Purification, characterization, and gene cloning of glycerol dehydrogenase from Hansenula ofunaensis, and its expression for production of optically active diol

Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 (Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD(+)-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only (R)-1,2-octanediol in 50 mM racemate (R:S=52:48), and produced (S)-1,2-octanediol (24 mM, <99.9% e.e.) after 24 h of incubation. The reaction product was suggested to be 1-hydroxy-2-octanone by GC-MS, which showed secondary hydroxyl groups oxidized. Glycerol dehydrogenase from H. ofunaensis could be useful for the production of long-chain optically active secondary alcohols.     

Formation of the spore clumps during heat treatment increases the heat resistance of bacterial spores

Effects of the clumping of bacterial spores on their heat resistance as a result of heat treatment were investigated. Spore suspensions of Bacillus cereus, Bacillus coagulans and Bacillus licheniformis were heated at 85 degrees C. Survivor curves of the three strains showed tailing in all treatments after 30 min. As the treatment time increased, the formation of spore clumps increased in all strains after 20 min. Relative hydrophobicity of the spore surface increased as a result of heat treatment. The effect of spore concentration on the inactivation of the B. licheniformis spores was investigated, and surviving curves showed no tailing below a concentration of 4.9 log CFU/ml.     

Inter-laboratory validation studies of biological method for determination of streptomycin in royal jelly

Inter-laboratory validation studies were conducted in 6 laboratories to validate the biological method for determination of streptomycin in royal jelly. Streptomycin spiked at the level of 0.2 and 1.0 ppm was analyzed. Mean recoveries were 89 and 96%, reproducibility relative standard deviations (RSD(R)) were 15.0 and 14.0%, HORRAT(R) values were 0.7 and 0.9. Samples containing residues at the levels of 0.25 and 0.80 ppm were analyzed. Mean recoveries were 113 and 99%, RSD(R) were 15.0 and 10.4%, and HORRAT(R) values were 0.8 and 0.6. The determination limit was 0.1 ppm. These results show that this method has good performance.     

Study of pesticide residues in agricultural products for the "Positive List" system

During a 3-year monitoring survey (April 2002-March 2005) of pesticide residues in agricultural products, 592 samples (324 domestic; 268 imported) collected in Hyogo prefecture, Japan were analyzed. The number of pesticides tested increased from 232 in FY 2002 to 323 in FY 2004. The purpose of the study was to clarify the residue status by accumulating information about pesticides detected frequently, to allow effective and efficient regulation under the new "Positive List" legislation to be implemented in FY 2006. Overall, 47% of domestic and 61% of imported samples contained detectable residues and ca. 60% of positive samples contained multiple residues. The limit of quantitation was set at 0.01 microg/g and the limit of detection was 0.001-0.003 microg/ g. Most of the residues were present at low concentrations: 80% of the detections in samples excluding imported citrus fruits were < 0.05 microg/g. More than 5 different pesticides (> 0.01 microg/g) were detected simultaneously in 13 samples. The sum of the ratios of residues to MRLs was calculated as one of the indexes to represent the risk of multiple residues, and they exceeded 100% in 3 imported frozen vegetables; baby kidney bean, spinach, Welsh onion. Samples in violation of the Food Sanitation Law were not found in our survey, but 1.9% of the samples might be in conflict with the new "Positive List" legislation.     

Family M42 aminopeptidase from the syntrophic bacterium Symbiobacterium thermophilum: characterization using recombinant protein

The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co2+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.     

Survey of pesticide residues in imported frozen vegetables and fruits (1989.4~2008.3)

A survey of pesticide residues in 595 imported frozen products on the Tokyo market from April 1989 to March 2008 was carried out. Forty three kinds of pesticides, including organophosphorus, organochlorine, carbamate, pyrethroid and others, were detected between levels of trace (below 0.01 ppm) and 4.6 ppm from 162 samples. Chlorpyrifos, cypermethrin and omethoate were frequently detected in green vegetables (komatsuna leaf and spinach), cypermethrin and methamidophos were detected in pods and seeds (green soybean and string pea), chlorpropham (CIPC) was detected in potato, and captan and carbaryl were detected in berries (blueberry, raspberry and strawberry). The hydrophilic pesticide methamidophos was detected in flesh of lychee. Residue levels of these pesticides were calculated as between less than 0.5% and 30% of their ADI values according to the daily intake of frozen products. Therefore, these frozen products should be safe when they were eaten in customary amounts.     

Analysis of trichothecenes in barley tea and beer by liquid chromatography/tandem mass spectrometry

A simple method for analysis of trichothecenes [Type A: diacetoxyscirpenol, neosolaniol, HT-2 toxin, and T-2 toxin, Type B: deoxynivalenol, nivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivarenol] in barley tea and beer using liquid chromatography tandem mass spectrometry (LC/MS/MS) was developed. Trichothecenes were extracted with ethyl acetate-methanol (19:1). The solvent was evaporated to dryness and the residue was dissolved in water-methanol (3:1) for injection into the LC/MS/MS. The LC separation was performed with an octadecylated silica column at a flow-rate of 0.2 mL/min, using a mobile phase consisting of water, methanol and acetonitrile. MS/MS was used in multiple reaction monitoring, employing electrospray ionization (ESI-MRM). The recoveries of trichothecenes from drinks at 1 microg/L (Type A) and 10 microg/L (Type B) were 52.5-115.2% (barley tea) and 68.1-127.5% (beer). Five barley tea and ten beer samples were analyzed by this method. Trichothecenes were not detected in them. This method may have applications in quality assurance.     

Determination of low levels of methanol and ethanol in licorice extract by large volume injection head-space GC

A large volume injection head-space GC method was established for measuring low levels of residual methanol and ethanol in licorice extract used a food additive. A vial was kept at 50 degrees C in the oven of the head-space sampler. Injection of the head-space gas for 0.75 min into a Poraplot Q GC column with a initial oven temperature of 35 degrees C, enabled the determination of low levels (5 micrograms/g) of methanol and ethanol. The standard deviations for five rounds of analysis of methanol and ethanol in licorice extracts were between 0.82 and 2.97. Methanol was found in 6 samples out of 9 collected in 1999, at concentrations exceeding 50 micrograms/g, the limit set by the Japanese Government, established in 1999 and coming into force on April 1, 2000. The highest concentration reached 10,000 micrograms/g. Methanol at a concentration exceeding 50 micrograms/g was found in 2 out of 9 samples collected in 2000. The highest concentration was 270 micrograms/g.