Kumar Ashwani, Sharma Jitender, Mohanty Ashok Kumar, Grover Sunita, Batish Virender Kumar
Division of Dairy Microbiology, National Dairy Research Institute, Karnal-132 001, India.
Comp Biochem Physiol B Biochem Mol Biol. 2006 Sep;145(1):108-13. doi: 10.1016/j.cbpb.2006.06.010. Epub 2006 Jun 29.
Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor prochymosin. It is responsible for hydrolysis of kappa-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat (Capra hircus) chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 degrees C with maximum activity at 30 degrees C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat chymosin increased with incubation time at 37 degrees C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.
凝乳酶是皱胃酶（牛奶凝结酶）的主要成分，是一种酸性蛋白酶，由以无活性前体凝乳酶原形式存在的哺乳期反刍动物（包括山羊和绵羊）的第四胃产生。它负责水解牛奶酪蛋白胶粒中的κ-酪蛋白链，因此，在奶酪制备中用作牛奶凝固剂。本研究旨在纯化和鉴定山羊（Capra hircus）凝乳酶作为牛奶凝固剂的适用性。该酶从羔羊皱胃组织中提取，并使用阴离子交换剂和凝胶过滤色谱法纯化了近30倍。山羊凝乳酶在通过DEAE-纤维素离子交换柱时分解为三个主要活性峰，表明可能存在异质性。在SDS-PAGE上，纯化后的酶分子量为36 kDa，这通过蛋白质印迹分析得到进一步证实。纯化后的酶制剂在55℃下稳定，在30℃时活性最高。随着pH值升高，牛奶凝固活性稳步下降，在pH 5.5时显示出最大活性。山羊凝乳酶的蛋白水解活性在37℃下随孵育时间增加。发现山羊凝乳酶比牛凝乳酶更耐热，并且与水牛凝乳酶稳定性相当。
