[background and overview][1][2]
phenylalanine is d, l-α-aminoβ-phenylpropionic acid, which is white or colorless flaky crystals with orthorhombic crystal system. it has a bitter almond flavor, solubility in water 29. 6 g/l (25 ℃), slightly soluble in alcohol, insoluble in ether, melting point 283 ℃. it was first discovered and isolated from lupine seedlings by schulze in 1879. then fischer successfully isolated it from animal proteins in the early 20th century. phenylalanine is an important pharmaceutical and food additive and one of the eight essential amino acids for the human body. since the 1980s, with the development of anti-cancer drug preparations and amino acid infusions as well as the rapid increase in the production of low-calorie sweetener aspartame, the market demand for phenylalanine has grown rapidly. according to statistics, the world’s l-phenylalanine consumption was 50t/a in 1981, increased to 9000t/a in 1990, reached about 11,000t/a in 1997, and reached about 100,000t/a in 2006. with the continuous improvement of people’s living standards and the continuous development of medicine and health care, the demand for phenylalanine in my country’s medicine and health products, food additives, feed additives, etc. is rapidly expanding, especially in amino acid infusions, various brands of amino acid capsules, amino acid additives and sweeteners based on amino acids are developing the fastest and have the largest demand. with the further opening of the domestic market and the improvement of people’s living standards, the domestic market for this low-calorie sweetener is also increasing. therefore, the market demand for phenylalanine also continues to grow.
[application][1]
1. highly effective sweetener—aspartame
worldwide, the main use of phenylalanine is the production of aspartame. aspartame, also known as aspartame, is commonly known as protein sugar in china. it is an excellent low-calorie sweetener. its sweetness is similar to that of sucrose, and its sweetness is 200 times that of sucrose. it is currently the leading sweetener in the international market. agent. aspartame was first used as a sweetener in 1974; in 1981, fao and who recommended that the intake of aspartame be 40 mg/person·day, and the relevant regulations in the united states during the same period were 50 mg/person·day. in 1996, the united states allowed aspartame to be used as a general sweetener in all foods without special regulations. the production and application of aspartame in china has just started, and its development potential is huge. in the late 1980s, china began to study aspartame. however, because there was no domestic l-phenylalanine production equipment at that time, imported raw materials were expensive, resulting in production costs that could not compete with imported products, so no industrialized production and market were formed. supplies are limited. at present, china consumes 7.6 million t/a of sucrose every year. if 5% of this is replaced by aspartame, it will require about 1,600 t/a of aspartame. correspondingly, the demand for l-phenylalanine will also grow.
2. pharmaceutical field
according to different uses in the pharmaceutical field, phenylalanine has different grades such as pharmaceutical grade, food grade, and feed grade. the prices among the grades vary greatly, ranging from us$18/kg to us$40/kg. in addition to being directly used in medical compound amino acid infusions and synthetic medicines, phenylalanine can also be used to prepare pharmaceutical culture media. medicines synthesized from phenylalanine include hiv protease inhibitors, chymosin, anti-tumor drugs p-fluorophenylalanine, brain neurotransmitters, etc. the consumer demand for phenylalanine in this field has grown steadily.
3. other fields
in recent years, the application fields of phenylalanine have continued to expand. for example, the technology of synthesizing chiral intermediates using phenylalanine and its derivatives as raw materials has been developed, and it has been discovered that phenylalanine can be added to unroasted cocoa. acid, leucine and degradable sugar can significantly improve the flavor of low-quality cocoa.
[biological metabolism][3]
phenylalanine is an essential amino acid for the human body. it cannot be synthesized by the human body itself and can only be synthesized by plants and microorganisms. the starting materials for phenylalanine synthesis are erythrose-4-phosphate (e4p), an intermediate product of the five-carbon sugar phosphate pathway, and phophoenolpyruvate, an intermediate product of the glycolysis process. , pep) are condensed to form a seven-carbon ketose open-chain phosphate compound called 3-deoxy-α-arabinoheptulonic acid-7-phosphate (dahp), and then dahp is converted into shikimic acid (shik), and then converted again it is chorismate (cha), and phenylpyruvic acid (ppy) is synthesized from chorismate. finally, phenylpyruvic acid undergoes transamination to generate phenylalanine, as shown in the figure:
【preparation】[3]
1. extraction method
extraction method refers to the method of producing phenylalanine using natural products as raw materials through separation and purification. industrially, defatted soybeans are hydrolyzed with hydrochloric acid, acidic amino acids are removed, phenylalanine and tyrosine are adsorbed with activated carbon or decolorizing resin, and then the phenylalanine is dissolved and separated using a solvent. another method is to convert phenylalanine in the hydrochloric acid hydrolyzate of defatted soybeans into 2,5-dibromobenzenesulfonate, and then use the difference in solubility to separate it from other amino acids. the process is complex, separation and extraction are difficult, product quality is unstable, and production costs are high. in addition, pig hair can also be used as raw materials for production, but the acid production rate is low, the investment is large, the energy consumption is large, the sources of raw materials are limited, and the output is not easy to expand.
2. chemical synthesis
1) condensation method of benzaldehyde and acetylglycine
generally, sweeten the sugar firstthe amino acid is acetylated to obtain acetylglycine, which is then condensed with benzaldehyde to form acetamidocinnamic acid lactone. the latter is hydrolyzed, electrolyzed, reduced, and enzymatically hydrolyzed to obtain phenylalanine. this method synthesizes phenylalanine as raw materials are easily available and the cost is low, but the process route is long, there are many steps, and the yield is low.
2) cyanamide method
as early as 1951, the cyanamide method was used to prepare phenylalanine, but the chemical yield and radiochemical yield were only about 28%. in 1978, c6h5ch214cn was used as raw material. due to the radiochemical synthesis steps (including from k14 sub>cn the radiochemical synthesis of c6h5ch214cn takes a long time, and the yield is still less than 60%. improve this method and use c6h5ch2cho as raw material to prepare d, phenylalanine-1 -14c, the chemical yield and radiochemical yield both reached 67%. then thermolysin enzymatically synthesizes z-l-ala-l-phe-om (1-14c) dipeptide from dl-phenylalanine-1-14c and z-l-alanine to achieve racemic benzene for the separation of alanine, the dipeptide is enzymatically reacted with thermolysin in n-methylmorpholine buffer solution to finally obtain phenylalanine. this method has relatively mild conditions in the introduction of k14cn and the subsequent three-step reaction, and the yield and purity of the final product are both high. the disadvantage is that the highly toxic k14cn is introduced during the reaction, making it difficult to treat the three wastes.
3) glycine method
the glycine method requires first protecting the carboxyl and amino groups of glycine to generate benzylidene aminoethyl acetate. the activity of the hydrogen atom of α-methylene is then used to carry out alkylation reaction under phase transfer conditions. finally, after hydrolysis reaction, dl-phenylalanine is obtained. the reaction conditions of this method are mild, the raw materials are easily available, the yield is high, there are no highly toxic raw materials, and there are no three waste problems that are difficult to deal with. the organic solvent used can be recycled. it is a promising chemical synthesis method.
3. production of phenylalanine by microbial fermentation
1) production using yeast
a strain with higher phenylalanine ammonia lyase (pal) activity was isolated from the natural environment. this strain was used as a starting point for uv and ems mutagenesis. the obtained strain had higher enzyme activity than the original strain. great improvement, and the bacteria grew well in the pilot test, with a cumulative yield of 58. 0 g l -1 .
2) use bacillus to produce phenylalanine
the conditions for producing phenylalanine were optimized based on the brevibacterium mutant strain fmp92814. the lactose-fermenting brevibacterium fmp92814 obtained through artificial mutation breeding can utilize relatively extensive agricultural and sideline products, such as starch hydrolyzed sugar, corn syrup, soybean cake powder hydrolyzate, and molasses as raw materials to ferment and produce phenylalanine, which can reduce production costs and is suitable for at present, various amino acid fermentation plants are promoting it.
3) production of phenylalanine using pseudomonas
pseudomonas sp. e4-106 cells will be screened from soil to conduct a phenylalanine production experiment. the results show that the optimal temperature for the transamination reaction is 35 to 40°c; the catalytic reaction does not change much in the ph range of ph 7 to ph 10; treating the cells with surfactant or adding mg2 + to the reaction solution can significantly improve transamination reaction rate. in this reaction system, the transaminase activity of e4-106 strain per unit wet weight of cells is 1039u/g; when the product phenylalanine mass concentration is 32. 2 g/l and 50. 4 g/l, the mole of phenylpyruvate the conversion rates were 97. 5% and 87. 2% respectively, and the product recovery rate was 81. 8%.
3. enzymatic production of l-phenylalanine
enzymatic production has the advantages of high product concentration, fewer purification steps, and strong production capacity. the disadvantage is the high cost of some raw materials. experts believe that the route from benzaldehyde → hydantoin → phenylpyruvic acid → phenylalanine or benzaldehyde → cinnamic acid → phenylalanine is the best solution.
1) one enzyme one acid method
the one-enzyme-one-acid method is the phenylpyruvate enzyme method using hyacinth as the raw material. the industrial development and research of this method has been started since the late 1980s.
2) two bacteria and two enzymes method
using the two-bacteria dual-enzyme method, escherichia coli ep8-10 is used to produce high-activity transaminase and low-activity aspartase after culture, while e. coli ea produces high-activity aspartase and very low activity. transaminase, coupled with the action of transaminase in ep8-10 and asp enzyme in ea-1, generates l-phe from phenylpyruvic acid and fumaric acid. the optimal conditions of this system are: the mass ratio of the two cells of ea-1 and ep8-10 is 0.6:1, n (ppa)/n (fu) =1:1.2 (molar ratio). when the ppa concentration is 0. 24 mol/l, the reaction can produce 38. 5 g/l phenylalanine for 6 hours.
5. genetic engineering method to produce l-phenylalanine
the phenylalanine synthesis pathway has multiple regulatory sites. the dahp synthase (ds), mycolate mutase (cm) and prephenylalanate dehydratase (pd) in the figure are the key enzymes that are regulated. ; aminotransferase (at) is the last step in the synthesis pathway to catalyze phenyl acetone.
[main reference materials]
[1] deng juxiang, wang zuyuan. production status and development prospects of phenylalanine[j]. anhui chemical industry, 2007, 33(4): 1-3.
[2] li yonghui, liu yun, xu qishou. research progress on phenylalanine biosynthesis[j]. biotechnology communications, 2002, 13(4): 296-300.
[3] li jixin, zhang chao. research progress on production and application of l-phenylalanine[j]. amino acids and biological resources, 2006, 28(2): 51-56.