| 7-aminocephalosporanic acid (7-ACA), a starting compound in the synthesis of cephalosporin antibiotics, can be obtained from cephalosporin C by either chemical or two-step enzymatic process. Compared with the traditional chemical method, the enzymatic process is a more simplified, high-yield and environmental-friendly technique for the production of 7-ACA. However, the cost of 7-ACA produced by the enzymatic method is rather high at present in the industry, due to the enzyme activity of the applied strain is not very satisfied.Based on the techniques of genetic engineering and bioinformatics, the recombinant strains with a high-level expression of the D-amino acid oxidase and glutaryl-7- aminocephalosporanic acid (GL-7-ACA) acylase were constructed, and a new catalysis-separation coupling process was further developed for the production of 7-ACA in this dissertation.The main findings are:1. A gene of D-amino acid oxidase (DAAO) was obtained by isolating the total RNA from Trigonopsis variabilis and then amplified by the reverse transcription polymerase chain reaction (RT-PCR). A recombinant plasmid pET-DAAO was constructed by cloning the DAAO gene into a prokaryotic expression vector, pET-28a. After the transformation and the screening, a recombinant Escherichia coli BL21(DE3)/ pET-DAAO was obtained. The induction conditions, such as IPTG concentration, the time of induction,the induction temperature and the dissolved oxygen level, were optimized. The possibility of using lactose as an alternative inducer of IPTG was studied. A high DAAO activity of 175 U·mL-1, the highest value in the kindred documents, was obtained by a fed-batch culture with lactose induction. Cloning and expression of DAAO in Pichia pastoris were also preliminarily studied. Unfortunately, the enzyme activities of the two recombinant Pichia (intracellular and secreted expression, respectively) were both very low.2. A novel method for rapidly obtaining the GL-7-ACA acylase genes from the soil was presented. By the selective culture of the microorganism in the soil, the Pseudomonas cells in the sample were accumulated and at the same time, most of the organic substances which inhibit the PCR amplification significantly, such as humic acid, were removed. The genomic DNA was extracted from the selective cultures and the GL-7-ACA acylase gene was directly amplified by PCR technique. In comparison with the conventional process, this method has the great advantage of the simplicity and speediness of operation. 3. To enhance the expression level of the recombinant GL-7-ACA acylase, a fragment of the gene, in which the sequence encoding the signal peptide was deleted by PCR method, was used to construct a recombinant plasmid, pET-ACY, and then the recombinant E. coli BL21(DE3)/ pET-ACY. The highest GL-7-ACA acylase activity of 3000 U·L-1, the superior expression level in the documents, was obtained by the optimization of the fed-batch culture and the induction conditions of the recombinant strain. Cloning and expression of GL-7-ACA acylase in Pichia pastoris were also studied. With the induction of the methanol, the enzyme activity of the intracellular expression recombinant strain GS115(pPIC3.5k-ACY)was 75 U·L-1only. And the intracellular and secreted expressed enzyme activity of GS115(pPIC9k-ACY)was 101 U·L-1 and 40 U·L-1, respectively, which was remarkably lower than that of the recombinant E. coli.4. A novel recombinant plasmid, pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed by subcloning of pET-DAAO and pET-ACY. A rather high DAAO activity of 95 U·mL-1 and GL-7-ACA acylase activity of 390 U·L-1 were simultaneously obtained by fed-batch culture of the recombinant strain BL21(DE3)/ pET-DA. It is possible to catalyze the cephalosporin C into 7-ACA directly with the two enzymes produced by the double-gene-coexpressed recombinant strain, although the enzyme activity is not as high as that in the strain where the two enzymes were solely expressed.5. A new idea was further presented and re...
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