Petzelbauer Inge, Splechtna Barbara, Nidetzky Bernd
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien (BOKU), Muthgasse 18, A-1190 Vienna, Austria.
Biotechnol Bioeng. 2002 Feb 15;77(4):394-404.
Hydrolysis of lactose by hyperthermophilic beta-glycosidases from the archaea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) was carried out at 70 degrees C in a continuous stirred-tank reactor (CSTR) coupled to a 10-kDa cross-flow ultrafiltration module to recycle the enzyme. Recirculation rates of > or =1 min(-1), reaction of proteins with reducing sugars, and enzyme adsorption onto the membrane are major "operational" factors of enzyme inactivation in the CSTR. They cause the half-life times of SsbetaGly and CelB to be reduced two- and eight-fold, respectively, the average value for both enzymes now being approximately 5 to 7 days. Using lactose at initial concentrations of 45 and 170 g/L, the CSTR was operated at a constant conversion level of approximately 80% for more than 2 weeks without the occurrence of microbial contamination. The productivities for the SsbetaGly-catalyzed conversion of lactose were determined at different dilution rates and initial substrate concentrations, and exceed by a factor of < or =2 those observed with CelB under otherwise identical conditions. This difference reflects the approximately eight-fold stronger product inhibition of CelB by D-glucose. While the maximum total galacto-oligosaccharide production (90-100 mM) at 170 g/L lactose in the CSTR was not different from that in the batch reactor (CelB) or was greater by approximately 25% (SsbetaGly), continuous and batchwise reactions with both enzymes differed markedly with regard to relative proportions of the individual saccharide components present at 80% substrate conversion. The CSTR yielded an up to four-fold greater ratio of disaccharides to trisaccharides concomitant with a 5- to 30-fold larger relative proportion of beta-D-Galp-(1-->3)-D-Glc in the product mixture. The results show that apart from continuous hydrolysis of lactose at 70 degrees C, a CSTR charged with SsbetaGly or CelB and operated at steady-state conditions could be a useful reaction system for the production of galacto-oligosaccharides in which composition is narrower and more easily programmable, in terms of the individual components contained, as compared to the batchwise reaction.
利用来自嗜热古菌嗜热栖热菌（SsbetaGly）和激烈火球菌（CelB）的超嗜热β-糖苷酶在70℃下于连续搅拌釜式反应器（CSTR）中对乳糖进行水解，该反应器与一个10 kDa错流超滤模块相连以实现酶的循环利用。再循环速率≥1 min⁻¹、蛋白质与还原糖的反应以及酶在膜上的吸附是CSTR中酶失活的主要“操作”因素。它们分别使SsbetaGly和CelB的半衰期缩短了两倍和八倍，两种酶的平均值现在约为5至7天。使用初始浓度为45和170 g/L的乳糖，CSTR在约80%的恒定转化率下运行了超过2周，未发生微生物污染。测定了在不同稀释率和初始底物浓度下SsbetaGly催化乳糖转化的生产率，在其他条件相同的情况下，其生产率比CelB观察到的高出≤2倍。这种差异反映了D-葡萄糖对CelB的产物抑制作用大约强八倍。虽然CSTR中170 g/L乳糖时的最大总低聚半乳糖产量（90 - 100 mM）与间歇反应器（CelB）中的产量没有差异，或者比（SsbetaGly）高出约25%，但两种酶的连续反应和间歇反应在底物转化率为80%时存在的各个糖类组分的相对比例方面有显著差异。CSTR产生的二糖与三糖的比例高达四倍，同时产物混合物中β-D-半乳糖基-(1→3)-D-葡萄糖的相对比例大5至30倍。结果表明，除了在70℃下连续水解乳糖外，装有SsbetaGly或CelB并在稳态条件下运行的CSTR可能是一种用于生产低聚半乳糖的有用反应系统，与间歇反应相比，就所含的各个组分而言，其组成更窄且更易于编程。
