Study On Preparation Of 7-Amino-Cephalosporanic Acid By Tri-Enzymatic System In One-Pot

Objective: Through the site-directed mutagenesis of the original strains NK703,the strain producing ?-ketone adipoyl-7-amino-cephalosporanic acid(AKA-7-ACA)acylase was screened out,which owned high substrate specificity and catalytic activity.AKA-7-ACA acylase enzyme with high catalytic activity was investigated on the high level expression,immobilization and enzymology.Combining D-amino acid oxidase(DAAO)and catalase(CAT),cephalosporin C(CPC)was conversed to7-ACA by tri-enzyme systems in one-pot.The high activity of AKA-7-ACA acylase producing strain will bring great changes to the current enzymatic process of 7-ACA,which will make production of7-ACA to be simple,more economic and environmental friendly.Methods: 1.The GA acylase gene from wild Pseudomonas sp.and enzyme protein were purified,sequenced and modeled the enzyme with3 D structure.Compared with BRENDA and PDB database data,the key amino acid in the center of enzyme activity was analyzed and confirmed,and the influence of mutation sites on the enzyme catalytic activity was determined.2.The GA nucleotide originated from Pseudomonas sp.was derived as template,different primers were designed for amplification of gene in vitro by the error prone PCR.The conditions of PCR reaction system was controlled strictly,which obtained genetic diversity library of the best frequency of mutation.The positive clones were obtained by screening inthe LB medium with kanamycin.3.The fermentation process and extraction method of enzyme were optimized.The enzyme protein separation was studied and purified.The enzyme molecular immobilization and the stability were assayed.4.The single enzymatic system,di-enzymatic system and tri-enzymatic system were used to transform CPC to 7-ACA in one-pot,respectively,and the 7-ACA yield of each enzyme method was analyzed.The advantages and disadvantages of the enzymatic processes were analyzed in the end of the research.Results: 1.The amino acid sequences and the characteristics of gene sequences of NK703 were confirmed.2.The homology modeling was used to identify the mutation sites included N21?M?N21?Q?P22?F?P22?N?P22?Y?H23?F?H23?R?T69?L?T69?N?V70?F?V70?L?N244?M?N244?Q?K374?R?K374?E?S377?M and S377?Y.The corresponding strains were obtained by the site directed mutagenesis.The GA activity of 4 mutant strains(V70?L?K374?R?S377?Y and S377?M)and the AKA-7-ACA acylase activity of6 mutant strains(V70?L ?V70?F? K374?R? K374?E ? S377?Y and S377?M)were higher than that of the original strain.When GL-7-ACA was used as the substrate to determine the Km,the Km values of the 4mutant strains(V70?L?V70?F?K374?E and S377?Y)were smaller than the original strain;using AKA-7-ACA as substrate to determine the Km,the Km values of the 5 mutant strains(V70?L?V70?F?K374?R?K374?E and S377?Y)were smaller than the original strain.3.The 3%(w/v)lactose was used as inducer.The GA and CAT activity of recombinant NK703 was reached the highest level,at 2800.12U/L and 124.28 U/L,which increased by 2.9 times and 1.3 times higherthan that induced by IPTG.By the feeding strategy,this fermentation was induced for 60 h in 5 L-fermentor,the biomass reached at 35.21 g/L.The recombinant GA and CAT activity reached the highest in 72 h,obtained7099.85 U/L and 15776.20 U/L,respectively.The biomass increased by4.3 times,and GA activity increased by 5.3 times,as well as CAT activity was enhanced by 8.5 times than these from the flask fermentation.4.The functionalized graphene oxide was used as an immobilization carrier and the immobilized process was optimized.Under the best conditions of the immobilized process,the p H value of the fixed solution system was 7 and the ratio of enzyme and carrier was 15 mg/g.The immobilized process was last for 60 min in 35 ?.The rate of immobilization and enzyme activity recovery was 86% and 52%,respectively.After immobilized on the carrier,the thermal stability of GA was 1.7 times higher than free enzyme.The value of Km of free GA and immobilized GA was 1.07 m M and 3.15 m M,respectively.And the value of Vmax was 6.76 ?mol/min and 5.61 ?mol/min,respectively.The increasing Km of immobilized enzyme indicated that the affinity of immobilized GA was lower than that of free enzyme.5.Under the same conditions,the yield of 7-ACA prepared by single enzymatic system,di-enzymatic system and tri-enzymatic system in one-pot were 54.80%,80.50% and 87.28%,respectively.The yield of7-ACA prepared by the tri-enzyme system in one-pot was the highest.Conclusions: 1.The target strain NK703 and mutagenesis were obtained.The six positive mutations were obtained in all mutant strains.2.The strain of NK703 was fermented and expressed under highly density condition in 5L-fermentor.The maximum enzyme activities of GA and CAT through optimal extracting process were 7099.85 U/L and15776.20 U/L,respectively.And these enzymes from this method could be used to produce 7-ACA.3.Functionalized graphene oxide carrier was used to immobilize GA.The rate of immobilization and enzyme activity recovery was 86%and 52%,respectively.The value of Km was 3.15 m M and the affinity was lower than the free enzyme.4.The yield of 7-ACA prepared by tri-enzymatic system in one-pot process was the highest.