Roskilde University, NSM, Biomaterials, Universitetsvej 1, DK-4000, Roskilde, Denmark.
Appl Biochem Biotechnol. 2011 Mar;163(5):626-35. doi: 10.1007/s12010-010-9068-z. Epub 2010 Aug 29.
Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end, development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis. In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about 500 pmol glucose s(-1). Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the complexity of the substrate. Dose-response experiments with a typical cellulase cocktail enabled a multidimensional analysis of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis rate of pretreated corn stover is limited initially by available attack points on the substrate surface (<10% conversion) but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced hydrolysis rate over time.
酶解高固体生物质（>10%干物质）在第二代生物乙醇生产中作为关键步骤变得越来越重要。为此，开发定量实时分析方法对于经验优化和详细的动力学分析都是可取的。在目前的工作中，我们研究了等温量热法在研究两种底物（预处理玉米秸秆和 Avicel）在高固体含量（高达 29%干物质）下的酶解动力学中的应用。结果发现，量热热流提供了水解速率的真实测量，检测限约为 500 pmol 葡萄糖 s(-1)。因此，量热法被证明是一种高灵敏度的实时方法，适用于高固体，并且与底物的复杂性无关。用典型的纤维素酶混合物进行剂量反应实验，能够对酶负荷与反应速率、时间和程度的相互关系进行多维分析。结果表明，预处理玉米秸秆的水解速率最初受限于底物表面的可用攻击点（<10%转化率），但在后期（>10%转化率）与酶剂量成正比。这种动力学模式被解释为随着水解的进行，聚合物末端浓度（CBH 的底物）增加，可能是由于酶混合物中的 EG 活性所致。最后，不可逆的酶失活似乎不是水解速率随时间降低的原因。
