The Study Of Limonene Biotransformation Into α-terpineol Using Penicillium Digitatum DSM 62840

Limonene is one of the most widely distributed terpene in nature, and is widely used as a flavor and fragrance additive in food. They proceed under mild conditions, have an elavated regio-and enantioselectivity, do not generate toxic wastes and the products obtained can be labeled as “natural”.Limonene can be transformed into a lot of more valuable oxygenated derivatives such as carveol, carvone,perilla alcohol and α-terpineol et al.This study aimed to compare the effects of three fungal strains(Fusarium oxysporum CICC41029,Penicillium digitatum DSM62840 and Penicillium sp.)on R-(+)-limonene biotransformation. Growth kinetics in liquid culture and the effect of fungal growth phase on biotransformation of Penicillium digitatum DSM 62840 were further studied. Several parameters such as substrate concentration,co-solvent selection,and cultivation conditions(temperature, p H, and agitation) were optimised to obtain the highest yield of α-terpineol. Finally,i TRAQ technology was used to investigate the differentially expressed protein of Penicillium digitatum DSM 62840 in different periods of limonene biotransforamtion.The main results are as follows:1. The biotransformation of R-(+)-limonene by three fungal strains was investigated,13 products was identified using SPME-GC-MS.Peritenone,isopiperitenone and isophorone were unique products of Fusarium oxysporum CICC41029;perilla alcohol and perilla aldehyde were unique products of Penicillium sp;γ-terpinene,α-terpinolene and α-terpineol were unique products of Penicillium digitatum DSM62840;p-Menth-1-en-9-al were pruducts of Fusarium oxysporum CICC41029 and Penicillium sp.;dihydrocarvone and carveol were pruducts of Fusarium oxysporum CICC41029 and Penicillium digitatum DSM 62840;1,3,8-p-Menthatriene were products of Penicillium sp.and Penicillium digitatum DSM 62840.All the three strains obtained carvone.Among these three strains tested, P. digitatum DSM 62840 was proved to be an efficient biocatalyst to transform R-(+)-limonene to α-terpineol.P. digitatum DSM 62840 could transformed limonene into high concentration of α-terpineol(202 ± 14.7mg/L) in 24 h.2. Among three strains tested, P. digitatum DSM 62840 was proved to be an efficient biocatalyst to transform R-(+)-limonene to α-terpineol.A series factors were optimised including the growth phase of P. digitatum DSM 62840,limonene concentration,organic co-solvent,temperature,p H and agitation.The maximum α-terpineolconcentration was obtained by adding R-(+)-limonene in 20% substrate/ethanol solution(final concentrationg of limonene=840mg/L) in 48 h preculture and by cultivating at 24 °C, 150 rpm, and p H 6.0 for 12 h,833.93± 18.67 mg/L α-terpineol was obtained under these conditions,which were 4-fold before optimization.The biotransformation represented a wide tolerance of temperature:α-terpineol concentration underwent no significant change at 8–32 °C. In addition, p H ranging from 5–8 had little effect on the concentration and selectivity of α-terpineol.3. Enatioselectivity and inducibility of Penicillium digitatum DSM 62840 enzyme responsible for this biotransformation were studied.The enzyme involved was enantioseletive,it can only biotransformed R-(+)-limonene.Addition of small amounts of R-(+)-limonene(84 mg/L) at the start of fungal log-phase obtained 1.5-fold yield of α-terpineol, indicating that the enzyme was inducible. Enzyme inhibitors phenanthroline and 8-hydroxyquinoline all inhibited this biotransformation.4. To investigate the mechanism of P. Digitatum DSM62840 R-(+)-limonene biotransformation.i TRAQ technology was used to identify the protein expression difference in different periods(0h, 12h)of biotransformation,178 differentially expressed protein was identified,including 76 up-regulated proteins and 102 down-regulated proteins.Go analysis showed that differentially expressed proteins were involved in 11 biological processes, mainly metabolic process and cellular process; most of differentially expressed proteins were in cell and cell part; molecular function were mainly catalytic activity and binding.These results laid foundation for further study of the mechanism of this biotransformation.