摘要
肌肽(β-Ala-L-His)是一种高效抗氧化剂,广泛应用于生物、化工、医药等领域。应用微水相酶促合成类肌肽,效率高价格低,且具有相似性质,开发前景广阔。本研究以L-丙氨酸和4,5-二羧酸咪唑制备类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑,正交实验下的最佳合成条件为:四氢呋喃:pH8磷酸缓冲溶液=10:1.6(V/V),L-丙氨酸:4,5-二羧酸咪唑=1:3(m/m),α-胰凝乳蛋白酶:底物=1:200(m/m),35oC下磁力搅拌1.5h。硅胶G60薄层色谱(TLC)分离反应产物,Rf=0.81处出现新斑点;刮下该点纯化后进行紫外扫描,高效液相色谱(HPLC)和核磁共振,紫外光谱253nm处吸收明显增强,310nm处出现新吸收峰;253nm、310nm、330nm高效液相色谱保留时间均为4.0min;13C核磁共振显示8组碳原子。结合胰凝乳蛋白酶的催化机理,得出产物结构为4(5)-丙氨酰胺-5(4)-羧酸咪唑。
Carnosine （β-Ala-L-His） has high antioxidant activity,and it is widely used in biology,chemical engineering,medicine and other fields.Its analogue syntheised in non-aqueous solvent and catalyzed by enzymes is high-effective but low-price,so it has great prospect.Here,we synthesized a carnosine analogue imidazole 4（5）-alanylamide-5（4）-carboxylic acid with imidazole-4,5-dicarboxylic acid and L-Alanine as substrates,α-chymotrypsin as catalyst in tetrahydrofuran （THF） solvent.Based on the orthogonal experiments,the optimized synthetic conditions are 4,5-dicarboxylic acid ： L-alanine = 1：3 （m/m）, α-chymotrypsin ： substrates （4,5-dicarboxyl acid and L-alanine ）=1：200 （m/m）, pH 8 phosphate buffer：THF = 1.6：10 （V/V）, reaction temperature 35℃, time 1.5 h. We separated the product with silica gel G60 thin-layer chromatography （TLC）, and a new spot appeared at Rf（ratio to front） = 0.81; then the new spot was purified and characterized with UV spectra, high performance liquid chromatogram （HPLC） and ^13C NMR （^13C nuclear magnetic resonance）. The UV spectra shows a new absorption peak at 310 nm, and the peak in 253 nm is largely strengthened; HPLC reserve times are all 4.5 min at 253 nm, 310 nm, 330 nm; ^13C NMR shows 8 carbons. Combing with the catalytic mechanism of α-chymotrypsin, structure of the analogue is confirmed, i.e. imidazole 4（5）-alanylamide-5（4）-carboxylic acid.
出处
《生物工程学报》
CAS
CSCD
北大核心
2009年第12期1940-1947,共8页
Chinese Journal of Biotechnology
