University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada.
Biotechnol Biofuels. 2014 Jun 10;7:87. doi: 10.1186/1754-6834-7-87. eCollection 2014.
There is still considerable debate regarding the actual mechanism by which a "cellulase mixture" deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis. In the work reported here we tried to determine the predominant mode of action, at the fiber level, of how a cellulase mixture deconstructs pretreated softwood and hardwood pulp fibers. Quantitative changes in the pulp fibers derived from different pretreated biomass substrates were monitored throughout the course of enzymatic hydrolysis to see if the dominant mechanisms involved either the fragmentation/cutting of longer fibers to shorter fibers or their "peeling/delamination/erosion," or if both cutting and peeling mechanisms occurred simultaneously.
Regardless of the source of biomass, the type of pretreatment and the chemical composition of the substrate, under typical hydrolysis conditions (50°C, pH 4.8, mixing) longer pulp fibers (fiber length >200 μm) were rapidly broken down until a relatively constant fiber length of 130 to 160 μm was reached. In contrast, shorter fibers with an initial average fiber length of 130 to 160 μm showed no significant change in length despite their substantial hydrolysis. The fragmentation/cutting mode of deconstruction was only observed on longer fibers at early stages of hydrolysis. Although the fiber fragmentation mode of deconstruction was not greatly influenced by enzyme loading, it was significantly inhibited by glucose and was mainly observed during initial mixing of the enzyme and substrate. In contrast, significant changes in the fiber width occurred throughout the course of hydrolysis for all of the substrates, suggesting that fiber width may limit the rate and extent of cellulose hydrolysis.
It appears that, at the fiber level, pretreated pulp fibers are hydrolyzed through a two-step mode of action involving an initial rapid fragmentation followed by simultaneous swelling and peeling/erosion of the fragmented fibers. This latter mechanism is the predominant mode of action involved in effectively hydrolyzing the cellulose present in pretreated wood substrates.
“纤维素酶混合物”解构纤维素材料的实际机制仍存在相当大的争议，在微观水平上底物的可及性是限制快速、完全纤维素水解的主要限制因素之一。在本报告中，我们试图确定纤维素酶混合物在纤维水平上解构预处理软木和硬木纸浆纤维的主要作用方式。在酶水解过程中监测来自不同预处理生物质底物的纸浆纤维的定量变化，以确定涉及较长纤维切割成较短纤维的主要机制，还是它们的“剥落/分层/侵蚀”，或者两种切割和剥落机制是否同时发生。
无论生物质的来源、预处理的类型和底物的化学组成如何，在典型的水解条件下（50°C，pH4.8，混合），较长的纸浆纤维（纤维长度>200μm）会迅速分解，直到达到相对恒定的纤维长度 130 至 160μm。相比之下，初始平均纤维长度为 130 至 160μm 的较短纤维尽管水解程度很大，但长度没有明显变化。在水解的早期阶段，只有较长的纤维才会观察到解构的碎片化/切割模式。虽然纤维解构的碎片化模式不会受到酶负荷的很大影响，但它会被葡萄糖显著抑制，主要发生在酶和底物的初始混合过程中。相比之下，所有底物在水解过程中纤维宽度都发生了显著变化，这表明纤维宽度可能限制纤维素水解的速率和程度。
在纤维水平上，预处理的纸浆纤维似乎通过两步作用机制进行水解，包括初始快速碎片化，然后是碎片化纤维的同时膨胀和剥落/侵蚀。在后一种机制中，纤维素酶混合物有效地水解预处理木材底物中存在的纤维素，这是主要作用方式。
