The Study On Pyruvate Production By Biocatalysis

α-keto acids (RC(O)COOH), especially their amino acid analogues, are important intermediates in metabolic pathways. For example, pyruvate is one of the key metabolites, connecting EMP and TAC; oxaloacetic acid, α-ketoglutaric acid, oxalocitric acid are significant intermediates of TAC. In 1835, the first α-keto acid, pyruvate, was prepared by Berzelius. Thereafter, a series of papers on the synthesis and characterization of α-keto acids were published. Interests in biotransformation research mainly focus on: as valuable chemical intermediates; as enzyme inhibitors and drugs; contribution to the studies of metabolic pathways and mechanism. As the representative of α-keto acids, pyruvate molecule comprises both the carbonyl and carboxyl groups with many reaction sites, and exhibits more special chemical characteristics than other compounds. Much attention has been given to pyruvate and its derivatives, for they are important intermediates in the process of organic and pharmaceutical syntheses. Preparation of pyruvate can be achieved by chemical catalytic synthesis, microbial fermentation and biocatalysis. Due to the simpleness of reaction mixture, high substrate-transforming rate and product purity, the low cost of extraction and simpleness of operation, preparation of pyruvate with the biocatalysis method has aroused the interest of many researchers.Biotechnological revolution has been brought about by the combination of industrial biocatalysis with pharmaceutical and agricultural technology. Aided by advanced biocatalysis technology both home and abroad and green chemistry, with the increasing demands for chemically synthesized pyruvate, preparation of pyruvate by the traditional chemical synthesis method proves inadequate. Therefore, development of a non-polltutive, low-cost, high efficient biocatalysis method to produce pyruvate is of great value and practical significance. Based on the previous research results, we attempted to investigate whole cells as the cheap catalyst. Aiming to fill the vacancy of pyruvate preparation by biocatalysis, we started with strain screening and proceeded to study the processes of biocatalyst preparation, biotransformation, separation and extraction of the product; and finally elaborated the eligible product. This paper described an innovative and industrially-promising method of pyruvate preparation.Aiming to fill the vacancy of pyruvate preparation using biocatalysis, we began withstrain screening. It mainly focused on:1. Using the relatively cheap substrate lactic acid as the sole source of carbon, a strain with high activity to transform substrate to pyruvate was screened out, capable of producing 500 mmol/L pyruvate, which was of great industrialization perspective. Physiological and biochemical tests as well as 16S rDNA identification was conducted on the two strains with highest transforming activity. They were identified as Acinetobacter sp. and Pseudomonas stutzeri, respectively.The following research was carried out on strain WLIS, the one with highest transforming activity. Results showed that WLIS could transform both D-sodium lactate and L-sodium lactate, exhibiting its potential as biocatalyst on industrial application.Optimal carbon and nitrogen sources, medium components, pH and saturation of dissolved oxygen (DO) were identified. Research on cultivation mode and cell stability were also performed. Sodium lactate and NH4Cl were selected as the carbon and nitrogen sources; the optimal culture medium was confirmed and designated as OCM: addition of 10 g l-1 sodium lactate and 2.0 g l-1 NH4Cl to minimum medium (MM). Growth of the strain was detected within a broad range of pH. When pH was 6.5-8.5, highest cell concentration and transforming activity were achieved. Results demonstrated that pH didn't make much difference, while DO had great impact on cell growth. No cell growth was dectected under low DO. When DO was too high, the cells could grow very well but result in no transforming activity. Thus, DO control was the key to obtainment of cells with high transforming activity. Primary research on the fed-batch culture technique was conducted in 5 L B.Braun bioreactor. The highest cell concentration and transforming activity were 9.56 g l-1 and 2.51 U ml-1, respectively, which almost doubled those of batch culture and proved the effectiveness of fed-batch cultivation. Studies on the stability of biocatalyst indicated that when stored at 4℃, the biocatalyst could be preserved for 10 d with almost no loss of activity. This good stability demonstrated its value for practical applicaton. Factors that influenced the transformation of lactic acid to pyruvate using whole cells were investigated, including pH, temperature, DO and so forth. The optimal transforming conditions were confirmed. Based on the results form flask experiments, culture in 5 L auto bioreactor was also performed and it proved the feasibilty of industrialization. The addition of EDTA and tetracycline could stabilize the product pyruvate and inhibit enzymes from degrading it as well. Optimal pH during transformation was 7.0-7.5. Under experimental conditions, the optimal cell concentration in the transformation process was 6 g/L, and the optimal temperature was 30℃. DO was the limiting factor of this process. By application of basic salt medium to 5 L B.Braun auto-bioreactor to culture cells and transformation of lactic acid to pyruvate in a distilled water system, the concentration of pyruvate could reach 0.507 M. The ratio of pyruvate to lactic acid was 0.72 g g-1. The pyruvate producing rate was 1.33 g l-1 h-1. Industrialization level was achieved. This study proved that cell culture and cell transformation could be coupled, which futher simplified the operation process and reduced the cost.Transformation of lactic acid to pyruvate using immobilized cells was investigated primarily. Attempts were made to prove the feasibility of successive production, including immobilization methods, immobilization carriers and conditions. 2.5% agar was used as the carrier to immobilize cells. Cell concentration was 15%. When 30% of immobilized cells were added, the best transformation results could be achieve. Some of the outstanding benefits ofapplying immobilized cells to biotransformation were that the components of the transforming liquid were simple, the liquid was lucid and downstream treatment was easy to conduct. Nevertheless, problems concerning actual application still remained. For example, how to increase the repeatability of immobilized cells was still a key to its practical application in the future;oxygen supply and so on.5. The single column-crystallization process was a new technique established to extract pyruvate, according to the simpleness of the transforming liquid in distilled water system. Application . of cation exchange resin to remove the inhibitor EDTA was developed. Technical conditions to crystallize sodium pyruvate were confirmed and 95% of sodium pyruvate was retrieved,with over 99% purity.6. Separation of lactic acid and pyruvate mixture by the fixed bed of double column ion exchange and extraction of pyruvate were developed. Among other resins, optimal anion resin LKTFF for static exchange capacity was selected to separate pyruvate from the transforming liquid. The optimal column rate and pH were determined. By this method, as much as 90% of pyruvate could be retrieved.7. Complexing extraction was applied to the pyruvate-lactic acid system to extract pyruvate, with triotylamine as the complexing agent. Preferable extraction and back-extraction techniques were extablished. When TOA was the complexing agent, extraction balance could be reached in a short time, and as the extraction time prolonged, the distribution coefficient of pyruvate remained the same. Extraction could be done in 10-15 min. Decrease of pH in the water phase contributed to the extraction of pyruvate. Using TOA as the complexing extraction reagent and ethyl butyrate as diluter, better result was obtained, with over 90% of pyruvate. When the ratio of TOA to pyruvate was 1:1, relatively high distribution coefficient and separation factor were detected. In the above extraction systems, the separation factor of pyruvate and lactic acid was over 10. back-extraction experiment demonstrated that the best percentage of back-extraction, which was above 80%, was acquired using 0.50 M NaOH Decreasing the temperature would contribute to extraction while increasing it to back-extraction.Among the previously reported techniques of pyruvate preparation, chemical synthesis and fermentation are the two widely adopted methods. Biotransformation of pyruvate from lactic acid, described in this paper, was a new technique that facilitated the process of production, with lower cost and less sophisticated equipment. Due to the complexity of this research, many details still need to be further studied. Both theoretical and practical investigations, especially pilot-scaled experiments and those with larger scale, are being arranged and carried out.