Lysine and peptide production by rumen bacteria and protozoa

本课题的主要目的是建立2，6-二氨基苯甲酸(DAP)三种立体异构体的分离、分离和定量测定方法，明确DAP-立体异构体(DAPSI)在瘤胃细菌细胞壁中的分布，以及检测瘤胃原生动物和细菌从DAPSI中产生赖氨酸的情况。1992年，采用手性柱MCI Gel CRS10W(Mitsubishi Kasei，Co.Ltd.)建立了DAPSI的分离和收集方法。(见J.Chromatogr.A.653：336.1993)。然后，我们尝试根据Zano和Gastaldo(1991)对DAPSI进行定量测定。然而，他们的方法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色谱柱，没有与它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的未知峰(物质)明显分开。1993年，我们…了许多种细菌中的DAPSI更加坚定。结果表明，威尔士绵羊和日本山羊瘤胃细菌的中位DAP含量分别为4.99和4.04 mg/g干物质，LL-DAP分别为0.72和0.97 mg/g干物质，均未检出DD型。5种纯化的瘤胃细菌中只含有中位型DAP，其中牛链球菌JB1含量最低，为1.08 mg/g干物质，解脂厌氧弧菌5S含量最高，为6.11 mg/g干物质，氨基酸含量为5：135.1993；SoC。坚果。物理。3：154.1994)。所有这些数据在世界上都是第一次。1994年，我们研究了瘤胃原生动物和细菌对DAPSI的代谢，在这些实验中，我们首先意识到应该使用灭菌的容器和缓冲液。结果表明：瘤胃原生动物悬浮液中0.29、0.16和0.12 mM(共0.57 mM)的Meso-DAP、LL-DAP和DD-DAP在39℃孵育12h后消失，培养液中赖氨酸增加0.44 mM，其余消失的DAP可转化为吡哌酸。当每个分离的DAPSI单独作为底物时，得到了相似的结果。这些结果表明，瘤胃原生动物可能存在下同分异构酶。在混合瘤胃细菌悬液中，0.29、0.17和0.12 mM的Meso-DAP、LL-DAP和DD-DAP分别从培养液中消失，25.9%的消失DAP以赖氨酸形式在细菌中积累。其余的可以转化为氨、醋酸盐和丁酸盐。当每个分离的DAPSI单独作为底物时，得到了相似的结果。较少

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