Studies On The Enzymatic Production Of¹⁵NL-phenylalanine And Purification Of Phenylalanine Ammonia-lyase

As a stable, nonradioactive, safe and available tracer,15NL-Phenylalanine (15NL-Phe) has been attached more importance in the fields of medicine, biology and chemistry. To date, only Hadener A. and Tamm Ch. reported the synthesis of L-phenyl-[2-13C,15N] alanine with a low purity of62.9%and yield of29%. Therefore, it is very valuable for commercial applications to improve the process of15NL-Phenylalanine to obtain a product with higher purity, abundance and yield than the traditional one. As a key enzyme in the biosynthesis of15NL-Phe, Phenylalanine ammonia-lyase(PAL) is also effective in the treatment of certain mouse neoplastic tumors and phenylketonuria. Therefore, the research on purification of PAL is imperative for the treatment of certain diseases and the production of L-Phe by immobilized PAL.Based on the primary study on the enzymatic reaction of trans-cinnamic acid (t-Ca) and ammonia to15NL-Phe by PAL in the Rhodotorula glutinis, a further and thorough research on this biosynthesis were conducted and a route applicable for a large scale production of 15NL-Phe was found. In addition, the release of the endoenzyme of PAL in the cell and the extraction of PAL by PEG/salt aqueous two-phase system were also investigated in the dissertation and a new way to purify the PAL was proposed. The details of the research are as follows:As a critical enzyme in the enzymatic reaction, the activity of PAL is very important to the yield of15NL-Phe. However, the PAL activity is very unstable in the reaction. Therefore, looking for an effective way to stabilize the PAL activity is favorable to increase the yield of15NL-Phe. The effects of polyethylene glycol (PEG)6000, D-sorbitol, trehalose, dithiothreitol(DTT), glycerol and glutarie dialdehyde on PAL activity in enzymatic reaction were investigated in this dissertation. By single factor experiment,0.5% PEG was found to stabilize PAL activity significantly by80.3%. By response surface experiment, it was found that the combination use of0.54% PEG6000,0.62% trehalose and0.31% DTT could effectively stabilize the PAL activity by92.9%.The substrate inhibition of t-Ca on PAL significantly limits the productivity of15NL-Phe. Therefore, looking for a way to relievate the inhibition is important to increase the yield of15NL-Phe. With the benzyl, t-Ca is suggested to be included by β-CD and to be released when the concentration of the t-Ca is greatly decreased. Therefore, the substrate inhibition by the t-Ca on PAL can be relievated with the addition of β-CD, which leads to a better conversion of the t-Ca. At the optimum molar ratio of β-CD to t-Ca of0.13, the lowest inhibitory concentration of t-Ca increases from135mmol/1to270mmol/1and the L-Phe productivity increases to266.7% of that without the addition of β-CD at270mmol/1t-Ca. Furthermore, with the fed-batch addition of t-Ca at270mmol/1t-Ca, the15NL-Phe productivity reaches45.9mmol/1,302.0% of that without the additions. The effect of β-CD on the substrate inhibition of t-Ca is depicted by kinetic parameters with the increasing γMAX and Ki and decreasing Km.The cells were removed by centrifugation and t-Ca was precipitated by adjusting pH to4.0with4%sulphate acid from the product mixture. The supernatant was prepared for the following separation of L-Phe by HP20resin. The resin can separate15NL-Phe from (15NH4)2SO4effectively and remove the nonpolar color matter as well. After adsorption and elution,91.2%15NL-Phe and95.2%15NH4+were recovered. Adjust the pH of the elution to5.5, condense under vacuum, and then dissolved in the hot ethanol at70℃. Put the crystal seed into the solution and crystallize the15NL-Phe at room temperature for4h and at4℃for24h, then freeze-dry it. The15NL-Phe with purity of99.3%, yield of71.1%and abundance of5.10%was obtained using5.34%(15NH4)2SO4and the82.1%of recovery yield of (15NH4)2SO4.The effects of freezing crack by liquid nitrogen, ultrasonic disruption, chemical penetration by L-glycine and L-alanine, surfactants on the release of Phenylalanine ammonia-lyase (PAL) in the cell of Rhodotorula glutinis were investigated in the paper. The experiments indicated that the joint use of ultrasonic disruption and TritonX-100could improve the release of the PAL significantly. Penetrating with TritonX-100for6h, then ultrasonic disruption under800W for30min, the release rate of protein got to76.7%, the PAL activity was stabilized to75.3%of the original total activity.Owing to simplicity and the effectiveness, aqueous two-phase system is employed in the study of purification of PAL. By investigating the effects of different molecular weight and concentration of PEG and various salts on the partitioning of PAL, a two-step purification of PAL in ATPS was obtained. With the application of11.0%PEG1000/14.0%Na2SO4(pH8.8) and11.0%PEG1000/14.0%Na2SO4/5.3%Na2CO3(pH8.8), the PAL with purification factor of9.3-fold and recovery yield of80.6%was obtained. The method enhances both the purity and the recovery yield in comparison with that of salting-out proteins by ammonium sulfate. The recovery yield is3.7-fold the highest purification factor reported in literature. Therefore, the two-step extraction offers a useful method for the study on PAL purification.