglyceride levels in cells subjected to SSV at frequencies ranging from 10 to 30 Hz increased compared with those measured in control cells. The expression of adipogenic genes, such as PPAR-γ and C/EBP-α, markedly increased in response to SSV at 20 Hz and 30 Hz during maturation.SignificanceThese results suggest that SSV affected adipogenic gene expression at 20 and 30 Hz.     

Role of the myristoylation site in expressing exogenous functional proteins in coxsackieviral vector

We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.     

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.     

Dual positional specificity and expression of non-traditional lipoxygenase induced by wounding and methyl jasmonate in maize seedlings

Lipoxygenases (LOXs) catalyze the formation of fatty acid hydroperoxides involved in responses to stresses. This study examines the expression of a non-traditional dual positional specific maize LOX in response to wounding or methyl jasmonate (MeJA). Full-length maize LOX cDNA was expressed in Escherichia coli, and recombinant LOX was purified and characterized enzymatically. RP-HPLC and GC-MS analysis showed that the purified LOX converts alpha-linolenic acid into 13-hydroperoxylinolenic acid and 9-hydroperoxylinolenic acid in a 6:4 ratio. LOX mRNA accumulated rapidly and transiently in response to wounding reaching a peak of expression about 3 h after wounding. This increase followed an initial increase in endogenous jasmonic acid (JA) 1 h after wounding (JA burst). However, the expression of LOX induced by MeJA lasted longer than the expression induced by wounding, and the MeJA-induced expression seemed to be biphasic pattern composed of early and late phases. The expression of LOX in the presence of inhibitors of JA biosynthesis was not completely inhibited, but delayed in wound response and the expression period was shortened in MeJA response. These results suggest that wound-responsive JA burst may trigger the early phase of LOX expression which facilitates biosynthesis of endogenous JA through its 13-LOX activity, and subsequently leads to the activation of the late phase LOX expression in MeJA-treated maize seedlings. Implications of dual positional specificity of maize LOX in the observed expression kinetics are discussed.     

Dual positional specificity of wound-responsive lipoxygenase from maize seedlings

Wound-responsive lipoxygenase full-length cDNA from Zea mays was used to heterologously express the lipoxygenase enzyme and positional specificity of the lipoxygenase reaction was determined. The purified lipoxygenase catalyzed the conversion of α-linolenic acid into both 13-hydroperoxylinolenic acid and 9-hydroperoxylinolenic acid with a ratio of 6 to 4. The phylogenetic tree analysis indicated that the lipoxygenase is a type 1-lipoxygenase and belongs to 9-lipoxygenase subfamily with exceptional positional specificity. Dual positional specificity of the wound-responsive lipoxygenase indicates the versatile utilization of a singular lipoxygenase species as both 13-lipoxygenase and 9-lipoxygenase.     

Characterization of a recombinant endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from the isolated bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The bacterium Bacillus licheniformis, which exhibits high hydrolytic activity toward arabinan, was isolated from soil, and its gene encoding endo-1,5-α-l-arabinanase was cloned and sequenced. The gene has an open reading frame that encodes 328 amino acids, including a signal peptide of 37 amino acids. Endo-1,5-α-l-arabinanase, a member of glycosyl hydrolase family 43, was expressed in Escherichia coli and purified as a 34-kD monomer with a specific activity of 27 U/mg. Optimal activity toward debranched arabinan (linear 1,5-α-l-arabinan) occurred at pH 6.0 and 35°C, with a k _cat of 160/sec and a K _m of 19 mg/mL.