A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.
a-Ionone, a-methylionone, and a-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from a-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-a-ionone, trans-3-hydroxy-a-ionone, and 3-oxo-a-ionone. 2,3-Dehydro-a-ionone, 3,4-dehydro-o-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from a-methylionone and a-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for a-ionones and elucidation of stereochemical features of the bioconversion. lonones and their derivatives are widely distributed in nature. They are important constituents of many kinds of essential oils and are presumably generated from carot-* Corresponding author.
Wehave been studying the bioconversion of terpenes by fungi. Terpenes containing a cyclohexane ring in their molecules, such as ionones,1 2) isophorone3) and oxoisophorone,4) were found to be converted into oxidative or reductive compounds by Aspergillus niger JTS 191. It was apparent that the main conversion reaction in the furigus was regio-and stereospecific hydroxylation. There have been reports5~7) on the microbial conversion of acyclic monoterpenes with one or more double bonds. However, there have been very few on the microbial conversion of /?-myrcene, an acyclic monoterpene.As /?-myrcene has conjugated double bonds and a gemdimethyl terminal in its molecule and it is a basic material for synthetic terpenes,8} we were interested in the conversion of this compound by the fungus. A. niger JTS 1911} was cultured in a 3-1 Erlenmeyer flask containing one liter of medium as previously described.4) To the resulting culture broth, one ml of /?-myrcene was added as the substrate. The culture was further incubated at 28°C with shaking. Ten ml of the broth was removed each day for analysis of the substrate and conversion products. Gas liquid chromatography (GLC) was performed as previously described,4) except that the oven temperature was programmed from 60 to 160°C (2°C/min).The total content of conversion products in the neutral fraction corresponded to less than 10% of the initially added substrate, for the incubation.As the contents of substrate and conversion products in the mediumwere low, those in the mycelia were analyzed.Mycelial pellets were separated from the culture broth by suction filtration and then weighed. The substrate in the mycelia was extracted twice with 100mlof ethyl acetate. The pooled extract was analyzed by GLCwithout concentration, to prevent loss due to vaporization of the substrate.As shown in Fig.
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