Parenicová L, Benen J A, Kester H C, Visser J
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
Eur J Biochem. 1998 Jan 15;251(1-2):72-80. doi: 10.1046/j.1432-1327.1998.2510072.x.
In the present study, the molecular and basic biochemical characterization of endopolygalacturonase E, the fourth Aspergillus niger N400 endopolygalacturonase, is reported. The entire endopolygalacturonase E gene consists of 1293 bp interrupted by three short introns (50, 50, and 59 bp, respectively) as concluded from the cDNA sequence. The deduced amino acid sequence comprises 378 residues that include 39 N-terminal amino acids of the prepropeptide. The calculated Mr and pI of the mature protein are 35,584 and 3.6, respectively. Compared with other endopolygalacturonases from A. niger N400, the mature protein endopolygalacturonase E has the highest sequence identity with endopolygalacturonase C (77.6%) followed by endopolygalacturonase I (57.6%) and endopolygalacturonase II (54.3%). For overproduction of endopolygalacturonase E, an A. niger multicopy strain was used that was transformed with a promoter gene fusion construct that directs expression from the glycolytic A. niger pyruvate kinase promoter. The enzyme was purified and characterized as an endopolygalacturonase based on product analysis after polygalacturonate hydrolysis and on bond cleavage frequencies of oligogalacturonates of different degree of polymerisation (n = 2-7). The pH optimum was 3.8. The Km and Vmax for polygalacturonate hydrolysis were 2.5 +/- 0.4 mg x ml(-1) and 1.3 +/- 0.2 microkat x mg(-1), respectively. A subsite map was calculated by the combination of the methods of Suganuma et al. [Suganuma, T., Matsuno, R., Ohnishi, M. & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 293-316] and Nitta et al. [Nitta, Y., Mizushima, M., Hiromi, K. & Ono, S. (1971) J. Biochem. (Tokyo) 69, 567-576]. This indicated that the enzyme was composed of at least five subsites.
在本研究中，报道了黑曲霉N400的第四个内切聚半乳糖醛酸酶——内切聚半乳糖醛酸酶E的分子和基础生化特性。根据cDNA序列推断，完整的内切聚半乳糖醛酸酶E基因由1293 bp组成，被三个短内含子（分别为50、50和59 bp）中断。推导的氨基酸序列包含378个残基，其中包括前原肽的39个N端氨基酸。成熟蛋白的计算分子量和pI分别为35,584和3.6。与黑曲霉N400的其他内切聚半乳糖醛酸酶相比，成熟蛋白内切聚半乳糖醛酸酶E与内切聚半乳糖醛酸酶C的序列同一性最高（77.6%），其次是内切聚半乳糖醛酸酶I（57.6%）和内切聚半乳糖醛酸酶II（54.3%）。为了过量生产内切聚半乳糖醛酸酶E，使用了一个黑曲霉多拷贝菌株，该菌株用一个启动子基因融合构建体进行转化，该构建体指导从糖酵解的黑曲霉丙酮酸激酶启动子进行表达。基于聚半乳糖醛酸水解后的产物分析以及不同聚合度（n = 2 - 7）的寡聚半乳糖醛酸的键断裂频率，该酶被纯化并鉴定为内切聚半乳糖醛酸酶。最适pH为3.8。聚半乳糖醛酸水解的Km和Vmax分别为2.5 +/- 0.4 mg x ml(-1)和1.3 +/- 0.2 microkat x mg(-1)。通过Suganuma等人[Suganuma, T., Matsuno, R., Ohnishi, M. & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 293 - 316]和Nitta等人[Nitta, Y., Mizushima, M., Hiromi, K. & Ono, S. (1971) J. Biochem. (Tokyo) 69, 567 - 576]的方法相结合计算出一个亚位点图谱。这表明该酶由至少五个亚位点组成。
