Enzymatic And Microbial Production Of Vanillin

Vanillin(3-methoxy-4-hydroxybenzaldehyde)is one of the most important flavours in the world.It has been widely used in food,pharmaceutical,medical,tobacco,cosmetic,electroplating and various other industries.The global annual demand of vanillin is about 15,000 tons,and there are three main ways to produce it: chemical synthesis,plant extraction and bioconversion.At present,most of the vanillin in the market comes from chemical synthesis,the raw material of it is abundant and the process is mature,but the product is not specific and not environment friendly.Vanillin obtained from plant extraction are healthy and pure,but the production is limited.The bioconversion strategy includes enzymatic transformation and microbial transformation,both possesing the advantages of mild reaction conditions,short reaction cycle,and low cost.Moreover,some legislatures of foreign countries(such as Europe and US)stipulate that the substances produced by living cells or their components(such as enzymes)can be referred to as natural products.Therefore,use of the bioconversion strategy to synthesize vanillin is promising and has a great market potential.In this paper,both enzymatic and microbial transformation methods have been used to investigate the bioconversion from isoeugenol to vanillin.In terms of the microbial transformation,Bacillus fusiformis CGMCC1347 was used as the catalyst,and the impacts of natural deep eutectic solvents(NADES)on the bioconversion were examined.In terms of the enzymatic method,lipoxygenase extracted from soybeans was used as the catalyst,and the effects of different reaction conditions(such as pH,temperature,reaction time,substrate concentration and enzyme dosage)and various additives(including surfactants,organic solvents,DESs and NADESs,denaturants,chelators on the production of vanillin were investigated.The main contents of this paper are as follows: 1.Impacts of NADESs on the microbial transformation from isoeugenol to vanillin13 different NADESs were screened as additives both on microbial and microbial-free transformation of vanillin.Most of NADESs showed the ability of improving the production in the two conditions.The toxicity of NADESs to the microbial cells was also investigated by the use of ultraviolet spectrophotometry,laser scanning confocal microscopy and flow cytometry.Results have shown that the presence of NADESs was indeed toxic to some degrees to the bacterial cells,causing a damage to the integrity of the cellular membrane and the release of some proteins and nucleic acids out of the cells.2.Enzymatic synthesis of vanillinBy using soybean lipoxygenase as the catalyst,isoeugenol as the substrate,and borate buffer(pH 9.0,0.2 M)as the reaction medium,the impact of various reaction conditions such as pH,temperature,substrate concentration and enzyme dosage was examined.The optimum reaction conditions is: 5 ml borate buffer(p H 9.0,0.2 M),substrate concentration 30 g/L,enzyme dosage 0.8 g,reaction temperature 28 ?,reaction time 84 h.Under the optimal condition,vanillin was produced with a yield as high as 2.68 g/L.After the reaction conditions were established,the impacts of different types of additives on the enzymatic synthesis of vanillin were investigated.These additives include various of surfactants,organic solvents,eutectic solvents(DESs),natural eutectic solvents(NADESs),chelating agents and denaturants.Especially,some chelators were able to significantly improve the yield of vanillin,up to 405% as compared with the control.This is the first time to report that addition of chelators can improve the enzymatic synthesis of vanillin,which may be related to the active site conformation and catalytic mechanism of the enzyme.In summary,vanillin can be synthesized by using both the enzymatic and whole-cell catalytic methods.Addition of some chelators are capable of sigficantly improving the enzymatic conversion.