Proteins differentially expressed during limonene biotransformation by Penicillium digitatum DSM 62840 were examined using iTRAQ labeling coupled with 2D-LC-MS/MS.

This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation.

Optimisation of α-terpineol production by limonene biotransformation using Penicillium digitatum DSM 62840.

BACKGROUND: In this study, (R)-(+)-limonene biotransformation using three fungal strains was compared. Penicillium digitatum DSM 62840 was distinguished for its capacity to transform limonene into α-terpineol with high regioselectivity. Growth kinetics in submerged liquid culture and the effects of growth phase and contact time on biotransformation were studied using this strain. Substrate concentration, co-solvent selection, and cultivation conditions were subsequently optimised. RESULTS: The maximum concentration of α-terpineol (833.93 mg L(-1)) was obtained when the pre-culture medium was in medium log-phase by adding 840 mg L(-1) substrate dissolved in ethanol and cultivation was performed at 24 °C, 150 rpm, and pH 6.0 for 12 h. Addition of small amounts of (R)-(+)-limonene (84 mg L(-1)) at the start of fungal log-phase growth yielded a 1.5-fold yield of α-terpineol, indicating that the enzyme was inducible. CONCLUSION: Among these three strains tested, P. digitatum DSM 62840 was proved to be an efficient biocatalyst to transform (R)-(+)-limonene to α-terpineol. Further studies revealed that the optimal growth phase for biotransformation was in the medium log phase of this strain. The biotransformation represented a wide tolerance of temperature; α-terpineol concentration underwent no significant change at 8-32 °C. The biotransformation could also be performed using resting cells.

Characterisation of free and bound volatile compounds from six different varieties of citrus fruits.

Free volatile compounds in six varieties of citrus juices were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry. Bound fractions were isolated and extracted with methanol and Amberlite XAD-2 resin and then hydrolyzed by almond ß-glucosidase. A total of 43 free and 17 bound volatile compounds were identified in citrus. Free volatile contents in sweet orange were the most abundant, followed by those in grapefruits and mandarins. Among free volatiles, terpenes were the most abundant in citrus juice. Sensory analysis results showed that the flavor of the same citrus cultivars was similar, but the flavor of different cultivars varied. Among bound volatiles, benzenic compounds were the most abundant in these citrus juices. Bound volatiles also significantly differed among cultivars. In addition, only p-vinylguaiacol were detected in all of the samples.

Characteristics of ß-glucosidase from oranges during maturation and its relationship with changes in bound volatile compounds.

BACKGROUND: The hydrolysis of glycosidically bound volatile compounds can release potential aromas in oranges during maturation. ß-Glucosidase is the key enzyme that influences the hydrolysis of bound volatiles. In this study the changes in ß-glucosidase and bound volatile compounds in Jincheng oranges during maturation were investigated. The relationship between ß-glucosidase activity and bound volatiles was analyzed. RESULTS: The optimal temperature and pH of ß-glucosidase from Jincheng oranges were 40 °C and 5-6 respectively. Its Km and Vmax values were 0.61 mmol L(-1) and 0.009 U mg(-1) respectively. The activity of ß-glucosidase was strongly inhibited by Zn(2+), Fe(2+), Cu(2+), Ag(+), Hg(2+) and Fe(3+). ß-Glucosidase activity in pulp increased gradually during maturation, while that in peel first increased and then decreased in November. In total, 12 and 14 bound volatiles were found in pulp and peel respectively of this orange during maturation. CONCLUSION: The concentration of bound volatiles in pulp and peel decreased with the rise in ß-glucosidase activity in pulp and peel during maturation. This indicated that bound volatiles in Jincheng oranges were released during maturation owing to the increase in ß-glucosidase.