Wofford N Q, Beaty P S, McInerney M J
J Bacteriol. 1986 Jul;167(1):179-85. doi: 10.1128/jb.167.1.179-185.1986.
Syntrophomonas wolfei is an anaerobic fatty acid degrader that can only be grown in coculture with H2-using bacteria such as Methanospirillum hungatei. Cells of S. wolfei were selectively lysed by lysozyme treatment, and unlysed cells of M. hungatei were removed by centrifugation. The cell extract of S. wolfei obtained with this method had low levels of contamination by methanogenic cofactors. However, lysozyme treatment was not efficient in releasing S. wolfei protein; only about 15% of the L-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase activity was found in the lysozyme supernatant. Cell extracts of S. wolfei obtained with this method had high specific activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. These activities were not detected in cell extracts of M. hungatei grown alone, confirming that these activities were present in S. wolfei. The acyl-CoA dehydrogenase activity was high when a C4 but not a C8 or C16 acyl-CoA derivative served as the substrate. S. Wolfei cell extracts had high CoA transferase specific activities and no detectable acyl-CoA synthetase activity, indicating that fatty acid activation occurred by transfer of CoA from acetyl-CoA. Phosphotransacetylase and acetate kinase activities were detected in cell extracts of S. wolfei, indicating that S. wolfei is able to perform substrate-level phosphorylation.
沃氏互营单胞菌是一种厌氧脂肪酸降解菌，只能与诸如亨氏甲烷螺菌等利用氢气的细菌共培养才能生长。通过溶菌酶处理选择性地裂解沃氏互营单胞菌的细胞，然后通过离心去除未裂解的亨氏甲烷螺菌细胞。用这种方法获得 的沃氏互营单胞菌细胞提取物中，产甲烷辅因子的污染水平较低。然而，溶菌酶处理在释放沃氏互营单胞菌蛋白方面效率不高；在溶菌酶上清液中仅发现约15%的L-3-羟酰基辅酶A（CoA）脱氢酶活性。用这种方法获得的沃氏互营单胞菌细胞提取物具有较高的酰基辅酶A脱氢酶、烯酰辅酶A水合酶、L-3-羟酰基辅酶A脱氢酶和3-酮酰基辅酶A硫解酶比活性。在单独培养的亨氏甲烷螺菌细胞提取物中未检测到这些活性，这证实这些活性存在于沃氏互营单胞菌中。当以C4而非C8或C16酰基辅酶A衍生物作为底物时，酰基辅酶A脱氢酶活性较高。沃氏互营单胞菌细胞提取物具有较高的辅酶A转移酶比活性，且未检测到酰基辅酶A合成酶活性，这表明脂肪酸活化是通过辅酶A从乙酰辅酶A转移实现的。在沃氏互营单胞菌细胞提取物中检测到磷酸转乙酰酶和乙酸激酶活性，这表明沃氏互营单胞菌能够进行底物水平磷酸化。
