Benen J A, Kester H C, Visser J
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
Eur J Biochem. 1999 Feb;259(3):577-85. doi: 10.1046/j.1432-1327.1999.00080.x.
Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3-7). Apparent Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mukat.mg-1, 36.5 mukat.mg-1 and 415 nkat.mg-1 for endopolygalacturonases I, II and C, respectively. K(m) values were < 0.15 mg.mL-1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes. Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated delta 4,5-unsaturated oligogalacturonates of degree of polymerization 4-8. Whereas endopolygalacturonase I showed a strong preference for generating the delta 4,5-unsaturated dimer, with endopolygalacturonase II the delta 4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the delta 4,5-unsaturated dimer and trimer were observed, although in different ratios.
从重组黑曲霉菌株中分离出的内切聚半乳糖醛酸酶I、II和C，针对其最适pH值、对聚半乳糖醛酸的活性、作用模式以及对不同链长（n = 3 - 7）的低聚半乳糖醛酸的动力学进行了表征。以内切聚半乳糖醛酸酶I、II和C为例，在最适pH值4.1下，以聚半乳糖醛酸为底物计算得到的表观Vmax值分别为13.8 μkat·mg-1、36.5 μkat·mg-1和415 nkat·mg-1。这三种酶的K(m)值均< 0.15 mg·mL-1。以聚半乳糖醛酸为底物进行的产物进程分析表明，这三种酶均呈现随机切割模式，且内切聚半乳糖醛酸酶I和C表现出持续作用行为。使用低聚半乳糖醛酸对作用模式进行分析，证实了这一结果。对于内切聚半乳糖醛酸酶I和C，当底物的聚合度分别超过5或6时，观察到了持续作用。通过对低聚半乳糖醛酸水解获得的键切割频率用于评估亚位点图谱。图谱表明，这三种酶的亚位点最少数量均为7个。使用不同酯化度的果胶表明，内切聚半乳糖醛酸酶II对甲酯的存在最为敏感。与内切聚半乳糖醛酸酶II一样，本研究这一部分中包含的内切聚半乳糖醛酸酶I、C和E均更倾向于非酯化的果胶酸。通过分析果胶酸裂解酶产生的聚合度为4 - 8的δ4,5 - 不饱和低聚半乳糖醛酸混合物水解反应产物，揭示了底物特异性的其他差异。内切聚半乳糖醛酸酶I强烈倾向于生成δ4,5 -不饱和二聚体，而内切聚半乳糖醛酸酶II积累的是δ4,5 -不饱和三聚体，这表明底物特异性存在进一步差异。对于内切聚半乳糖醛酸酶C和E，均观察到了δ4,5 -不饱和二聚体和三聚体，尽管比例不同。
