摘要
为研究粘红酵母酪氨酸解氨酶(Rg TAL)的酶学性质,利用生物信息学分析方法对Rg TAL的氨基酸序列和蛋白结构进行分析;利用大肠杆菌BL21(DE3)和p ET-32a表达系统获得重组Rg TAL;用高效液相色谱(HPLC)检测酶的催化活性。结果:克隆获得完整的Rg TAL编码基因,氨基酸同源性分析发现,与已有的Rg TAL序列的同源性只有74%,序列具有活性结构区域位点Ala217、Ser218、Gly219和底物特异性决定位点His143。重组的Rg TAL最适反应温度40℃,最适合反应p H值5.0。对Rg TAL的动力学分析表明,催化底物L-酪氨酸的活力不高,有待进一步的研究。本文测定了重组Rg TAL的酶学性质,为今后利用Rg TAL生产香豆酸的和为进一步改造Rg TAL奠定了基础。
Objective： To investigate the characteristic of tyrosine ammonia-lyase （TAL） from Rhodotorula glutinis. Methods： To explore the amino acid sequence and protein structure of RgTAL by bioinformatics analysis; Rgtal was ex- pressed in Escherichia coli BI221 （DE3） and pET-32a system. High performance liquid chromatography （HPLC） analyses were carried out to measure the catalytic activity of RgTAL. Results： In this report, a tal gene from Rhodotorula glutinis was cloned. Amino acid analysis showed that only 74% homology with existing RgPAL. RgTAL contained the active site residue for important for catalytic structure （Ala217, Ser218, Gly219） and for substrate selection （His143）. The optimum temperature for the recombined RgTAL activity was 40℃, and the optimum pH for the activity of this enzyme was 5.0. The kinetic analysis of RgTAL showed that its catalytic efficiency was low and further study was needed. Conclusions： The characteristic of RgTAL were measured, which lays a foundation for the p-coumaric bioproduction and rational en- zyme modification research.
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2017年第9期213-219,共7页
Journal of Chinese Institute Of Food Science and Technology
基金
国家自然科学基金项目(31272217
31071837)
广东省科技计划项目(2013B010404041)
关键词
粘红酵母
酪氨酸解氨酶
对香豆酸
Rhodotorula glutinis
tyrosine ammonia-lyase
p-coumaric acid
