Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

深红酵母转化反式肉桂酸生成L-苯丙氨酸的研究

研究了深红酵母Ａｓ２．２７９产生Ｌ－苯丙氨酸解氨酸（ＰＡＬ）的条件、转化反式 桂酸（ｔＣａ）生成Ｌ－苯丙氨酸（Ｌ－Ｐｈｅ）的条件以及几种因素对ＰＡＬ稳定性的影响,结果表明,最佳转化条件为：１．０％ｔ－Ｃａ,８ｍｏｌ／Ｌ氨,ｐＨ１０．０,３０℃。在转化液中加入还原剂和充入Ｎ２有利于提高酶的稳定性,在此条件下可一次转化６４％的ｔ－Ｃａ,保留６０％的酶活。生成Ｌ－Ｐｈｅ浓度为５．８ｇ／Ｌ。     

Regulation of phenylalanine ammonia lyase in Rhodotorula glutinis.

In the red yeast Rhodotorula glutinis, phenylalanine ammonia lyase (PAL) was induced 10-fold during carbon starvation even in the absence of exogenous phenylalanine, although maximal induction occurred when phenylalanine was the nitrogen (40-fold) or carbon (100-fold) source. Apparent regulatory mutations that affected the expression of PAL were isolated by selecting mutants resistant to the analog p-fluoro-D,L-phenylalanine (PFP). One such mutant, designated FP1, could use phenylalanine as a nitrogen source but not as a carbon source. Similarly, FP1 failed to utilize intermediates of the phenylalanine degradative pathway, namely, benzoate, p-hydroxybenzoate, or 3,4-dihydroxybenzoate, as carbon sources. Although the PFP-resistant mutant contained a low level of PAL, no increase was found when it was grown with phenylalanine as the nitrogen source. A derivative of FP1, FP1a, was isolated that simultaneously regained an inducible PAL and the ability to use phenylalanine, benzoate, p-hydroxybenzoate, and 3,4-dihydroxybenzoate as carbon sources. In addition, when p-hydroxybenzoate was the carbon source, PAL was induced in the mutant FP1a but not in the PFP-sensitive parental strain. We propose that the mutation to PFP resistance occurred in a regulatory gene that controls the entire phenylalanine degradative pathway. Secondary mutations at this locus, as found in strain FP1a, not only restored expression of this pathway, but also altered the induction of PAL by metabolites of this pathway.     

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.     

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg^?1 and the k _cat/K _m was 1.9 × 10⁴ mM^?1 s^?1, exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.     

Phenylalanine ammonia lyase production by gamma irradiated and analog-resistant mutants of Rhodotorula glutinis

Mutants resistant to phenylalanine analogs (L-tyrosine, p-fluoro-D, L-phenylalanine (PFP) and trans-cinnamic acid) were isolated from a wild type strain of Rhodotorula glutinis A-97 by mutagenic treatment with gamma radiation and screened for phenylalanine ammonia lyase (PAL) production. One such mutant, gammaT11 (resistant to L-tyrosine), exhibited four times the PAL activity of the parent wild strain A-97. Mutant isolate gammaTFP5.6 which was selected as L-tyrosine and PFP resistant isolate, produced inducible PAL activity at levels 5.94-fold higher than the wild-type A-97 and 2.66-fold higher than its parent mutant isolate gammaT5 which was resistant to L-tyrosine. The mutant isolate gammaTC5d which was resistant to L-tyrosine and trans-cinnamic acid, exhibited 3.48 and 1.56-fold increase in PAL activity compared to the parent wild strain A-97 and its parent mutant isolate gammaT5, respectively. Different media have been examined for the induction of PAL.     

Kinetics of growth and fatty acid production of Rhodotorula glutinis

Summary The effect of culture conditions, especially nitrogen/carbon (N/C) ratio and temperature, on acyl lipid profiles in the oleaginous yeast Rhodotorula glutinis NRRL Y 1091 have been investigated. Cultivation of the microorganism under N-limited conditions (N/C < 0.14 g/g) resulted in enhanced fatty acid (FA) cell content but a reduced relative amount of polyunsaturated fatty acids (PUFA). The maximal FA productivity was obtained for a 0.025 N/C ratio resulting from the arrangement between the specific rate of FA synthesis and the concentration of lipid-free biomass. Under nitrogen-non-limiting conditions, cells grown at lower temperatures had a higher PUFA content and the maximal productivity of -linolenic acid was obtained by shifting the temperature of the culture from 30° C to 25° C. Offprint requests to: A. Pareilleux     

Purification and stabilization of phenylalanine ammonia lyase fromSporidiobolus pararoseus

The purification of phenylalanine ammonia-lyase (PAL) from S. pararoseus is described: PAL is recovered from cellular extracts by acid precipitation of contaminating proteins, nucleic acids and carotenoids; concentrated by ultrafiltration and purified by ion exchange chromatography in DEAE-Sephacel, with 80% yield and 32.6 fold purification. Elimination of carotenoids and stabilization with glycerol are introduced in the methodology to increase stability and purification yield.     

L-phenylalanine ammonia-lyase (maize, potato, and Rhodotorula glutinis). Studies of the prosthetic group with nitromethane.

Highly purified enzyme (EC 4.1.3.5) from Rhodotorula glutinis was shown by sodium dodecyl sulfate gel electrophoresis to have subunits which if not identical are closely similar in molecular weight. Like the enzyme from maize and potato [Havir, E. A., and Hanson, K. R. (1973), Biochemistry 12, 1583] it is a tetramer of molecular weight similar to 4 times 83,000. Enzyme from all three sources inactivated and labeled at the active site with 14-CH3NO2 gave on HCl hydrolysis 14-CO2, H-14-CO2H, D- and L-[14-C]aspartic acid, and unidentified radioactive products. In addition, the labeled R. glutinis enzyme gave [1,2-14-C2]glycine. The formation of the first three products is compatible with the hypothesis that the electrophilic prosthetic group of the enzyme contains the dehydroalanine imine system greater than C equals to N minus C-alpha(equals to C-beta-H2)COminus and inactivation involves attack on C-beta. The second-order rate constants for CH3NO2 inactivation varied with pH as a simple titration curve. The pKa values calculated from the curves for the three enzymes differed and were lower than the pKa of CH3NO2 by at least 1 pH unit. Apparently the inactivation process is enzyme catalyzed. Both inactivation and addition of the substrate amino group may occur with attack on C-beta.     

Lead uptake and potentiometric titration studies with live and dried cells of Rhodotorula glutinis

The ability of live cells (LC), freeze dried cells (FDC) and oven dried cells (ODC) of the yeast Rhodotorula glutinis to remove lead from aqueous solution has been studied. Discernible differences were found between the biosorption properties of LC and the other two types of cell preparation. The LC preparation exhibited an uptake level of about 12 mg g⁻¹ in a batch contactor with a biomass dosage of 2 g l⁻¹ and an initial lead concentration of 100 mg l⁻¹. This compared with, respectively, about 26 and 30 mg g⁻¹ for the FDC and ODC biosorbents under the same experimental conditions. It is seen that the level of lead uptake by the two latter biosorbents was increased to, respectively, 2.2- and 2.5-fold of the level observed for the LC preparation. The superior performance of the FDC and ODC biosorbents in the lead binding process was attributed to the presence of additional binding sites on their cell wall surfaces as indicated by potentiometric titration data. These binding sites were ascribed to carboxylic and phosphoric groups, which are the primary sites of divalent metal complexation. Modeling of the titration data revealed that subjecting R. glutinis biomass to freeze drying or oven drying increased its proton binding site concentration by a factor of 3. It appears that the two simple physical treatments were able to compromise the R. glutinis cell wall structure in such a way as to make sites normally inaccessible to become active in proton and lead binding.     

Manipulation of temperature and illumination conditions for enhanced beta-carotene production by mutant 32 of Rhodotorula glutinis

AIMS: Enhancement in the production of beta-carotene by the hyper producer mutant 32 of Rhodotorula glutinis by manipulation of temperature and illumination. METHODS AND RESULTS: Growth and beta-carotene production was investigated in a 1 litre fermenter at different temperature and illumination conditions. The optimum temperature for growth and beta-carotene production was 30 and 20 degrees C, respectively. At 30 degrees C, beta-carotene production was 125 +/- 2 mg l-1 and accounted for 66% of the total carotenoids in 72 h; at 20 degrees C, it was 250 +/- 7 mg l-1 and accounted for 92% of total carotenoid content. Continuous illumination of the fermenter by 1000 lx white light hampered growth as well as carotenoid synthesis. At 30 degrees C, illuminating the fermenter in late logarithmic phase resulted in a 58% increase in beta-carotene production with a concurrent decrease in torulene; at 20 degrees C, however, it showed no appreciable increase. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper manipulation of culture conditions enhanced beta-carotene production by R. glutinis which makes it a significant source of beta-carotene.     

Loss of cell components during rehydration of dried Rhodotorula glutinis and its implications for lead uptake

Microbial cells are routinely dried and ground before they are used in metal biosorption studies. In this work, a metal biosorbent was prepared by drying biomass of the yeast Rhodotorula glutinis in an oven at 70°C for 24 h followed by grinding. Two forms of the prepared biosorbent particles, washed and unwashed, were examined for their ability to remove lead from solution. It was found that the unwashed biosorbent exhibited higher lead uptake than the washed biosorbent. Analysis of the supernatant of washed cells incubated in water and that of unwashed cells incubated in lead solution revealed the presence of protein, carbohydrates, organic acids and inorganic phosphate. Overall, the washed and unwashed cells leached, respectively, 14.5 and 13.4% of their initial dry weight (100 mg). Acid‐base titration data revealed that the leached components contained several potential binding sites for metal cations with carboxyl and phosphoryl groups being particularly important. The higher level of lead uptake exhibited by the unwashed biomass was attributed to the fact that it leached smaller amounts of cell constituents with proton binding sites relative to the washed cells.     

Monitoring and rapid quantification of total carotenoids in Rhodotorula glutinis cells using laser tweezers Raman spectroscopy

Rhodotorula glutinis is known to accumulate large amounts of carotenoids under certain culture conditions, which have very important industrial applications. So far, the molecular mechanism of regulating carotenogenesis is still not well understood. To better understand the carotenogenesis process, it requires methods that can detect carotenogenesis rapidly and reliably in single live cells. In this paper, a method based on laser tweezers Raman spectroscopy (LTRS) was developed to directly detect carotenoids, as well as other important biological molecules in single live R. glutinis cells. The data showed that the accumulation of carotenoids and lipids occurred mainly in the late exponential and stationary phases when the cell growth was inhibited by nutrient limitation. Meanwhile, the carotenoid concentration changed together with the concentration of nucleic acids, which increased in the first phase and decreased in the last phase of the culture. These data demonstrate that LTRS is a rapid, convenient, and reliable method to study the carotenogenesis process in vivo.     

Optimal conditions for bioconversion of ferulic acid into vanillic acid by Pseudomonas fluorescens BF13 cells

Pseudomonas fluorescens BF13 is especially capable of promoting the formation of vanillic acid during ferulic acid degradation. We studied the possibility of enhancing the formation of this intermediary metabolite by using suspensions of cells at high density. The bioconversion of ferulic into vanillic acid was affected by several parameters, such as the concentration of the biomass, the amount of ferulic acid that was treated, the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with 6 mg/ml cells pre-grown on p-coumaric acid and 2 mg/ml ferulic acid. Under these conditions the bioconversion rate was 95% in 5 h. Therefore BF13 strain represents a valid biocatalyst for the preparative synthesis of vanillic acid. Received: 1 July 1997 / Received revision: 28 October 1997 / Accepted: 16 November 1997     

Exogenous application of L-phenylalanine and ferulic acid enhance phenylalanine ammonia lyase activity and accumulation of phenolic acids in pea (Pisum sativum) to offer protection against Erysiphe pisi

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of L-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100, 150 ppm) of L-phenylalanine caused increased activity of PAL activity in comparison to control. In pea leaves treated with 50 ppm L-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but it further increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves compared to control. Minimum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt after 24 h at 50 ppm and then increased with time. Treatment with both compounds significantly increased the accumulation of phenolic acids and salicylic acid and reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. Conidial germination on L-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid treated leaves 34% compared to control (46%). Foliar application of different concentrations of L-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum enzyme activity in terms of the accumulation of cinnamic acid (79.3 and 83.5 μg/g fresh wt) was observed following the application of L-phenylalanine after 24 and 48 h respectively. At 50 ppm, cinnamic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt and 74.3 and 86.5 μg/g fresh wt at 100 ppm.     

The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis

Rhodotorula glutinis is a yeast that produces copious quantities of lipids in the form of triacylglycerols (TAG) and can be used to make biodiesel via a transesterification process. The ester bonds in the TAG are broken leaving behind two products: fatty acid methyl esters and glycerol that could provide an inexpensive carbon source to grow oleaginous yeast R. glutinis. Described here are the effects of different growth substrates on TAG accumulation and fatty acids produced by R. glutinis. Yeast cultured 24h on medium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or dextrose and glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry weight basis, respectively. Lipids were extracted from R. glutinis culture and transesterified to form fatty acid methyl esters. The results show a difference in the degree of saturation for the carbon sources tested. Cells cultivated on glycerol alone had the highest degree of unsaturated fatty acids at 53% while xylose had the lowest at 25%. R. glutinis can be cultivated on all sugars tested as single carbon substrates or in mixtures. Glycerol may be used as secondary or primary carbon substrate.     

透性化嗜酸乳杆菌细胞转化亚油酸为共轭亚油酸的反应动力学

本实验旨在研究透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的反应动力学。探讨了细胞浓度、底物浓度、反应体系pH值和温度等因素对生物转化共轭亚油酸反应速度的影响;建立了透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的动力学模型。结果表明,透性化嗜酸乳杆菌细胞有利于共轭亚油酸的生物转化,最适细胞浓度、pH值和反应温度分别为10×1010ufc/mL、4.5和45℃;生物转化共轭亚油酸存在底物抑制现象,当亚油酸的浓度为0.6mg/mL时,反应速度达到最大值17.8μg/(mL·min)。在低亚油酸浓度下,反应初始阶段的反应规律与经典米氏方程相符,而在高亚油酸浓度下,存在底物抑制现象。在最适反应条件下建立了动力学模型,模型基本反映了共轭亚油酸的生物转化特性。