Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable beta-glycosidases.
Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable beta-glycosidases.
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DOI：
10.1002/(sici)1097-0290(20000720)69:2
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发表时间：
2000-07
影响因子：
3.8
通讯作者：
I. Petzelbauer;R. Zeleny;A. Reiter;K. D. Kulbe;B. Nidetzky
中科院分区：
文献类型：
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作者：
I. Petzelbauer;R. Zeleny;A. Reiter;K. D. Kulbe;B. Nidetzky
在70 ℃下的乳糖转化过程中，当由来自古生硫化叶菌（Sulfolobus solfataricus）（SsbetaGly）和激烈火球菌（Pyrococcus furiosus）（CelB）的β-糖苷酶催化时，半乳糖基转移到除水以外的受体与底物的完全水解有效竞争。该过程导致一系列新产品的瞬时形成，主要是二糖和三糖，并显示出对初始底物浓度和乳糖转化率的显著依赖性。用毛细管电泳和高效阴离子交换色谱对寡聚体进行了定量分析。初始乳糖浓度为270 g/L时，在乳糖转化率为80%时，其最大浓度为86 g/L。对于这两种酶，三糖与二糖的摩尔比在反应的早期阶段是最大的，并且与增加的底物转化率成正比地降低。总的来说，CelB比SsbetaGly多产生约6%的水解副产物。然而，SsbetaGly的产物谱更富含三硫键，这与SsbetaGly和CelB催化半乳糖基转移的稳态动力学分析结果一致。SsbetaGly和CelB的主要转半乳糖基化产物已被鉴定。它们是β-D-Galp-（1->3）-Glc和β-D-Galp-（1->6）-Glc，以及β-D-Galp-（1->3）-乳糖和β-D-Galp-（1->6）-乳糖，并且它们的形成和降解已显示依赖于乳糖转化。这两种酶在反应后期积累β（1->6）-连接的糖苷，特别是异乳糖。因为高寡糖浓度占优势直到约80%乳糖转化，所以热稳定的β-糖苷酶对于从乳糖生产寡糖是有效的。因此，它们被证明是乳糖利用的稳定和通用催化剂。
During lactose conversion at 70 degrees C, when catalyzed by beta-glycosidases from the archea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB), galactosyl transfer to acceptors other than water competes efficiently with complete hydrolysis of substrate. This process leads to transient formation of a range of new products, mainly disaccharides and trisaccharides, and shows a marked dependence on initial substrate concentration and lactose conversion. Oligosaccharides have been analyzed quantitatively by using capillary electrophoresis and high performance anion-exchange chromatography. At 270 g/L initial lactose, they accumulate at a maximum concentration of 86 g/L at 80% lactose conversion. With both enzymes, the molar ratio of trisaccharides to disaccharides is maximal at an early stage of reaction and decreases directly proportional to increasing substrate conversion. Overall, CelB produces about 6% more hydrolysis byproducts than SsbetaGly. However, the product spectrum of SsbetaGly is richer in trisaccharides, and this agrees with results obtained from the steady-state kinetics analyses of galactosyl transfer catalyzed by SsbetaGly and CelB. The major transgalactosylation products of SsbetaGly and CelB have been identified. They are beta-D-Galp-(1-->3)-Glc and beta-D-Galp-(1-->6)-Glc, and beta-D-Galp-(1-->3)-lactose and beta-D-Galp-(1-->6)-lactose, and their formation and degradation have been shown to be dependent upon lactose conversion. Both enzymes accumulate beta(1-->6)-linked glycosides, particularly allolactose, at a late stage of reaction. Because a high oligosaccharide concentration prevails until about 80% lactose conversion, thermostable beta-glycosidases are efficient for oligosaccharide production from lactose. Therefore, they prove to be stable and versatile catalysts for lactose utilization.