利用新型红酵母苯丙氨酸解氨酶制备低苯丙氨酸酪蛋白

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

生物技术通报 ›› 2024, Vol. 40 ›› Issue (8): 309-319.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0092

利用新型红酵母苯丙氨酸解氨酶制备低苯丙氨酸酪蛋白

张阿娜¹(), 韩雪²^,³, 谷天一²^,³, 辛凤姣²^,³(), 王钰璐²^,³()

1.山西农业大学食品学院，晋中 030600
2.中国农业科学院农产品加工研究所，北京 100193
3.中国农业科学院农产品加工与营养健康研究院（沧州），沧州 061001

收稿日期:2024-01-24 出版日期:2024-08-26 发布日期:2024-06-27
通讯作者: 王钰璐，女，博士，助理研究员，研究方向：生物大分子结构与功能；E-mail: wnewyx@163.com；
辛凤姣，女，博士，研究员，博士研究生导师，研究方向：食品酶学与合成生物学；E-mail: 2002hongzhi30@163.com
作者简介:张阿娜，女，硕士研究生，研究方向：食品酶学；E-mail: zhangana123@163.com
第一联系人：韩雪为本文共同第一作者
基金资助:
中央级公益性科研院所基本科研业务费专项(S2022JBKY-13);中国农业科学院农产品加工研究所创新工程院所重点任务(CAAS-ASTIP-G2022-IFST-07)

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

ZHANG A-na¹(), HAN Xue²^,³, GU Tian-yi²^,³, XIN Feng-jiao²^,³(), WANG Yu-lu²^,³()

1. College of Food Seience, Shanxi Agricultural University, Jinzhong 030600
2. Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193
3. Institute of Food Science Technology Nutrition and Health（Cangzhou）, Chinese Academy of Agricultural Sciences, Cangzhou 061001

Received:2024-01-24 Published:2024-08-26 Online:2024-06-27

摘要/Abstract

摘要：

【目的】挖掘高活性、高稳定性的苯丙氨酸解氨酶（EC 4.3.1.24; penylalanine ammonia-lyase, PAL），为后续在制备无（低）苯丙氨酸特膳食品的应用奠定基础。【方法】从胶红酵母（Rhodotorula mucilaginosa）和双倒卵形红酵母（Rhodotorula diobovata）中克隆到基因RmPAL和RdPAL，并通过生物信息学分析两个酶的序列和结构特征；在大肠杆菌中异源表达纯化RmPAL和RdPAL蛋白，测定最适反应条件和底物特异性；通过高效液相色谱和苯丙氨酸试剂盒测定了RmPAL和RdPAL转化酸解酪蛋白（casein acid hydrolysate, CAH）中苯丙氨酸（L-phenylalanine, L-Phe）的能力。【结果】RmPAL和RdPAL是真菌来源的PAL，分别由3个结构域组成：MIO结构域（MIO domain）、核心结构域（core domain）和屏蔽结构域（shielding domain），活性中心具有催化氨基酸Tyr和底物特异性特征氨基酸His；RmPAL和RdPAL在溶液中均以四聚体形式存在，两个酶的最适pH和最适温度均为8.9和50℃，且具有较宽泛的pH和温度稳定性，优于黏红酵母来源的商用PAL酶；此外，两种酶均能催化L-Phe和酪氨酸（L-tyrosine, L-Tyr）反应，且对L-Phe的催化效率较高，约为L-Tyr的5倍，脱除酸解酪蛋白中L-Phe的转化率分别为88%和93%。【结论】RmPAL和RdPAL具有较强稳定性和L-Phe水解偏好性，可从食源蛋白中有效去除L-Phe。

关键词: 苯丙酮尿症, 苯丙氨酸解氨酶, 低L-Phe蛋白, 催化活性, 热稳定性, L-Phe转化率, 特医食品

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

张阿娜, 韩雪, 谷天一, 辛凤姣, 王钰璐. 利用新型红酵母苯丙氨酸解氨酶制备低苯丙氨酸酪蛋白[J]. 生物技术通报, 2024, 40(8): 309-319.

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.

图/表 11

图1 PAL对 L-苯丙氨酸的脱氨作用

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

表1 RmPAL和RdPAL的基本性质

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

图2 RmPAL和RdPAL的多序列比对分析的PAL包括：RgPAL（黏红酵母，XP_018274290.1）、RtPAL（圆红冬孢酵母，XP_016272209.1）、SbPAL（高粱，XP_002454198.1）、NpPAL（点形念珠藻，WP_012408693.1）、PbPAL（巴西浮霉状菌，WP_150106093.1）、AtPAL（拟南芥，NP_190894.1）、AvPAL（多变鱼腥藻，WP_011320679.1）、KkPAL（康氏菌，WP_015781593.1）。催化氨基酸Tyr用三角形标出，MIO辅基（Ala-Ser-Gly）和底物特异性关键氨基酸His/Phe用黑框标出

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

图3 不同来源PAL系统发育树分析本研究挖掘的酶RmPAL和RdPAL加粗表示

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

图4 RmPAL的结构预测 A：RmPAL的一级结构预测；粉色为MIO结构域、青色为核心结构域、绿色为屏蔽结构域；B：AlphaFold2模拟RmPAL的三级结构，粉色为MIO结构域、青色为核心结构域、绿色为屏蔽结构域；C：L-Phe/L-Tyr与RmPAL活性位点结合的局部展示；催化氨基酸Tyr 116、底物特异性氨基酸His 143和MIO（217Ala-Ser-Gly219）为绿色棒状；内盖环为小麦色；底物L-Phe和L-Tyr分别为粉色棒状和灰色棒状；氢键显示为蓝色虚线

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

图5 RmPAL（A）和RdPAL（B）的蛋白纯化

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

图6 RmPAL和RdPAL的最适反应条件 A：在pH 5.0-11.0范围内，pH对RmPAL和RdPAL活性的影响；B：在10-80℃测定温度对RmPAL和RdPAL活性的影响

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℃

图7 RmPAL和RdPAL的温度及pH稳定性 A, B：RmPAL（A）和RdPAL（B）在10、20、30、40、50、60、70和80℃ 预孵育0-6 h后测定热稳定性；C：在不同pH值（pH 4.0-11.0）的缓冲液中预处理1 h后测定RmPAL和RdPAL的pH稳定性

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）

图8 RmPAL和RdPAL催化反应的Michaelis-Menten图以L-Phe为底物RmPAL（A）和RdPAL（C）的Michaelis-Menten图；以L-Tyr为底物RmPAL（B）和RdPAL（D）的Michaelis-Menten图

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.

表2 RmPAL 和 RdPAL对L-Phe和L-Tyr的动力学参数

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]

图9 HPLC分析RmPAL 和 RdPAL水解CAH的能力 A：RmPAL水解CAH反应24 h产物t-CA吸光值；B：RdPAL水解CAH反应24 h的产物t-CA吸光值；C, D：RmPAL（C）和 RdPAL（D）分别在不同反应时间（4、8、16、24 h）水解CAH生成产物t-CA量的折线图；E：L-Phe标准曲线；F：RmPAL 和 RdPAL水解CAH反应24 h L-Phe的脱除率

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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利用新型红酵母苯丙氨酸解氨酶制备低苯丙氨酸酪蛋白

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

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