Mechanism of Cellulase Reaction on Pure Cellulosic Substrates
Mechanism of Cellulase Reaction on Pure Cellulosic Substrates
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DOI：
10.1002/bit.22195
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发表时间：
2009-04-15
影响因子：
3.8
通讯作者：
Lee, Y. Y.
中科院分区：
文献类型：
--
作者：
Gupta, Rajesh;Lee, Y. Y.
使用以下纯纤维素底物研究纤维素酶反应机理：微晶纤维素（Avicel）、α-纤维素（Sigma）、滤纸、棉和非结晶纤维素（NCC）。NCC是我们实验室通过浓硫酸处理制备的无定形纤维素。当用纤维素酶水解时，NCC产生大量的作为反应中间体的纤维寡糖（COS）沿着葡萄糖和纤维二糖。根据聚合度（DP）的不同，将纤维素酶产生的COS分为两类：低DP（小于7）COS（LD-COS）和高DP COS（HD-COS）。内切葡聚糖酶（Endo-G）在NCC上快速反应，将其DP降低至3060，之后Endo-G与NCC的反应停止。HD-COS是由NCC通过Endo-G的作用产生的，而LD-COS是由外切葡聚糖酶（Exo-G）产生的。β-葡萄糖苷酶（β-G）水解LD-COS以产生纤维二糖，但它不水解HD-COS。NCC的DP以这样的方式影响Exo-G的作用，即对于高DP NCC，总产率高。这与先前的发现一致，即Exo-G的底物识别需要结合在DP为10的β-葡聚糖链上以进行水解。因此，DP小于10的固体内的单个纤维素链残基保持未反应。对于高DP NCC，未反应部分的百分比将较低，这导致高的总转化率。底物的表面积和反应位点的数量有利于酶的吸附，因此有利于水解的初始速率。然而，纤维素的总转化程度主要由其固有特性如DP和结晶度控制。
Cellulase reaction mechanism was investigated with the use of following pure cellulosic substrates: Microcrystalline cellulose (Avicel), alpha-cellulose (Sigma), filter paper, cotton, and non-crystalline cellulose (NCC). NCC is amorphous cellulose prepared in our laboratory by treatment with concentrated sulfuric acid. When hydrolyzed with cellulase, NCC produces significant amount Of cello-oligosaccharides (COS) as reaction intermediates along with glucose and cellobiose. The COS produced by cellulase were categorized into two different moieties based upon their degree of polymerization (DP): low DP (less than 7) COS (LD-COS) and high DP COS (HD-COS). Endo-glucanase (Endo-G) reacts rapidly on the NCC reducing its DP to 3060, after which the Endo-G reaction with NCC ceases. HD-COS is produced from NCC by the action of Endo-G, whereas LD-COS is produced by exo-glucanase (Exo-G). beta-Glucosidase (beta-G) hydrolyzes LD-COS to produce cellobiose, but it does not hydrolyze HD-COS. DP of NCC affects the action of Exo-G in such a way that the overall yield is high for high DP NCC. This is in line with previous findings that substrate-recognition by Exo-G requires binding on beta-glucan chain with DP of 10 for the hydrolysis to take place. The individual cellulose chain residues within solid having DP less than 10 therefore remain unreacted. The percentage of the unreacted portion would be lower for high DP NCC, which results high overall conversion. The surface area and the number of reactive sites on the substrate facilitate adsorption of enzyme therefore the initial rate of the hydrolysis. The overall extent of conversion of cellulose, however, is controlled primarily by its inherent characteristics such as DP and crystallinity.