Isolation, Purification, Properties And Its Outer Factors Of Phenylalanine Ammonia Lyase(PAL) In D. Huoshanense

The herb of Dendrobium huoshanense is a kind of precious traditional Chinese medicinal materials in Anhui Province and is protected by government for its medicinal value.Because it has affluent amylose and alkaloids,it can diminish inflammation,clear the sight and improve the immunity of human body.But because of its low alkaloids content,it has been unable to meet the needs of the market.So strengthening the secondary metabolites's synthesize control,increasing alkaloids content and accelerating the improvement of medicinal value become emphases in Dendrobium's study.Phenylalanine ammonia-lyase (PAL,EC4.3.1.5) mostly catalyzes the spontaneous nonoxidative deamination of L-phenylalanine(L-Phe) to trans-Cinnamic acid, which provides precursors for alkaloids,phenolics,flavanoids and isoflavanoids.PAL is the key enzyme and rate-limit enzyme in phenylpropanoid pathway.Some researches indicated that the differentiation of Dendrobium's alkaloids content related to PAL activity.PAL may be the key factor for the synthesize of Dendrobium's alkaloids.Study on D. huoshanense PAL and its properties can interpret the function of PAL in synthesize of alkaloids,and can lay the base for artificially creating higher alkaloids content D. huoshanense by routine breeding or genetic engineering.In this research,PAL was isolated and purified from D. huoshanense and the characterization of the PAL and its outer factors was studied.The main results were as followes:1.After series isolating and purifying process of precipitation between 35% and 75% ammonium sulfate saturation, dialysis, concentration by PEG20000,DE-52 anion- exchange chromatography and Sephadex G-150 filtration column,the recovery of protein was 1.29%,and that of PAL was 10.38%, and the purification multiple was 8.2.The molecular weight of the subunit was measured by SDS-PAGE, and one protein band was observed, which proved that the PAL was purified to homogeneity.The molecular weight of the subunit was measured was 50KD by the Rf of the subunit according to the standard line obtained from the Rf of the standard proteins. And it was reported that PAL was made up of four subunits in the same size,so it was estimated that the molecular weight of D. huoshanense PAL was about 200 KD.3.PAL,which was purified,had strong absorbance at 220nm and 284nm by UV, speculating that it has conjugate structure and contain amino acid groups of phenyl-containing. 4.The chart of PAL reaction showed that the output and reaction time was linear in 3h.Its optimum pH was 8.4 in boracic acid buffer and PAL can remain stable in pH8.2-8.6. After being treated with pH4.0 and pH10.0 buffer,the PAL activities fell because of the molecular structure of enzyme changed.The optimum temperature of PAL was 45℃and the PAL can be able to bear higher temperature,its activity remained about 50% under 20 min at 60℃.Along with the augment of L-Phe's concentration,the activity of PAL was increased gradually,and the optimum substrate concentration was 2mmol/L. When L-Phe was replaced by D-Phe or L-Tyr as substance on PAL activity,the effects between L-Phe and D-Phe or L-Tyr were extremely significant.While along with the augment of PAL's concentration,the reaction rate was increased gradually,and when achieving stated concentration the reaction rate quickened abruptly but was slowdown after achieving a higher concentration.By the Lineweaver-Burk plot for Km,there are two Km values:Km1=0.4425×10-3mol/L, Km2= 0.847×10-4mol/L.PAL had better be reserved in low temperature with glycerol.5.Heavy metal salts AgNO3 and HgCl2 all inhibited PAL activities in different degree,and compared with contral the effects were extremely significant. EDTA and EDTA-Na2 inhibited PAL activities,but compared with contral the effect of EDTA was extremely significant while EDTA-Na2 was not. AlCl3,ZnSO4,MnSO4 and MgSO4 enhanced the PAL activities in diferent degree and these effect were not obvious compared with contral.The effect ranked was AlCl3>ZnSO4>MgSO4>MnSO4 and the most effective concentrations in the present experiment were as followed: AlCl3 1mmol/L,ZnSO4 1mmol/L,MgSO4 2mmol/L and MnSO4 1mmol/L.However, CuSO4 and CaCl2 (except 0.5mmol/L) inhibited the PAL activities.Compared with contral the effect of CuSO4 was extremely significant but was not obvious for diferent concentrations CaCl2. The effects of different Al3+ salts on PAL activities were different. AlCl3 and Al2(S04)3 enhanced the PAL activities and the effects were significant, while AlCl3'effects were higher than Al2(S04)3'.Al(N03)3 inhibited PAL activities, and the effect was not obvious.6.L-Tyr,L-His,L-cystine,L-Cys and D-Phe were all PAL'inhibitor,and the effects were extremely significant compared with contral.And the results also showed that the inhibitory effects increased with the rise in the concentration of the L-cystine.