Enzyme Activities Related to Protein Catabolism and Km Value of Glutamate Dehydrogenase System in Rumen Bacteria and Protozoa of Sheep

XIONG Wenxiu; ZHAI Weishuang; Tunnisa Maitisaiyidi; WU Xin; YANG Kailun

doi:10.3969/j.issn.1006-267x.2017.01.037

2017 , Vol. 29 >Issue 1: 325 - 332

DOI: https://doi.org/10.3969/j.issn.1006-267x.2017.01.037

SHORT COMMUNICATIONS

Enzyme Activities Related to Protein Catabolism and K_m Value of Glutamate Dehydrogenase System in Rumen Bacteria and Protozoa of Sheep

XIONG Wenxiu ,
ZHAI Weishuang ,
Tunnisa Maitisaiyidi ,
WU Xin ,
YANG Kailun

Expand

Xinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk Production, Xinjiang Agricultural University, Urumqi 830052, China

Received date: 2016-07-04

Online published: 2017-01-16

Fold

Abstract

This study was to conducted to determine enzyme activities related to protein catabolism and K_m value of glutamate dehydrogenase (GDH) system in rumen bacteria and protozoa of sheep, to provide enzymology reference to explain protein catabolism in rumen bacteria and protozoa of sheep. Six 1-year-old healthy Chinese merino sheep (Xinjiang type)[average body weight was (32.00±1.36) kg] were chosen. The sheep were installed with permanent fistula in rumen. Dietary forage to concentrate ratio was 30:70. Rumen fluid was collected sequentially before feeding (0 h) and 1.5, 3.0, 6.0, 9.0 and 12.0 h after feeding, respectively, which were repeated 3 times. After centrifugation, crushing liquid of rumen bacteria and protozoa was prepared to determine the activities of enzymes and Km values of GDH system. The results showed as follows:1) the activities of protease, glutamic-pyruvic transaminase (GPT), glutamic oxal(o)acetic transaminase (GOT) and GDH showed an initial increase and then a decrease as the feeding time passed by, and generally reached the maximum at 1.5 h after feeding in rumen bacteria and protozoa crushing liquid of sheep; the contents of glutamate and ammonia (NH₄⁺) contents showed similar tendency. The activities of the four enzymes involved in protein metabolism in rumen protozoa crushing liquid were significantly higher than those in rumen bacteria (P<0.01). 2) Glutamate content of rumen protozoa crushing liquid was significantly higher than that of bacteria (P<0.01). Ammonia content of rumen protozoa crushing liquid was significantly higher than that of bacteria at 1.5, 6.0, 9.0 and 12.0 h (P < 0.05 or P < 0.01). 3) K_m values of GDH to NAD in bacteria and protozoa crushing liquid were 2.60×10^-7 and 1.48×10^-7 mol/L, respectively; those to glutamate in bacteria and protozoa crushing liquid were 8.41×10^-6 and 4.91×10^-6 mol/L, respectively; those to NADH in bacteria and protozoa crushing liquid were 3.80×10^-8 and 2.70×10^-8 mol/L, respectively; those to α-oxoglutarate in bacteria and protozoa crushing liquid were 1.16×10^-6 and 2.07×10^-6 mol/L, respectively; those to ammonia in bacteria and protozoa crushing liquid were 2.97×10^-5 and 1.40×10^-5 mol/L, respectively. The results indicate that the activities of protease, GPT, GOT and GDH generally reach the maximum at 1.5 h after feeding, then gradually decrease in rumen bacteria and protozoa of sheep; meanwhile, the enzyme activities in protozoa are significantly higher than those in bacteria, and protein catabolism in protozoa is more active than in bacteria; there is not only a transamination mechanism of glutamate, but also a re-using ammonia mechanism for synthesis of amino acids in rumen protozoa.

Key words： rumen; bacteria; protozoa; protein catabolism; enzyme activity; K_m value

Cite this article

XIONG Wenxiu , ZHAI Weishuang , Tunnisa Maitisaiyidi , WU Xin , YANG Kailun . Enzyme Activities Related to Protein Catabolism and K_m Value of Glutamate Dehydrogenase System in Rumen Bacteria and Protozoa of Sheep[J]. Chinese Journal of Animal Nutrition, 2017 , 29(1) : 325 -332 . DOI: 10.3969/j.issn.1006-267x.2017.01.037

References

[1] 冯仰廉.反刍动物营养学[M].北京:科学出版社,2004:35.
[2] RUSSELL J B,ONODERA R,HINO T.Ruminal protein fermentation:new perspectives on previous contradictions[M]//TSUDA T,SASAKI Y,KAWASHIMA R.Physiological aspects of digestion and metabolism in ruminants.Tokyo:Academic Press,1991:681-697.
[3] JOUANY J P,USHIDA K.The role of protozoa in feed digestion[J].Asian-Australasian Journal of Animal Sciences,1999,12(1):113-128.

[4] BOGUHN J,KLUTH H,RODEHUTSCORD M.Effect of total mixed ration composition on amino acid profiles of different fractions of ruminal microbes in vitro[J].Journal of Dairy Science,2006,89(5):1592-1603.

[5] LENG R A.Dynamics of protozoa in the rumen of sheep[J].British Journal of Nutrition,1982,48(2):399-415.

[6] WALLACE R J,ONODERA R,COTTA M A.Metabolism of nitrogen-containing compounds[M]//HOBSON P N,STEWART C S.The rumen microbial ecosystem.Netherlands:Springer,1997:283-328.
[7] WALLACE R J.Ruminal microbial metabolism of peptides and amino acids:altering ruminal nitrogen metabolism to improve protein utilization[J].The Journal of Nutrition,1996,126(4):1326S-1334S.
[8] WALLACE R J,BRODERICK G A,BRAMMALL M L.Protein degradation by ruminal microorganisms from sheep fed dietary supplements of urea,casein,or albumin[J].Applied and Environmental Microbiology,1987,53(4):751-753.
[9] FIRKINS J L,YU Z,MORRISON M.Ruminal nitrogen metabolism:perspectives for integration of microbiology and nutrition for dairy[J].Journal of Dairy Science,2007,90(Suppl.1):E1-E16.
[10] HINO T,RUSSELL J B.Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts[J].Applied and Environmental Microbiology,1985,50(6):1368-1374.
[11] 张龙翔,张庭芳,李令媛.生化实验方法和技术[M].2版.北京:高等教育出版社,1997:135-140.
[12] PALMQUIST D L,BALDWIN R L.Enzymatic techniques for the study of pathways of carbohydrate utilization in the rumen[J].Applied and Environmental Micobiology,1966,14(1):60-69.
[13] 吉尔鲍特G G.酶法分析手册[M].缪辉南,陈石根,译.上海:上海科学技术出版社,1983:89-96.
[14] CHEN G,RUSSELL J B,SNIFFEN C J.A procedure for measuring peptides in rumen fluid and evidence that peptide uptake can be a rate-limiting step in ruminal protein degradation[J].Journal of Dairy Science,1987,70(6):1211-1219.

[15] 王梦芝.山羊瘤胃原虫与细菌吞噬关系和微生物AA变化机制的研究[D].博士学位论文.扬州:扬州大学,2008:56-59.
[16] 翟卫爽.不同精粗比日粮时绵羊瘤胃液细菌、原虫内与葡萄糖代谢相关酶活力的比较研究[D].硕士学位论文.乌鲁木齐:新疆农业大学,2014:34-39.
[17] SHIIO I,OZAKI H.Regulation of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Brevibacterium flavum,a glutamate-producing bacterium[J].Journal of Biochemistry,1970,68(5):633-647.
[18] KUJO C,OHSHIMA T.Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents[J].Applied and Environmental Microbiology,1998,64(6):2152-2157.
[19] NEWBOLD C J,MCEWAN N R,CALZA R E,et al.An NAD⁺-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan,Entodinium caudatum[J].FEMS Microbiology Letters,2005,247(2):113-121.

[20] WILLIAMS A G,COLEMAN G S.The rumen protozoa[M].New York:Springer,1992:135-138.

Options

Outlines

模态框（Modal）标题

Abstract

Cite this article

References