IFP, Biotechnology Department, Avenue de Bois-Préau 92852 Rueil-Malmaison, France.
Biotechnol Biofuels. 2010 Feb 11;3(1):3. doi: 10.1186/1754-6834-3-3.
The enzymatic hydrolysis of cellulose is still considered as one of the main limiting steps of the biological production of biofuels from lignocellulosic biomass. It is a complex multistep process, and various kinetic models have been proposed. The cellulase enzymatic cocktail secreted by Trichoderma reesei has been intensively investigated. beta-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. beta-glucosidase (BGL1) is very poorly secreted by Trichoderma reesei strains, and complete hydrolysis of cellulose often requires supplementation with a commercial beta-glucosidase preparation such as that from Aspergillus niger (Novozymes SP188). Surprisingly, kinetic modeling of beta-glucosidases lacks reliable data, and the possible differences between native T. reesei and supplemented beta-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1.
A comparative kinetic analysis of beta-glucosidase from Aspergillus niger and BGL1 from Trichoderma reesei, purified using a new and efficient fast protein liquid chromatography protocol, was performed. This purification is characterized by two major steps, including the adsorption of the major cellulases onto crystalline cellulose, and a final purification factor of 53. Quantitative analysis of the resulting beta-glucosidase fraction from T. reesei showed it to be 95% pure. Kinetic parameters were determined using cellobiose and a chromogenic artificial substrate. A new method allowing easy and rapid determination of the kinetic parameters was also developed. beta-Glucosidase SP188 (Km = 0.57 mM; Kp = 2.70 mM) has a lower specific activity than BGL1 (Km = 0.38 mM; Kp = 3.25 mM) and is also more sensitive to glucose inhibition. A Michaelis-Menten model integrating competitive inhibition by the product (glucose) has been validated and is able to predict the beta-glucosidase activity of both enzymes.
This article provides a useful comparison between the activity of beta-glucosidases from two different fungi, and shows the importance of fully characterizing both enzymes. A Michaelis-Menten model was developed, including glucose inhibition and kinetic parameters, which were accurately determined and compared. This model can be further integrated into a cellulose hydrolysis model dissociating beta-glucosidase activity from that of other cellulases. It can also help to define the optimal enzymatic cocktails for new beta-glucosidase activities.
纤维素的酶解仍然被认为是木质纤维素生物质生物生产生物燃料的主要限制步骤之一。这是一个复杂的多步骤过程，已经提出了各种动力学模型。里氏木霉分泌的纤维素酶酶促混合物已被深入研究。β-葡萄糖苷酶是几种纤维素酶之一，它催化从抑制性纤维二糖中释放葡萄糖的最后一步。β-葡萄糖苷酶（BGL1）在里氏木霉菌株中分泌得很差，通常需要补充来自黑曲霉（诺维信 SP188）的商业β-葡萄糖苷酶制剂才能完全水解纤维素。令人惊讶的是，β-葡萄糖苷酶的动力学建模缺乏可靠的数据，并且没有考虑到天然里氏木霉和补充的β-葡萄糖苷酶之间可能存在的差异，这可能是因为 BGL1 难以纯化。
使用新的高效快速蛋白质液相色谱协议对来自黑曲霉的β-葡萄糖苷酶和来自里氏木霉的 BGL1 进行了比较动力学分析。该纯化方法的特点是包括两个主要步骤，包括将主要的纤维素酶吸附到结晶纤维素上，最终的纯化因子为 53。对里氏木霉产生的β-葡萄糖苷酶级分进行定量分析表明，其纯度为 95%。使用纤维二糖和显色人工底物测定了动力学参数。还开发了一种新的易于快速测定动力学参数的方法。β-葡萄糖苷酶 SP188（Km=0.57 mM；Kp=2.70 mM）的比活性低于 BGL1（Km=0.38 mM；Kp=3.25 mM），并且对葡萄糖抑制也更敏感。验证了一种整合产物（葡萄糖）竞争性抑制的米氏-门坦模型，并且能够预测两种酶的β-葡萄糖苷酶活性。
本文提供了两种不同真菌来源的β-葡萄糖苷酶活性的有用比较，并表明充分表征两种酶的重要性。建立了包括葡萄糖抑制和动力学参数的米氏-门坦模型，并对其进行了准确测定和比较。该模型可以进一步整合到纤维素水解模型中，将β-葡萄糖苷酶的活性与其他纤维素酶的活性分离。它还可以帮助确定新的β-葡萄糖苷酶活性的最佳酶促混合物。
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