Keiko Yamada-Onodera

Degradation of polyethylene by a fungus, Penicillium simplicissimum YK

Abstract We isolated a strain of Penicillium simplicissimum , YK, for use in the biodegradation of polyethylene, characterizing the fungus and examining how to treat the polyethylene before cultivation to make degradation more complete. Degradation was monitored by high-temperature gel-permeation chromatography of the molecular weight distribution of polyethylene before and after the fungus was cultivated with it. Polyethylene with starting molecular weights of 4000 to 28,000 had lower molecular weights after 3 months of liquid cultivation with hyphae of the fungus. UV irradiation of polyethylene or its incubation with nitric acid at 80°C for 6 days before cultivation caused functional groups to be inserted into the polyethylene. The strain grew better on a solid medium with 0.5% polyethylene when it was irradiated for 500 h than when it was not irradiated. Polyethylene with a molecular weight of 100,000 or higher after nitric acid treatment had lower molecular weight after 3 months of liquid cultivation with hyphae of the fungus. The efficiency of polyethylene degradation depended on the growth phase in pure cultivation of the fungus. Functional groups inserted into polyethylene aided biodegradation. Bioremediation of polyethylene may become possible.

Expression of the Gene of Glycerol Dehydrogenase from Hansenula polymorpha Dl-1 in Escherichia coli for the Production of Chiral Compounds

For the production of chiral compounds, an Escherichia coli HB 101 strain was transformed. A chiral column that could resolve the three 2,3-butanediol isomers and the two acetoin isomers was used to demonstrate the stereospecificity of the enzyme. The H.polymorpha DL-1 gene product, glycerol dehydrogenase, catalyses the NAD + -dependent oxidation of 2,3-butanediol to acetoin as well as the corresponding reverse reactions. The recombinant E. coli HB101 strain harbouring the expression plasmid produced (3R)-acetoin [99% yield, >99.9% enantiomeric excess (e.e.)] from 110 mM (2R, 3R)-2,3-butanediol and (3S)-acetoin (99% yield, >99.9% e.e.) from 110 mM meso-2,3-butanediol after 24 h of incubation, showing specificity towards the secondary alcohol in R-configuration. From a racemate of 110 mM 2,3-butanediol (the molar ratio of 17 (2R, 3R): 15 (2S, 3S): 78 (meso)), (2S,3S)-2,3-butanediol (15 mM, 92% e.e.) was obtained in the resting-cell reaction without any additive to regenerate NAD + from NADH.

Degradation of long-chain alkanes by a polyethylene-degrading fungus, Penicillium simplicissimum YK

Abstract This study investigated the removal of long-chain alkanes, which are released as the result of shipping activities and fuel oil spills, with Penicillium simplicissimum YK, a fungus that degrades polyethylene. Two branched alkanes (pristane and squalane) and a phosphoric ester (Plysurf A210G) were used separately as a dispersant. Yeast extract and malt extract were added as a supplementary nutrient, respectively, to enhance cell growth. Squalane was found to be more favorable to the long-chain n- alkane degradation than pristine when the cell density was higher. When 0.001% (w/v) Plysurf A210G was used as the dispersant and 0.3% malt extract was added as the supplementary nutrient, about 26% to 51% of an initial concentration of 0.10% (w/v) of n -alkanes 30 to 40 carbons long was degraded in two weeks. In this case, no apparent correlation was observed between the molecular weights of the long-chain n- alkanes and their degradation rates. In a separate experiment, it was confirmed that the fungus used pristane, squalane, and n -alkanes with a chain of 20 and 50 carbons long for growth. This report demonstrated the biodegradability of the long-chain straight and branched alkanes by Penicillium simplicissimum YK.

Purification and characterization of an enzyme that has dihydroxyacetone-reducing activity from methanol-grown Hansenula ofunaensis.

An intracellular enzyme having reduction activity towards dihydroxyacetone (DHA), and that was induced by DHA, was purified and characterized from a methanol-grown yeast, Hansenula ofunaensis. After harvesting cells grown in a 1% methanol medium until the early stationary phase, the enzyme was purified through ammonium sulfate fractination and a series of ion-exchange, hydrophobic, and gel-filtration column chromatographies. SDS-PAGE and HPLC showed the enzyme to be a homo dimer composed of two identical subunits, each with a molecular mass of 38 kDa. The optimum pHs for DHA reduction and glycerol oxidation were 6.0 and 7.0, respectively. The optimum temperature for enzyme activity was 55 degrees C. The enzyme reduced several other compounds, including acetaldehyde, acetol, 2-butanone and 3-methyl-2-butanone, more effectively than it did DHA, while its oxidation activity was higher towards ethanol, 2-propanol, 1,2-propanediol, 2,3-butanediol and 1,3-butanediol than towards glycerol. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The purified enzyme had high K(m) values for glycerol and DHA and low K(m) values for 2-butanol and butanone, although physiologically it had a role in DHA metabolism. There were similarities between the purified enzyme and sec-alcohol dehydrogenases reported previously in their behavior towards inhibitors and metal ions, as well as in their K(m) values for 2-butanol and 2-butanone, but differences in their subunit molecular masses and activities for ethanol. At pH 9.8, the oxidative activity of the purified enzyme for l-2-butanol was about eleven times higher than that for d-2-butanol.

Characterization of a Flavinogenic Mutant of Methanol Yeast Candida boidinii and Its Extracellular Secretion of Riboflavin

A flavinogenic mutant was derived from Candida boidinii by mutagenesis. The mutant was smaller than the wild type, did not grow on a minimal medium, and required l-tryptophan, l-leucine, inositol, and nicotinate for growth. The mutant was defective in the oxidative pentose phosphate pathway, lacking glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The specific activities of the transaldolase and transketolase of the mutant were higher than those of the wild type. These high activities might direct the flux of the carbon source to the nonoxidative pathway with formation of a large amount of pentose phosphates, increasing riboflavin synthesis. Under microaerobic conditions at 25 degrees C, 90 mg/l riboflavin was obtained.