Immobilization of penicillin amidase on cotton and nylon fiber

Abstract

Penicillin amidase, an enzyme that hydrolyzed benzylpenicillin to yield 6-APA, was produced by culture of $\mbox{\underline{Bacillus}}$ $\mbox{\underline{megaterium}}$ (ATCC 14945) and purified 70 fold by means of adsorption on Celite followed by dialysis or ammonium sulfate precipitation. Little attempts have been made to utilize synthetic nylon fiber and natural cotton for enzyme immobilization. New enzymatic process for the preparation of 6-APA was developed by employing the enzyme immobilized onto these fibers as carriers. The feasibility of utilizing these materials as enzyme carriers was studied by evaluating four different immobilization methods. Cotton was activated with CNBr or $TiCl_4$, followed by coupling of enzyme. The optimal pH for activation was found in the range of 11.5 to 12, and the amount of protein coupled to cotton was as high as 163 mg/g of cotton matrix, showing a good possibility of employing natural cellulosic fiber as an enzyme carrier. Activation with $TiCl_4$ was not recommended because of degradation of fiber structure by strongly acidic $TiCl_4$ solution. Native nylon polymer has few end groups, and it must be pretreated in order to generate potentially reactive groups that can be covalently bound with enzyme molecule. Therefore, the nylon fiber was treated with N,N-dimethylaminopropylamine of HCl to generate reactive amino groups on it, and the enzyme was coupled to it by crosslinking with glutaraldehyde. Only 11\% of enzyme activity was retained by employing N,N-dimethylaminopropylamine, so the partial hydrolysis with HCl was further investigated. The optimal hydrolysis time was found about 5 to 10 minutes at 45$^\circ$C when 3.75N-HCl solution was used. Under these conditions, the amount of enzyme coupled onto the nylon was up to 63mg/g nylon matrix, and the activity retention was as high as 72\%. From the experiments on the effect of enzyme loading, two interesting features were found. First, the maximum coupling efficiency w...