Goosen Coenie, Yuan Xiao-Lian, van Munster Jolanda M, Ram Arthur F J, van der Maarel Marc J E C, Dijkhuizen Lubbert
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Eukaryot Cell. 2007 Apr;6(4):674-81. doi: 10.1128/EC.00361-06. Epub 2007 Feb 9.
A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40 degrees C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent K(m), K(i), and V(max) of 2.0 +/- 0.2 mM, 268.1 +/- 18.1 mM, and 6.6 +/- 0.2 mumol min(-1) mg(-1) of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides.
此前在真菌基因组中已鉴定出一个假定的细胞内转化酶新亚家族(糖苷水解酶家族32)。在此,我们报告了黑曲霉中鉴定出的两种新型细胞内转化酶之一SucB的系统发育、分子和生化特性。sucB基因在大肠杆菌和酿酒酵母的转化酶阴性菌株中表达。从大肠杆菌裂解物中纯化的酶经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析判断,分子量为75 kDa。其水解蔗糖的最适pH和温度分别确定为5.0和37至40摄氏度。除蔗糖外,该酶还能水解1-蔗果三糖、棉子糖和水苏糖,但不能水解菊粉和左聚糖。SucB分别从蔗糖和1-蔗果三糖产生1-蔗果三糖和棉子糖。以棉子糖为底物时,观察到聚合度高达4的产物。SucB表现出典型的米氏动力学,对蔗糖有底物抑制作用(表观K(m)、K(i)和V(max)分别为2.0±0.2 mM、268.1±18.1 mM和6.6±0.2 μmol min(-1) mg(-1)蛋白质[总活性])。在蔗糖浓度高达400 mM时,转果糖基化(FTF)活性约占总活性的20%至30%。在较高蔗糖浓度下,FTF活性增加至总活性的50%。黑曲霉中sucB的缺失导致在含有各种碳源的固体培养基上孢子形成提前开始,而在液体培养基中未观察到生长变化。因此,SucB在黑曲霉的菊粉或蔗糖分解代谢中不发挥重要作用,但可能是蔗糖在细胞内转化为果糖、葡萄糖和小寡糖所必需 的。