The Research Of Immobilized Porcine Pancreatic Lipase Hydrolysis Phenylhydantoin In Two-phase Systems

D-hydroxyphenylglycine is an important intermediate raw material of synthetic broad-spectrum antibiotic amoxicillin amoxicillin,cefadroxil,at present,there are many pharmaceutical companies has been preparation for chiral type hydroxyphenylglycine by chemical synthesis method.but,chemical synthesis D-hydroxyphenylglycine pollution is serious and structural specificity is not strong.Biosynthesis D-hydroxyphenylglycine:mainly through the two-step enzymatic hydrolysis 4-Hydroxyphenylhydantoin to D?-?-4-Hydroxyphenylglycine,First,D-hydantoinase catalyzed 4-Hydroxyphenylhydantoin to N-N-formyl D-hydroxyphenylglycine,and then through the N-methyl amide hydrolase Synthesis of D-hydroxyphenylglycine,the conversion rate of D-hydroxyphenylglycine and biological activity are very highly.But,the source of D-case is more less and it is difficult to separation and extraction,so it cannot be application for industrial.In order to solve above problem,the wide range of liapse of applicated to replaces D-case,the main contributions were as follow:First,Porcine pancreatic lipase-catalyzed hydroxyphenyl hydantoin amide bond hydrolysis,instead of the D-case which is very expensive.Second,the structural of 4-Hydroxyphenylhydantoin is instability,its phenol groups under the alkaline conditions easily to be oxidized,so the reaction of porcine pancreatic lipase catalysis must be control the pH.In this study,the immobilizated porcine pancreatic lipase on the chitosan to reduce the enzyme optimum pH to optimize the immobilization conditions.Third,the research of the immobilized porcine pancreatic lipase catalyzed hydrolysis of the hydroxy hydantoin amide bond synthesis the hydroxyphenylglycine reaction system and process parameters.Immobilization of porcine pancreatic lipase process as follows:the optimum of dissolution pH is 8.0,chitosan concentration is 3%,the optimum amount of enzyme 15mg/mL,the usage of 1.4%TPP is 6%,the adsorption time 15min,the best adsorption rate of pig pancreatic lipase was 75%,porcine pancreatic lipase in the fixed optimum pH from 8.5 down to 8.0.This paper explores the two-phase system sigma porcine pancreatic lipase-catalyzed hydroxy hydantoin amide bond hydrolysis reaction,the best reaction medium is NaH₂PO₄-Na2HPO4/isopropanol biphasic system,the initial pH is 6.6,buffer?NaH₂PO₄-Na2HPO4?/isopropanol volume is 8:10,the immobilized enzyme/substrate mass ratio is 2.2:1.When the reaction temperature is 45 ?,reaction time is 60h,under the optimum reaction conditions the yelid of N-N-formyl D-hydroxyphenylglycine rate is 47.12%.Domestic pig pancreatic lipase-catalyzed hydroxy hydantoin amide bond hydrolysis reaction,get the best reaction medium of NaH₂PO₄-Na2HP04/isopropanol biphasic system,the initial pH was 6.9,the yelid of N-N-formyl D-hydroxyphenylglycine rate is 27%.The conclusion of the experiment:first,proposed the application of lipase-catalyzed amide bond hydrolysis,and provides an experimental basis;second,compared with the free porcine pancreatic lipase,immobilized porcine pancreatic lipase enzymatic activity improved and the hydrolysis of the amide bond capacity significantly increased.