Study On Immobilization Of D-Lactonohydrolase

D-lactonohydrolase which could catalyze the stereospecific hydrolysis of DL-pantolacon to generate D-pantoic acid, has attracted increasing attention. D-pantoic acid is a very important intermediate in pharmaceutical industy. In this study, D-lactonohydrolase was extracted from mycelium of Fusarium proliferatum var. proliferatum AS3.4738. The crude enzyme, which was immobilized by the way of adsorption-crosslinking, was used to hydrolysis the DL-pantolacone. Finally, the DL-Pantolactone was hydrolysised with prepared immobilized D-lactonohydrolase. In the experiment, the method of extracting crude enzyme, the carrier for immoblization, the immobilization conditions and the conversion process with immolized enzyme were studied, and the enzymatic properties of immobilized enzyme were defined.The mycelium was squeezed with three different kinds of way:liquid nitrogen grinding, sonication and high pressure homogenization. Results showed that the activities of crude enzyme were57.58%,56.97%and53.78%, respectively. Then the crude enzyme solusion was treated with ultrafiltration. The loss of enzyme activity was only3.5%.In the experiment, D-lactonohydrolase was immobilized on six kinds of industrial absorption and ion exchange resins. Among them, D380showed a excellent result as the carrier for D-lactonohydrolase immobilization. Recovery coefficient of enzyme active and immobilized enzyme activity were66.7%and4U/g, respectively.Immobilization conditions were optimized by single factor experiment. The maximum activity recovery rate is66.7%when the amount of enzyme6U/g resin, adsorption pH7.5, adsorption temperature30℃, the adsorption time4h, concentration of glutaraldehyde0.1%, crosslinking time1h, cross-linking temperature4℃.The property of immobilized enzyme was determined by single factor experiment. Results showed that the optimum temperature of immobilized D-lactonohydrolase is40℃, increased by10℃than free enzyme. Its thermal stability and pH stability had improved to some extent. The Km of immobilized enzyme is28.2mmol/L. and the Km of free enzyme is3.6mmol/L, increasing by6.8times.Single factor and orthogonal experiment were applied to optimize hydrolysis conditions with immobilized enzyme. The best condition as follows:the optimum temperature is35℃; optimum pH is7.5; optimum substrate concentration is20%;the optimal concentration of immobilized enzyme was1%; speed180r/min; the reaction time of12h. It was found that pH is most significant factor on the hydrolysis by orthogonal experiments.