论文部分内容阅读
本试验旨在研究绵羊瘤胃细菌、原虫蛋白质分解代谢相关酶活力及谷氨酸脱氢酶体系的米氏常数(Km)值,为解释绵羊瘤胃细菌、原虫蛋白质分解代谢特征提供酶学依据。选用6只1岁左右安装永久性瘤胃瘘管的中国美利奴(新疆型)绵羊[平均体重为(32.00±1.36)kg],饲喂精粗比为30∶70的饲粮,依次采集饲喂前(0 h)和饲喂后1.5、3.0、6.0、9.0、12.0 h 6个时间点的瘤胃液,重复采集3次。分离和制备细菌、原虫破碎液,分别测定相关酶活力及谷氨酸脱氢酶体系的Km值。结果显示:1)绵羊瘤胃细菌、原虫破碎液中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力随饲喂时间的延长均呈现先升高后降低的动态变化规律,总体在饲喂后1.5 h达到峰值;谷氨酸和氨含量也呈现相似的变化规律。原虫破碎液中参与蛋白质分解代谢的这4种酶的活力在各时间点均极显著高于细菌(P<0.01)。2)原虫破碎液谷氨酸含量极显著高于细菌(P<0.01);原虫破碎液氨含量在1.5、6.0、9.0和12.0 h显著或极显著高于细菌(P<0.05或P<0.01)。3)绵羊瘤胃细菌、原虫谷氨酸脱氢酶对烟酰胺腺嘌呤二核苷酸(NAD)的Km值分别为2.60×10~(-7)、1.48×10~(-7)mol/L;细菌、原虫谷氨酸脱氢酶对谷氨酸的Km值分别为8.41×10~(-6)、4.91×10~(-6)mol/L;细菌、原虫谷氨酸脱氢酶对还原型烟酰胺腺嘌呤二核苷酸(NADH)的Km值分别为3.80×10~(-8)、2.70×10~(-8)mol/L;细菌、原虫谷氨酸脱氢酶对α-酮戊二酸的Km值分别为1.16×10~(-6)、2.07×10~(-6)mol/L;细菌、原虫谷氨酸脱氢酶对氨的Km值分别为2.97×10~(-5)、1.40×10~(-5)mol/L。结果提示,总体上,绵羊瘤胃细菌、原虫中蛋白酶、谷氨酸脱氢酶、谷丙转氨酶、谷草转氨酶的活力在饲喂后1.5 h达到峰值,之后逐渐降低;绵羊瘤胃原虫中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力均极显著高于细菌,原虫中蛋白质分解代谢更旺盛;瘤胃原虫中不仅存在谷氨酸转氨机制,还可能存在利用氨重新合成氨基酸的机制。
The purpose of this study was to investigate the Km value of protease catabolism related to rumen bacteria and protozoal in sheep and to provide an enzymological basis for explaining the protein catabolism characteristics of sheep rumen bacteria and protozoa. Six Chinese Merino (Xinjiang-type) sheep with a permanent rumen fistula at the age of 1 were selected [mean body weight (32.00 ± 1.36) kg] and fed with a ration of 30:70 Before (0 h) and 1.5, 3.0, 6.0, 9.0, 12.0 h after feeding six time points rumen fluid, repeated three times. Isolation and preparation of bacteria, protozoa broken liquid, respectively, determination of enzyme activity and glutamate dehydrogenase system Km value. The results showed that: 1) The activities of protease, alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase in sheep rumen bacteria and protozoa crush fluid increased first and then decreased with the prolongation of feeding time, 1.5 h after feeding peaked; glutamate and ammonia content also showed a similar pattern of changes. The activity of the four enzymes involved in protein catabolism in protozoal crushed liquid was significantly higher than that of bacteria at all time points (P <0.01). 2) The content of glutamic acid in the protozoa crushed liquid was significantly higher than that in the bacteria (P <0.01). The ammonia content of protozoa crushed liquid was significantly or extremely significantly higher than that of the bacteria at 1.5, 6.0, 9.0 and 12.0 h (P <0.05 or P <0.01) . 3) The Km values of rumen bacteria and protozoal glutamate dehydrogenase for nicotinamide adenine dinucleotide (NAD) were 2.60 × 10 -7 and 1.48 × 10 -7 mol / L, respectively The Km values of glutamic acid and glutamic acid dehydrogenase of bacteria and protozoa were 8.41 × 10 -6 and 4.91 × 10 -6 mol / L, respectively. Bacteria and protozoa glutamate dehydrogenase The Km values of NADH were 3.80 × 10 -8 and 2.70 × 10 -8 mol / L, respectively. Bacteria and protozoal glutamate dehydrogenase had the highest K Km value of ketoglutarate were 1.16 × 10 -6 and 2.07 × 10 -6 mol / L, respectively. The Km values of ammonia and glutamic acid dehydrogenase of bacteria and protozoa were 2.97 × 10 ~ (-5), 1.40 × 10 ~ (-5) mol / L. The results suggest that, in general, the activities of protease, glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase in sheep rumen bacteria and protozoa reached the peak at 1.5 h after feeding, and then decreased gradually. In sheep rumen protozoal proteases, Aminotransferase, aspartate aminotransferase and glutamate dehydrogenase activity were extremely significantly higher than the bacteria protozoan protein catabolism is more vigorous; rumen protozoa not only exists glutamate transaminase mechanism, there may be a mechanism of using ammonia to re-synthesize amino acids .