Talbot G, Sygusch J
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Quebec, Canada.
Appl Environ Microbiol. 1990 Nov;56(11):3505-10. doi: 10.1128/aem.56.11.3505-3510.1990.
Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.
嗜热栖热芽孢杆菌分泌能够水解半乳甘露聚糖底物的β-甘露聚糖酶和α-半乳糖苷酶活性。在刺槐豆胶存在的情况下，半纤维素酶活性的表达是相继发生的，甘露聚糖酶活性先于α-半乳糖苷酶活性的表达。通过硫酸铵分级分离、凝胶过滤、疏水相互作用色谱以及离子交换和聚焦色谱技术的组合，将半纤维素酶活性纯化至同质。纯化的β-D-甘露聚糖酶是一种二聚体酶（162千道尔顿），由分子量相同（73,000）的亚基组成。最大活性在pH 5.5至7.5之间没有变化。β-D-甘露聚糖酶活性表现出热稳定性，在70℃和pH 6.5下孵育24小时后仍保留几乎全部活性。该酶对半乳甘露聚糖底物具有高度特异性，未检测到二级木聚糖酶或纤维素酶活性。刺槐豆胶的水解产生与底物解聚的内切模式相容的短寡糖。在70℃和pH 6.5下，甘露聚糖酶活性的初始速率显示出底物抑制作用，Vmax和Km的估计值分别为455±60 U/mg和1.5±0.3 mg/ml。α-半乳糖苷酶活性对应于一种三聚体酶（247千道尔顿），其亚基分子量相同（82,000）。α-半乳糖苷酶在pH 7至7.5时具有最大活性，在60℃孵育24小时后仍保留全部活性。与对硝基苯基α-D-半乳糖的水解相比，该酶对半乳甘露聚糖底物的活性有限。对硝基苯基α-D-半乳糖水解的动力学产生线性倒数图，在60℃和pH 7下，Vmax和Km分别为195±10 U/mg和0.25±0.02 mM。甘露聚糖酶活性的表征与其在软木浆酶法漂白中的潜在用途一致。
