Loop B3对GH7内切纤维素酶功能的影响机制

Affecting Mechanism of Loop B3 on the Function of GH7 Endoglucanase

糖苷水解酶第七家族(GH7)包含内切和外切两类纤维素酶,其中对外切纤维素酶的研究较为成熟,但对内切纤维素酶的研究相对较少。本研究从嗜热真菌Myceliophthora thermophila的基因组中鉴定出一个新型GH7内切纤维素酶Mt Cel7b,其在60℃,pH 5.0时表现出最佳酶活力。在90℃孵育1 h后,Mt Cel7b仍能保留40%以上的活性。经过序列统计分析发现,Mt Cel7b loop B3存在长链型和短链型的进化差异,为了探究loop B3对内切纤维素酶结构和功能的影响,将MtCel7b的长链型loop B3进行截短,构建了B3^(cut)突变体。结果显示,B3^(cut)突变体在高温下的稳定性较野生型提高了约9%-44%,而其对3种纤维素底物的比活性降低了34%-74%。借助分子动力学模拟进一步分析显示,在突变体B3^(cut)中,其loop B3的截短导致催化裂隙两端的loop A3和loop B1发生了显著位移,缩小了催化口袋的空间结构,加强了催化位点周围的氢键作用网络,从而导致酶在高温下更加稳定。本研究阐明了loop B3在GH7内切纤维素酶中的重要作用,为酶分子的改良工作提供了新的参考。

Glycoside hydrolase family 7(GH7)contains two kinds of cellulases,cellobiohydrolase and endoglucanase,of which the study of cellobiohydrolase is more mature,however few studies on endoglucanase.A thermophilic endoglucanase of GH7 from the genome of the M.thermophila,Mt Cel7b,was identified and characterized with the maximum activity at 60℃,pH 5.0.After 1 h incubation at 90℃,Mt Cel7b retained over 40% activity.The loop B3 of Mt Cel7b,can be divided into short- and long-chains based on amino acid sequence comparison.In order to explore the effect of loop B3 on the structure and function of,endoglucanase,the mutant B3cut was formed by truncating 14 amino acids on long-chain B3 loop of Mt Cel7b.At high temperatures,the activity of B3cut was 9%-44% higher than that of Mt Cel7b;however,the hydrolysis ability of B3cut to different substrates was reduced by 34%-74%.Additional analysis with the assistance of molecular dynamics simulations demonstrated that in the mutant B3cut,the truncation of its B3 loop led to a significant displacement of loop A3 and loop B1 at both ends of the catalytic cleft,narrowing the spatial structure of the catalytic pocket and strengthening the network of hydrogen bonding interactions around the catalytic site,resulting in a more stable enzyme at high temperatures.This study sheds light on the role of loop B3 in GH7 endoglucanase and provides insight into the improvement of the enzyme molecule.