|Title||Lignin peroxidase mediated biotransformations useful in the biocatalytic production of vanillin|
|Author(s)||Have, R. ten|
|Source||Agricultural University. Promotor(en): J.A.M. de Bont; J.A. Field. - S.l. : S.n. - ISBN 9789058081698 - 90|
|Publication type||Dissertation, internally prepared|
|Keyword(s)||vanilline - lignine - peroxidase - vanillin - lignin - peroxidase|
|Categories||Food Biotechnology / Industrial Microbiology / Biochemistry|
This research concentrates on lignin peroxidase (LiP) mediated biotrans-formations that are useful in producing vanillin.
In order to obtain this extracellular enzyme, the white-rot fungus Bjerkandera sp. strain BOS55 was cultivated on nitrogen rich medium. This procedure resulted in a successful LiP production of 600 U/L. Peptone in the culture medium was shown to interfere with the standard LiP assay in which the formation of veratraldehyde (VAD) from veratryl alcohol (VA) is monitored. Removal of peptone by (NH 4 ) 2 SO 4 precipitation minimized the interference.
BOS55 excreted at least seven LiP isozymes of which two were purified and characterized. The predominant LiP isozyme (LiP-2) oxidized VA to VAD in the pH range from 2.5 to 6.5. The VA oxidizing activity was optimal at the lowest pH. The K m for H 2 O 2 was strongly depended on the pH. At pH 5.0, a physiological pH, the Km for H 2 O 2 was similar to the extracellular H 2 O 2 concentration measured in cultures of BOS55.
A model based on calculated ionisation potentials (IP) was developed to predict which potential vanillin precursor would be oxidized by LiP. By testing a series of non-phenolic aromatic compounds, of which the IP was calculated, an IP-threshold value of 9.0 eV was determined. This value was used to select compounds with a lower IP like O -acetyl coniferyl alcohol, and O -acetyl isoeugenol (isoeugenyl acetate, IEA). Indeed, these compounds were consumed and in part converted into vanillyl acetate, the acetyl ester of vanillin.
IEA was studied to elucidate mechanisms of its oxidation andcleavage of the propenyl side chain in IEA into vanillyl acetate. It was found that IEA was consumed via redox mediation. IEA was only oxidized in the presence of the redox mediator VA. The latter was first oxidized by LiP to the radical cation (VA · +) which in its turn oxidized IEA to its corresponding radical cation IEA · +. The followingcleavage reaction was the result of O 2 -dependent chemical reactions which resulted in vanillyl acetate and ethanal.
The isoeugenol methyl ether (DMPP) was also used to investigate the consumption and thecleavage mechanism. DMPP was not only consumed enzymatically, but also by O 2 -dependent self-propagating reactions. Mn 2+inhibited this chemical consumption. Since Mn 2+also inhibited the molar yield of the predominantcleavage product, VAD, it was concluded that this product was formed also during these chemical reactions. An other VAD producing route was discovered by incubating a DMPP oxidation side product, 1-(3',4'-dimethoxyphenyl)-2-propanone (DMPA) with LiP. Interestingly, VAD was only formed in the presence of O 2 . Without O 2 , solely DMPA dimers were detected by GC-MS.