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

作　　者：张阿娜 韩雪 谷天一[2,3] 辛凤姣 王钰璐[2,3] ZHANG A-na;HAN Xue;GU Tian-yi;XIN Feng-jiao;WANG Yu-lu(College of Food Seience,Shanxi Agricultural University,Jinzhong 030600;Institute of Food Science and Technology,Chinese Academy of Agricultural Sciences,Beijing 100193;Institute of Food Science Technology Nutrition and Health(Cangzhou),Chinese Academy of Agricultural Sciences,Cangzhou 061001)

机构地区：[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

出　　处：《生物技术通报》2024年第8期309-319,共11页Biotechnology Bulletin

基　　金：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)。

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。【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.

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 

分 类 号：Q78[生物学—分子生物学] TS201.21[轻工技术与工程—食品科学]