Traditional methods to produce tryptophan include chemical synthesis and proteolysis. With the constant improvement of production technology, the method occupying a dominant market position is the production of L-tryptophan by microbial method adopted by many companies. Microbiological methods mainly include enzymic method, direct fermentation methods and microbial transformation methods.
The enzymatic method mainly refers to using the catalytic function of the tryptophan biosynthesis enzymes in microorganisms to produce L-tryptophan. The main enzymes used in the enzymatic production of tryptophan include tryptophan synthase, serine racemase, etc. According to the number of microorganisms that provide the required enzymes, enzymatic methods are mainly divided into single-bacteria enzymatic methods and double-bacterial enzymatic methods.
Now the engineering bacteria for industrial production of L-tryptophan include escherichia coli, achromobacter hyaluronicum, corynebacterium glutamicum, pseudomonas putida, etc. The method of producing tryptophan is to directly add cell wall-dissolving enzyme in the fermentation process to destroy the cell wall, and then directly use the enzyme lysed from the bacterial cell. Similarly, the desired enzyme can be immobilized and purified before use.
Moreover, the precursors required in the enzymatic production of tryptophan do not have to be natural raw materials, and even chemically synthesized precursors can be used as raw materials for production, which greatly reduces the production cost; at the same time, the enzymatic synthesis also has the advantages of high purity of product, few by-products, high yield and easy operation. When L-tryptophan is produced by an enzymatic method using pyruvate, indole and ammonia as raw materials, due to the low price of alanine, and indole, as one of the substrates, has little effect on the enzyme activity, this method has certain application prospects and development potential.
The carbon source donors of the direct fermentation method are mainly cheap raw materials such as glucose and sugarcane molasses, and high-efficiency tryptophan essentials are used to produce strains, and the fermentation conditions are controlled to directly accumulate L-tryptophan. Therefore, the direct fermentation method is also a microbial method. However, because the regulation mechanism of the gradual metabolism of glucose to L-tryptophan synthesis in microorganisms is also relatively complicated, it has been difficult to obtain high-yield tryptophan-producing strains directly from nature. Therefore, selecting and breeding excellent strains are the key to the success of the direct fermentation method.
In addition, in microorganisms, the metabolic flow of tryptophan biosynthesis pathway is very weak, and the synthesis process requires continuous addition of various precursors, such as L-serine and glutamine. Only precursor is maintained at a higher ratio, can the tryptophan production achieve the purpose. With the development of the theory of metabolic engineering and the continuous improvement of the fermentation technology, the efficiency of direct fermentation to produce tryptophan is getting higher and higher, and now this method is gradually entering the stage of industrialization.
The microbial transformation method can also be called the precursor transformation method, which mainly refers to using glucose as a carbon source donor, and then culturing the microorganism, during which several precursors for tryptophan synthesis will be produced such as Benzoic acid, L-serine, indole, etc., and then microorganisms will use these precursors to synthesize tryptophan under the action of their own tryptophan synthase.
Enterprises that produce tryptophan using industrial method first used this method to obtain L-tryptophan on a large scale. In order to over-accumulate tryptophan, the feedback inhibition of various substrates in the metabolic process of aromatic amino acids needs to be relieved. In the process of producing L-tryptophan by this method, it is not advisable to add too many precursors, which is prone to inhibiting feedback. Therefore, most manufacturers use the fed-bitch to continuously add precursors. The transformation method of microbial precursors also has shortcomings. The price of precursors is expensive, which is not conducive to controlling costs. Moreover, when the concentration of the precursor is too high, it will inhibit in reverse the activity of the enzyme in the synthesis process and affect the accumulation efficiency of tryptophan.