Preparation method of fumaric acid
At present, fumaric acid is mainly prepared by chemical synthesis and biosynthesis in industry. These two synthesis methods will be introduced and compared in detail below. Chemical synthesis methods the chemical synthesis methods of fumaric acid mainly include maleic anhydride (maleic anhydride) isomerization method and furfural oxidation method. Maleic anhydride is widely used in synthetic resin, coating, pesticide, lubricating oil additive, medicine, paper treatment agent, food additive, stabilizer, etc. it is an organic raw material with large dosage . Furfural is an important organic chemical raw material obtained by hydrolysis of agricultural and forestry by-products. It is widely used in pharmaceutical, veterinary drugs, pesticides, dyes, spices, coatings, plastics, electrical insulating materials, additives, reagents, solvents, adhesives, feed additives, food, synthetic resin, synthetic rubber and synthetic fiber monomers . The preparation process, advantages and disadvantages of the two preparation methods are as follows
The main problems in the preparation of fumaric acid by chemical synthesis are the shortage of fossil resources, the increase of production cost and environmental pollution. As a non renewable resource, the storage capacity of oil is limited. The shortage of oil has caused a serious imbalance between supply and demand in the world oil market, resulting in the current soaring oil price, which directly led to the sharp rise in the price of bulk chemicals such as benzene, toluene, ethylene and ethylene glycol, and greatly increased the cost burden of downstream manufacturing industry. Therefore, it is urgent to find new resources and energy and establish a new bulk chemical synthesis platform, which is related to the sustainable development of the whole human civilization.
The biosynthesis of fumaric acid includes enzymatic conversion and microbial fermentation. The enzymatic conversion method appeared in the mid-1990s generally uses maleic acid as substrate to prepare fumaric acid under the action of maleic acid isomerase. This method has high conversion efficiency and yield. If combined with aspartase, aspartic acid can also be prepared directly from maleic acid. Japan has done a lot of work in this regard, and it is found that some maleic acid isomerases can withstand the high temperature of 50 ℃, which can greatly improve the enzyme catalytic conversion efficiency. The research on fumaric acid production by mold fermentation began in the early 20th century. With the progress of science and technology, the yield of fumaric acid once reached 30% ~ 40%. However, due to the limitation of technical level, the understanding of microbial growth and metabolism was not deep enough in the early stage, and the industrial production has not been carried out. With the emergence of the oil crisis in the 1970s, people increased the research on the preparation of fumaric acid by microbial fermentation. During this period, the microorganisms used to ferment fumaric acid were mold, yeast and bacteria. Among them, Rhizopus is also the focus of research because of its simple nutritional requirements, strong environmental adaptability and rapid growth, including Rhizopus oryzae, Rhizopus arrhizus and Rhizopus nigricans.
During this period, Chinese scientists also explored the research work on the production of fumaric acid by microbial fermentation. Among them, Bai Zhaoxi of Shanxi Institute of Microbiology screened Rhizopus oligorhizopus with the ability to produce fumaric acid. When oscillatory cultured in a medium containing 12% glucose, the yield of fumaric acid was 5315g / L, the yield was 45%, and more by-products such as malic acid were produced. Subsequently, Federici et al. Studied the fermentation of Rhizopus. It was found that the immobilization of Rhizopus could significantly improve the stability and production efficiency of the bacteria; The appropriate stirring rate and the addition of an appropriate amount of neutralizer are conducive to acid production. In 1996, Professor Tsao of Purdue University in the United States designed a rotating biofilm contactor coupled adsorption column device for Rhizopus oryzae fermentation and fumaric acid adsorption and collection by using the self fixation ability of Rhizopus oryzae. The fermentation system is continuously fed and recycled, Fumaric acid with an average yield of 85g / l can be produced from glucose with a concentration of 100g / L in the supplement of Volume 25 of quality ・ 28 ・ modern chemical industry within 20h.
2 Application of metabolic engineering in biosynthesis of fumaric acid in 1991, Professor Bailey of California Institute of technology, a famous expert in biochemical engineering, first discussed metabolic engineering in detail, marking the birth of a new discipline of metabolic engineering. In this short decade, metabolic engineering has developed by leaps and bounds. Its combined application with cell biology, genetic engineering and other technologies has become an essential means to study and control microbial growth and metabolism. Wright et al. Obtained the glucose metabolism model in Rhizopus oryzae by 14C isotope labeling method, and analyzed and proved that Rhizopus oryzae can ferment to produce both lactic acid and fumaric acid.
Longacre et al. Analyzed the metabolic flow of glucose in Rhizopus oryzae by metabolic engineering method, and finally increased the yield of lactic acid from 65% to 75% ~ 86%. Bai Dongmei of Tianjin University in China mutated and bred Rhizopus oryzae and obtained lactic acid producing bacteria. The changes of metabolic flux in the process of mutation were analyzed in detail. It was concluded that the theoretical yield of lactic acid produced by Rhizopus oryzae fermentation was 98.12%. Similarly, the author believes that metabolic engineering can certainly improve the yield of fumaric acid produced by Rhizopus oryzae fermentation and reduce its production cost, so as to improve its market competitiveness and make an important contribution to the realization of large-scale biological substitution of petroleum based bulk chemicals.
1-pyruvate carboxylase; 2-malate dehydrogenase (cytosol); 3-fumarase (cytosol); 4-lactate dehydrogenase; 5-pyruvate decarboxylase; 6-alcohol dehydrogenase; 7-pyruvate dehydrogenase complex; 8-citrate synthase; 9-succinate dehydrogenase; 10 fumarase; 11 - malate dehydrogenase NHD - nicotinamide adenine dinucleotide; Nhdh2 - reduced NHD; ATP adenosine triphosphate; TCA - tricarboxylic acid cycle; EMP glycolysis pathway Figure 1 glucose metabolism model in roryzae in the glucose metabolism model of Rhizopus oryzae (see Figure 1), there are two fumaric acid synthesis pathways. One way is through the tricarboxylic acid cycle in mitochondria. Succinic acid generates fumaric acid under the action of succinate dehydrogenase. Fumaric acid can enter the cytosol through the mitochondrial membrane through the transport system, or continue to react under the catalysis of fumarase to produce malic acid. Another pathway exists in cell fluid. Pyruvate reacts with CO2 under the action of pyruvate carboxylase to produce oxaloacetic acid. Oxaloacetic acid produces malic acid and malic acid under the action of malate dehydrogenase
Fumaric acid is produced through the catalysis of fumarase. The metabolic pathways of fumaric acid production from cytosol were analyzed and compared. It can be seen that the theoretical yield of cytosol pathway is much higher than that of mitochondrial pathway. Therefore, it is reasonable to think that cytosol pathway is the main synthetic pathway of fumaric acid. In order to improve the actual yield of fumaric acid, it is necessary to increase the distribution of metabolic flow in the cytosolic pathway as much as possible, but it should be noted that the mitochondrial pathway cannot be excessively reduced, because it provides most of the energy required for cell growth and synthesis. On the basis of ensuring the provision of sufficient energy, the whole metabolic network should be regulated to improve the conversion rate by increasing the metabolic flux of cytosolic pathway.
Comparison of two fumaric acid metabolic pathways in Rhizopus oryzae mitochondrial pathway cytoplasmic pathway theoretical reaction equation C6H12O6 + 3O2 C4H4O4 + 2co2 + 4H2O C6H12O6 + 2co22c4h4o4 + 2H2O energy balance (assuming that glucose completely enters the mitochondrial or cytoplasmic pathway) common glycolysis process, a total of 2molatp and 2molnadh are produced, and 1mol pyruvate is produced through the tricarboxylic acid cycle, 1mol ATP, 1mol NADH2 and 3mol NADH are produced. Converted to 1mol glucose, ATP production capacity is 24mol.
1mol pyruvate needs 1mol NADH and 1mol ATP to generate fumaric acid, which is converted into 1mol glucose. The energy consumption is just offset by the capacity of glycolysis. Theoretical molar yield 12 theoretical mass yield 6414% 12910% 3 conclusion fumaric acid is an important four carbon platform compound, whether it is to study its preparation methods, especially to produce fumaric acid by combining biological high and new technologies such as metabolic engineering technology with modern chemical technology and mature industrial technology, or to study its downstream products.