IAN P.Armste AFRC草地環境研究所遺伝育種部, 研究員
JOHN R.Ling ウェイルズ大学, 生命科学部, 講師
長澤 孝志 岩手大学, 農学部, 助教授 (80189117)
ARMSTEAD Ian AFRC's INSTITUTE OF GRASSLAND AND ENVIRONMENTAL RESEARCH,WALES, 遺伝育種部, 研究員
LING John R. ウェイルズ大学, 生物科学研究所, 講師
富田 純史 宮崎大学, 農学部, 助教授 (70113230)
TOMITA Yoshifumi FACULTY OF AGRICULTURE,MIYAZAKI UNIVERSITY
LING John r. INSTITUTE OF BIOLOGICAL SCIENCES,THE UNIVERSITY OF WALES
|Budget Amount *help
¥5,300,000 (Direct Cost : ¥5,300,000)
Fiscal Year 1994 : ¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1992 : ¥1,600,000 (Direct Cost : ¥1,600,000)
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
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.