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Lysine and peptide production by rumen bacteria and protozoa

瘤胃细菌和原生动物产生赖氨酸和肽

基本信息

批准号：
04044137
负责人：
ONODERA Ryoji
金额：
$ 3.39万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

This project mainly aimed to establish the methods to separate and collect, and to quantitatively determine the three stereoisomers of 2,6-diaminopimelic acid (DAP) , to clarifiy the distribution of DAP-stereoisomers (DAPSI) in rumen bacterial cell walls, and to examine the lysine production from each DAPSI by rumen protozoa and bacteria.In 1992, a separation and collection method for DAPSI was established using a chiral column, MCI GEL CRS10W (Mitsubishi Kasei, Co.Ltd.) (see J.Chromatogr.A.653 : 336.1993). Then we tried to quantitatively determine DAPSI according to Zanol and Gastaldo (1991). Their method, however, did not work well for separation of DAPSI in bacterial cell walls, and we established a different method using a column, Merck Lichrospher 100 RP-18, without gradient mobile phase different from them, which showed that each peak of DAPSI was clearly separated from unidentified peaks (substances) contained in bacterial hydrolysates.In 1993, DAPSI in many kinds of bacteria we … More re determined. The results showed that the rumen bacteria collected from Welsh sheep and Japanese goats contained 4.99 and 4.04 mg/g dry matter of meso-DAP and 0.72 and 0.97 mg/g dry matter of LL-DAP,respectively, DD-form was not detected in those of both countries. Five purified rumen bacteria were also analyzed and shown to contain only meso-form of DAP.The lowest and highest values were found in Streptococcus bovis JB1 (1.08 mg/g dry matter) and Anaerovibrio lipolytica 5S (6.11 mg/g dry matter), respectively (Amino Acid 5 : 135.1993 ; Proc. Soc. Nutr. Physiol. 3 : 154.1994). All these data are for the first time in the world.In 1994, metabolism of DAPSI by rumen protozoa and bacteria was examined and in these experiment, it was realized at first that we should use sterilized vessels and buffers. Then following results were obtained : In rumen protozoal suspension 0.29,0.16 and 0.12 mM (total 0.57 mM) of meso-, LL-and DD-DAP,respectively, disappeared from the medium, and 0.44 mM of lysine increased in the medium during 12 h-incubation at 39*C.The rest of the disappeared DAP may be converted to pipecolic acid. When each separated DAPSI was singly used as a substrate, similar results were obtained. These results suggested that rumen protozoa might have epimerases. In mixed rumen bacterial suspension, 0.29,0.17 and 0.12 mM of meso-, LL-and DD-DAP,respectively, disappeared from the medium, and 25.9% of the disappeared DAP was accumulated as lysine in bacteria. The rest may be converted to ammonia, acetate and butyrate. When each separated DAPSI was singly used as a substrate, similar results were obtained. Less

本课题主要旨在建立2,6-二氨基苯甲酸（DAP）的三种立体异构体的分离、收集和定量测定方法，阐明DAP立体异构体（DAPSI）在瘤胃细菌细胞壁中的分布，并检测瘤胃原生动物和细菌从每种DAPSI中产生的赖氨酸。1992年，采用手性色谱柱MCI GEL CRS10W （Mitsubishi Kasei, Co.Ltd.）建立了一种分离收集DAPSI的方法。(见J.Chromatogr.A。653: 336.1993)。然后我们尝试根据Zanol和Gastaldo（1991）定量确定DAPSI。然而，他们的方法并不能很好地分离细菌细胞壁中的DAPSI，我们建立了一种不同的方法，使用Merck Lichrospher 100 RP-18柱，没有与他们不同的梯度流动相，这表明DAPSI的每个峰都与细菌水解物中含有的未知峰（物质）清晰分离。1993年，我们对多种细菌中的DAPSI进行了更多的测定。结果表明，威尔士绵羊和日本山羊瘤胃细菌中分别含有4.99和4.04 mg/g中位磷酸二铵干物质，0.72和0.97 mg/g中位磷酸二铵干物质，两国瘤胃细菌中未检出dd型磷酸二铵。对5种纯化的瘤胃细菌也进行了分析，结果显示它们只含有中位形式的DAP。在牛链球菌JB1 （1.08 mg/g干物质）和脂溶性厌氧弧菌5S （6.11 mg/g干物质）中含量最低和最高(氨基酸5:135.1993;Proc. Soc。减轻。生理学。3:154.1994)。所有这些数据在世界上都是第一次。1994年，研究了瘤胃原生动物和细菌对DAPSI的代谢，在这些实验中，首次意识到我们应该使用灭菌的容器和缓冲液。结果表明：在39*C条件下培养12 h，瘤胃原虫悬浮液中meso-、ll -和DD-DAP的含量分别从培养基中消失0.29、0.16和0.12 mM（总含量0.57 mM），赖氨酸含量增加0.44 mM。其余消失的DAP可能转化为细果酸。当每个分离的DAPSI单独用作底物时，得到类似的结果。这些结果提示，瘤胃原生动物可能存在外膜酶。在混合瘤胃细菌悬浮液中，培养基中消失了0.29 mM、0.17 mM和0.12 mM的中膜磷酸酶（meso-）、ll -磷酸酶（ll -）和dd -磷酸酶（dd -磷酸酶），其中25.9%的磷酸酶以赖氨酸的形式在细菌中积累。其余的可转化为氨、醋酸盐和丁酸盐。当每个分离的DAPSI单独用作底物时，得到类似的结果。少