Preparation of Low-phenylalanine Casein by Novel Phenylalanine Ammonia-lyases Derived from Rhodotorula

doi:10.13560/j.cnki.biotech.bull.1985.2024-0092

Abstract

Abstract:

【Objective】To explore the phenylalanine ammonia-lyase（EC 4.3.1.24; PAL）with high activity and stability, and to lay a foundation for the subsequent preparation of special dietary foods with no（low）phenylalanine.【Method】The genes RmPAL and RdPAL were cloned from Rhodotorula mucilaginosa and Rhodotorula diobovata, respectively, and the sequence and structural features of the two enzymes were analyzed by bioinformatics. The two enzymes were heterologously expressed and purified in Escherichia coli, and the optimal reaction conditions and substrate specificity were determined. In addition, the ability of RmPAL and RdPAL to convert L-Phe in casein acid hydrolysate（CAH）was determined by high-performance liquid chromatography（HPLC）and phenylalanine assay kit.【Result】The fungal-derived RmPAL and RdPAL contained three domains: the MIO domain, the core domain, and the shielding domain, with the catalytic amino acid Tyr and the substrate-specific amino acid His in the active pocket. Both RmPAL and RdPAL existed as tetramers in solution. The optimal pH and temperature of the two enzymes were 8.9 and 50℃, respectively, and they demonstrated broad pH and temperature stability, which was superior to commercial PALs derived from Rhodotorula. Furthermore, both enzymes catalyzed L-Phe and L-Tyr, with a higher catalytic efficiency towards L-Phe, approximately five times that of L-Tyr. The conversion rates of L-Phe removal from CAH were 88% and 93% for RmPAL and RdPAL, respectively.【Conclusion】RmPAL and RdPAL have strong stability, L-Phe hydrolysis preference, and ability to remove L-Phe from food-derived protein.

Key words: phenylketonuria, phenylalanine ammonia-lyase, low L-Phe protein, catalytic activity, thermal stability, conversion rates of L-Phe, specialized medical foods

ZHANG A-na, HAN Xue, GU Tian-yi, XIN Feng-jiao, WANG Yu-lu. Preparation of Low-phenylalanine Casein by Novel Phenylalanine Ammonia-lyases Derived from Rhodotorula[J]. Biotechnology Bulletin, 2024, 40(8): 309-319.

Figures/Tables 11

Fig. 1 Deamination of L-phenylalanine by PAL (phenylalanine ammonia-lyase)

Table 1 Basic properties of RmPAL and RdPAL

蛋白名称 Protein name	来源 Source	基因号 Accession number	氨基酸数目 Number of amino acids	分子量 Molecular weight/kD	等电点 pI
RmPAL	Rhodotorula mucilaginosa	P10248.2	713	76	6.72
RdPAL	Rhodotorula diobovata	TNY17356.1	720	77	6.64

Fig. 2 Multiple sequence alignment of RmPAL and RdPAL The analysed PALs include: RgPAL（Rhodotorula glutinis, XP_018274290.1）, RtPAL（Rhodotorula toruloides, XP_016272209.1）, SbPAL（Sorghum bicolor, XP_002454198.1）, NpPAL（Nostoc punctiforme, WP_012408693.1）, PbPAL（Planctomyces brasiliensis, WP_150106093.1）, AtPAL（Arabidopsis thaliana, NP_190894.1）, AvPAL（Anabaena variabilis WP_011320679.1）, KkPAL（Kangiella koreensis, WP_015781593.1）. The catalytic amino acid Tyr is marked with a triangle. The MIO cofactor（Ala-Ser-Gly）and the substrate specific key amino acid His/Phe are marked with a black box

Fig. 3 Phylogenetic tree analysis of PALs from different sources RmPAL and RdPAL mined in this study are shown in bold

Fig. 4 Structure prediction of RmPAL A: Primary structure prediction of RmPAL; the MIO domain, core domain and shielding domain are colored by pink, cyan and green, respectively. B: AlphaFold2-based three-dimensional structure building of RmPAL, using the same coloring scheme as in A. C: Cartoon representation of L-Phe/L-Tyr binding to the active site of RmPAL. The catalytic amino acid Tyr 116, substrate switch His 143 and MIO（217 Ala-Ser-Gly 219）are depicted as green sticks. The inner active site loop is colored in wheat. The substrate L-Phe and L-Tyr are shown as pink and white sticks, respectively. Hydrogen bonding force between His 143 and L-Tyr is shown as a blue dashed line

Fig. 5 Protein purification of RmPAL（A）and RdPAL（B）

Fig. 6 Optimal conditions of RmPAL and RdPAL A: Effects of pH on the activities of RmPAL and RdPAL in the range of pH 5.0-11.0. B: Effects of temperature on the activities of RmPAL and RdPAL were determined at 10-80℃

Fig. 7 Temperature and pH stability of RmPAL and Rd-PAL A, B: Thermal stability of RmPAL（A）and RdPAL（B）was determined after pre-incubated for 0-6 h at 10, 20, 30, 40, 50, 60, 70 and 80℃. C: The pH stability of RmPAL and RdPAL was determined after 1 h pretreatment in buffers of different pH values（pH 4.0-11.0）

Fig. 8 Michaelis-Menten plots of RmPAL and RdPAL-catalyzed reactions Michaelis-Menten plots of RmPAL（A）and RdPAL（C）with L-Phe as the substrate. Michaelis-Menten plots of RmPAL（B）and RdPAL（D）with L-Tyr as the substrate.

Table 2 Kinetic parameters of RmPAL and RdPAL in the hydrolysis of L-Phe and L-Tyr

蛋白名称Protein name	底物Substate	K_m/（mmol·L^-1）	k_cat/（s^-1）	k_cat/K_m（mmol·L^-1·s^-1）	倍数Fold	参考文献Reference
RmPAL	L-Phe	1.10±0.05	5.83±0.13	5.30	5	This study
RmPAL	L-Tyr	0.96±0.18	1.02±0.06	1.06	5	This study
RdPAL	L-Phe	0.72±0.04	4.47±0.11	6.21	4.7	This study
RdPAL	L-Tyr	0.91±0.16	1.20±0.07	1.32		This study
RgPAL	L-Phe	0.29 ± 0.01	0.22 ± 0.02	0.76	0.003	[24]
	L-Tyr	0.028± 0.002	7.12 ± 0.9	254		[24]
RtPAL	L-Phe	0.54 ± 0.04	5.99 ± 0.06	11.03	2.24	[25]
	L-Tyr	0.21 ± 0.01	1.02 ± 0.01	4.92		[25]

Fig. 9 HPLC analysis of the degradation of CAH by RmPAL and RdPAL A: Product t-CA of CAH hydrolysis by RmPAL for 24 h. B: Product t-CA of CAH hydrolysis by RdPAL for 24 h. C, D: The time dependence absorption profiles for the amount of product t-CA in CAH hydrolysis by RmPAL（C）and RdPAL（D）. E: The standard curve of L-Phe. F: The removal rate of L-Phe after 24 h hydrolysis of CAH by RmPAL and RdPAL

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