Purification And Some Characterization Of Cathepsin L From Yak Meat

Tenderness is the key factor to determine the edible quality of beef. Maturity is the mosteffective way to improve the tenderness, which is currently recognized worldwide. Thechanges of tenderness are caused by endogenous proteases hydrolyzing myofibrillar protein inbeef. Cathepsin L is a kind of endogenous proteases closely related to tenderizing of beef,which plays an important role in the maturation process after the slaughter of livestock andpoultry. Therefore, it is necessary to purify cathepsin L which plays a key regulatory role tothe quality of beef, laying the theoretical foundation for further study of the maturemechanism of beef and improving the quality in the actual production.In this study, Longissimusdorsi of yak from Qinghai Datong was chosen as raw material.Separation and purification process conditions of cathepsin L from yak meat was studiedusing response statistical methods such as surface analysis, orthogonal experimental designand uniform design. Cathepsin L after purity identification was studied on its enzymaticcharacteristics. The test results are as follows:1. Assay of enzyme activity: Specific fluorescent substrate of cathepsin L from yak meatwas Z-Phe-Arg-AMC and its optimal reaction concentration was25μmol/L. The reaction timebetween enzyme and substrate was30min.2. Extraction process: The significant factors include pH of extraction buffer solution,L-Cys and ammonium sulfate saturation, and had a positive effect on enzyme activity.Primary and secondary order was as follows: pH of extraction buffer solution, ammoniumsulfate saturation and L-Cys. The best extraction process were: pH5.70of extraction buffersolution,6.46mmol/L L-Cys and76.39%ammonium sulfate saturation. Under theseconditions, specific activity of the enzyme was90.33U/mg, and extraction rate was0.153g/mg.3. Purification process: the best process parameters of DEAE-Sephacel anion exchangechromatography were: sample sizes was1mL, volumetric flow rate was0.6mL/min, elutionconcentration of NaCl was0.2～2.0mol/L. Primary and secondary order affecting specificactivity of the cathepsin L was as follows: volumetric flow rate, elution concentration of NaCland sample volume. Under optimum conditions, the measured specific activity was194.80U/mg, purification fold was9.90and recovery rate was28.52%. The optimal conditions ofSephacryl S-100gel filtration chromatography were: volumetric flow rate was0.6mL/min andsample volume was1.2mL. Primary and secondary order affecting specific activity as follows: volumetric flow rate and sample volume. Under this program, specific activity of cathepsin Lwas294.12U/mg. The enzyme was purified up to15.03times with a recovery of23.11%. Thepurity had reached Electrophoresis purity identified by SDS-PAGE and molecular weight wasabout36ku.4. Enzymatic characteristics: Optimum pH and temperature of the enzyme were pH5.5and40℃, respectively. It was high stable at pH5.0～5.5and20～40℃. Specific fluorescentsubstrate of the enzyme was Z-Phe-Arg-AMC. Metal ions including Ca2+、Cu2+、Mn2+、K+、Fe2+、Fe3+and Zn2+inhibited the enzyme in various degree. L-Cys and β-ME could increasethe activity of the protease while the enzyme was partially inhibited by EDTA and EGTA.Preliminary concluded that cathepsin L from yak meat is a kind of acid resistance andcysteine protease contains mercapto.In summary, cathepsin L was purified to Electrophoresis purity from yak meat utilizing thetechniques of ammonium sulfate precipitation, Salting, dialysis, ultrafiltration, DEAE-Sephacelanion exchange chromatography and Sephacryl S-100gel filtration chromatography. Theenzyme hydrolyzed specific fluorescent substrate Z-Phe-Arg-AMC, had acid and heat resistance,was inhibited by metal ions, whose molecular weight was about36ku and belonged to cysteineproteinase containing thiol.