Biocatalytic Synthesis Of Semisynthetic ?-Lactam Antibiotics From Penicillin V In Full Water Phase

At present,the production of the main semi-synthetic ?-lactam antibiotics such as amoxicillin is usually involved to the hydrolysis and synthesis of penicillin G acylase.The parent nucleus 6-Aminopenicillanic acid(6-APA)was yielded by use of penicillin G acylase(PGA)with penicillin G as substrate.Then the 6-APA is acted with a new side chain to synth a new ?-lactam antibiotics under the synthesis of penicillin G acylase(SPGA).Via the analysis of previous study data,there are some advantages(higher substrate concentration and transformation rate)to catalyze the production of ?-lactam semi-synthetic antibiotics by use of penicillin V acylase(PVA)with penicillin V as substrate.Therefore,the production of semi-synthetic ?-lactam antibiotics with penicillin V as a starting material will change the traditional process and produce better social and economic benefits.The directed evolution of PGA and SPGA and the process study of synthesis of amoxicillin in full water phase were performed in our study.The PVA and SPGA mutants with higher catalytic efficiency and better enzymatic property were obtained.Moreover,the synthesis process of amoxicillin in full water phase was established by combination of PVA and SPGA mutants.The main research results of this paper were summarized as follows:1.In order to obtain high catalytic efficiency mutant,the mutation study of BsPVA-wt derived from Bacillus sphaericus(gene accession number:M15660.1)was carried out by directed evolution strategy.The results showed that after multiple rounds of mutation screening,the triple mutant BsPVA-3(T63S/N198Y/S110C)with specific activity and catalytic efficiency increased by 12.4 and 11.3 times,respectively,was obtained.The enzymatic properties of BsPVA-wt and BsPVA-3 were studied.It was found that the optimum pH of both was 6.0,while the optimum temperature of BsPVA-wt was 50 ? and the optimum temperature of BsPVA-3 was 60.Analysis of protein activity showed that the specific activity of BsPVA-3 was increased by 9 times and the immobilization activity was increased from 92 U/g to 820 U/g.The residual activity of BsPVA-3 after incubated for 1 hour at a concentration of 30%penicillin V potassium salt was 89.1%.The reaction time of immobilized BsPVA-3 for preparing 6-APA using 25%penicillin V as substrate at pH 6.0 and 25 was shortened from 180 minutes to 60 minutes.The substrate conversion rate was reached up to 99.5%or more.The continuous use repeats of BsPVA-3 were more than 600 batches with no significant activity lost which indicated it had good operational stability.Using homology modeling and analysis,it was found that two new hydrogen bonds were formed inside the BsPVA-3 protein molecule,which maybe result in an increase in activity.The BsPVA-3 mutant with good performance will greatly reduces the production costing and increases production efficiency,which make it be suitable for industrial applications.2.In order to improve the synthetic activity of PGA,SPGA derived from Achromobacter xylosoxidans was taken for study(gene accession number:AF490005).The computer simulation design and site-directed mutagenesis were combined with high-throughput screening to carry out the directed evolution study of the enzyme.Finally,the mutant SPGA-4 was obtained.Subsequently,the protein expression and purification and immobilization for the SPGA-4 mutant were also investigated.The results showed that the synthetic activity of SPGA-4 mutant enzyme was increased by 5.6 times and the hydrolysis activity was decreased by 8.7 times.The synthetic hydrolysis ratio(S/H)value was increased by 8 times.The 79%of SPGA-4 residual activity was obtained at pH 2.0 for incubating 60 min.The substrate conversion rate reached up to 99%or more when the immobilized enzyme SPGA-4 was used to catalyze the synthesis of amoxicillin.The repeat use of immobilized enzyme was more than 300 batches.All the above results indicated that the SPGA-4 had higher synthesis activity and stability and acid resistance and lower hydrolysis activity,which will make it be candidate for industrial production.3.A new method for the synthesis of amoxicillin in full water phase was developed.The synthesis reaction of amoxicillin was carried out by BsPVA-3 and SPGA-4.The best process conditions for synthesizing amoxicillin in the full water phase were as follows.The 20%of penicillin V potassium and pH 6.2 and 15? were used in the reaction.The molar ratio of 6-APA to D-p-hydroxyphenylglycine methyl ester(D-HPM)was 1:1.03.The optimum catalytic reaction time was 75-85 min.Under the best reaction conditions,the molar yield of amoxicillin was as high as 91%with purity of 99.8%.Amoxicillin can be synthesized in one step in the full water phase by using our method.The organic solvent was lost in the production process and the by-product phenoxyacetic acid could be repeated use.Therefore,the amoxicillin obtained from our method has better quality and higher purity compared to the conventional chemical process.