丝蛋白肽醒酒活性组分分离纯化及其构效关系研究

Isolation,purification and structure-activity relationship of silk fibroin peptides with anti-alcoholism activity

以废蚕丝为原料,采用40%CaCl_2溶液将其溶解、透析后得蚕丝蛋白,优化其酶解工艺条件,并将制得的丝蛋白肽经凝胶过滤色谱(HPGFC)和反相液相色谱(RP-HPLC)分离纯化,利用瓦勒-霍赫法考察了不同浓度和组分丝蛋白肽的体外醒酒活性。结果表明,最佳蚕丝蛋白酶解工艺条件为时间240 min、加酶量([E]/[S]) 2. 0%、pH 8. 5、温度60℃、底物浓度5%,该工艺下可获得水解度为23. 22%的丝蛋白肽;体外醒酒活性试验表明有较高ADH激活率的丝蛋白肽浓度为2 mg/mL;经HPGFC和RP-HPLC分离纯化,可分别得到3个和17个组分,其中SFP-II-8组分体外醒酒活性最高,其ADH激活率可达44. 51%;将SFP-II-8组分通过液相色谱-四极杆-飞行时间串联质谱联用分析(UPLC-Q-TOF-MS)后得出其氨基酸序列为L-G-G-V-G-A。

Waste silk as raw material was dissolved with 40% CaCl_2 solution at first,and silk fibroin was extracted after dialysis; Then the enzymolysis conditions of silk fibroin was optimized. Besides,silk fibroin peptides was fractionated by using HPGFC and separated ulteriorly by RPHPLC,then their anti-alcoholism activity in vitro was detected by ValleHoch＇s method. The experiment results showed that the optimal enzymatic hydrolysis conditions of silk fibroin were as followed： time of 240 min,enzyme dosage of 2%,substrate concentration of 5%,pH 8. 5 and temperature of 60 ℃,and the optimal degree of hydrolysis（DH） was 23. 22%. In addition,the better alcohol dehydrogenase（ADH） activation rate of SFP-II-8 obtained by HPGFC and RP-HPLC was measured under the concentration of 2 mg/mL,and it can up to 44. 51%. UPLC-QTOF-MS analysis showed that its amino acid sequence was L-G-G-V-G-A.