Patel Basant Kumar, Jagannadham Medicherla V
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India.
J Agric Food Chem. 2003 Oct 8;51(21):6326-34. doi: 10.1021/jf026184d.
A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.
从海南狗牙花（夹竹桃科）的乳汁中纯化出一种半胱氨酸蛋白酶，其半胱氨酸含量高，且氨基末端序列与ervatamins B和C具有高度同源性。这种被命名为海因宁（M(r)=23 kDa）的酶具有相对较高的半胱氨酸含量（11个）、较高的等电点（10.8），并且对pH（2.5 - 11.5）、温度（63℃，15分钟）、强变性剂和有机溶剂具有高稳定性。该酶对酪蛋白和偶氮白蛋白等天然底物具有高比活性。最适pH和温度分别为pH 8.0 - 8.5和52±2℃。与ervatamins和木瓜蛋白酶相比，合成底物的水解和牛血清白蛋白的消化证实了海因宁具有独特的特异性。此外，通过酶联免疫吸附测定和奥克特洛尼双免疫扩散监测发现，海因宁具有明显的免疫原性。β-巯基乙醇、二硫苏糖醇等还原剂对酶有强烈激活作用，而E-64和碘乙酸等巯基蛋白酶抑制剂对酶有强烈抑制作用，这证明海因宁是一种半胱氨酸蛋白酶。高稳定性、比活性以及易于纯化可能使海因宁成为食品和生物技术应用中的一种潜在蛋白酶。
