凡纳滨对虾肌肉中组织蛋白酶L提取工艺优化及分离纯化

优化凡纳滨对虾肌肉中组织蛋白酶L提取工艺,分离纯化组织蛋白酶L并验证其对肌原纤维蛋白的降解作用。以凡纳滨对虾肌肉为原料,采用单因素试验、Plackett-Burman试验和双因素等重复试验对肌肉中组织蛋白酶L的提取工艺进行了优化。采用Tris-HCl缓冲液浸提、硫酸铵沉淀、Q-Seharose F.F阴离子交换层析、Sephacryl S-100凝胶过滤层析和SDS-PAGA等方法对组织蛋白酶L粗酶液进行分离纯化,并验证提纯后的组织蛋白酶L对肌原纤维蛋白的降解作用。最终确定对虾肌肉中组织蛋白酶L最佳提取工艺为:缓冲液体系(Tris-HCl缓冲液浓度40 mmol/L,半胱氨酸浓度6 mmol/L,苯甲基磺酰氟浓度0.4 mmol/L),料液比1∶8(g∶mL),pH 6.0。优化后组织蛋白酶L总酶活力值升至378.36 U,酶活力得率达到150.1 U/g,纯化后的组织蛋白酶L的比活力从0.34 U/mg提高到了101.15 U/mg,纯化倍数达到298.28倍,得率为16.50%,分子质量为46.4 kDa,并进一步验证了提纯后的组织蛋白酶L对肌球蛋白重链和肌动蛋白有降解作用。发现对...

Optimization of extraction and purification of cathepsin L from Litopenaeus vannamei muscle

The purpose of the study was to optimize the extraction condition of cathepsin L from shrimp muscle(Litopenaeus vannamei), to isolate and purify, cathepsin L in extraction solutions, and to verify the effect of cathepsin L on the degradation of myofibril protein. The shrimp muscle was used as raw materials. The extraction condition of cathepsin L from muscle was optimized by single-factor tests, Plackett-Burman test and double-factor test. The cathepsin L in extraction solutions was isolated and purified by Tris-HCl buffer extraction, ammonium sulfate precipitation, Q-Seharose F.F anion exchange chromatography, SephacrylS-100 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effect of cathepsin L on the degradation of myofibril protein was verified by the confirmatory experiment. The optimal extraction conditions of cathepsin L from shrimp muscle were determined as follows: buffer system containing Tris-HCl buffer of 40 mmol/L, L-Cys 6 mmol/L and PMSF 0.4 mmol/L, the ratio of solid to liquid at 1∶8(g∶mL), pH 6.0. Under the optimized conditions, the total enzyme activity value of cathepsin L increased to 378.36 U, and the yield of cathepsin L was 150.1 U/g. The specific activity of purified cathepsin L increased from 0.34 to 101.15 U/mg, the purification multiple was 298.28 times, the yield was 16.50%, and the molecular weight was 46.4 kDa. The confirmatory experiment further confirmed that the purified cathepsin L had significant influences on the degradation of actin and myosin heavy chain. cathepsin L had significant effects on the degradation of myofibril protein. It provides a theoretical basis for further study on the related properties of cathepsin L and its degradation mechanism in shrimp muscle.