Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, West Bengal, India.
Bioengineering and Environmental Sciences Lab, Department of Energy and Environmental Engineering (DEEE), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad-500007, India.
Appl Microbiol Biotechnol. 2022 Sep;106(18):6059-6075. doi: 10.1007/s00253-022-12117-4. Epub 2022 Aug 11.
One of the critical steps in lignocellulosic deconstruction is the hydrolysis of crystalline cellulose by cellulases. Endoglucanases initially facilitate the breakdown of cellulose in lignocellulosic biomass and are further aided by other cellulases to produce fermentable sugars. Furthermore, if the endoglucanase is processive, it can adsorb to the smooth surface of crystalline cellulose and release soluble sugars during repeated cycles of catalysis before dissociating. Most glycoside hydrolase family 9 (GH9) endoglucanases have catalytic domains linked to a CBM (carbohydrate-binding module) (mostly CBM3) and present the second-largest cellulase family after GH5. GH9 endoglucanases are relatively less characterized. Bacillus licheniformis is a mesophilic soil bacterium containing many glycoside hydrolase (GH) enzymes. We identified an endoglucanase gene, gh9A, encoding the GH9 family enzyme H1AD14 in B. licheniformis and cloned and overexpressed H1AD14 in Escherichia coli. The purified H1AD14 exhibited very high enzymatic activity on endoglucanase substrates, such as β-glucan, lichenan, Avicel, CMC-Na (sodium carboxymethyl cellulose) and PASC (phosphoric acid swollen cellulose), across a wide pH range. The enzyme is tolerant to 2 M sodium chloride and retains 74% specific activity on CMC after 10 days, the highest amongst the reported GH9 endoglucanases. The full-length H1AD14 is a processive endoglucanase and efficiently saccharified sugarcane bagasse. The deletion of the CBM reduces the catalytic activity and processivity. The results add to the sparse knowledge of GH9 endoglucanases and offer the possibility of characterizing and engineering additional enzymes from B. licheniformis toward developing a cellulase cocktail for improved biomass deconstruction. KEY POINTS: • H1AD14 is a highly active and processive GH9 endoglucanase from B. licheniformis. • H1AD14 is thermostable and has a very long half-life. • H1AD14 showed higher saccharification efficiency than commercial endoglucanase.
木质纤维素解构的关键步骤之一是纤维素酶对结晶纤维素的水解。内切葡聚糖酶最初有助于木质纤维素生物质中纤维素的分解，并在其他纤维素酶的进一步帮助下产生可发酵糖。此外，如果内切葡聚糖酶是进行性的，它可以吸附在结晶纤维素的光滑表面上，并在反复催化循环中释放可溶性糖，然后再解离。大多数糖苷水解酶家族 9（GH9）内切葡聚糖酶的催化结构域与碳水化合物结合模块（CBM）（主要是 CBM3）相连，是仅次于 GH5 的第二大纤维素酶家族。GH9 内切葡聚糖酶的特征相对较少。地衣芽孢杆菌是一种嗜温土壤细菌，含有许多糖苷水解酶（GH）酶。我们鉴定了一个内切葡聚糖酶基因 gh9A，它在地衣芽孢杆菌中编码 GH9 家族酶 H1AD14，并在大肠杆菌中克隆和过表达了 H1AD14。纯化的 H1AD14 在广泛的 pH 范围内对内切葡聚糖酶底物（如β-葡聚糖、lichenan、Avicel、CMC-Na（羧甲基纤维素钠）和 PASC（磷酸膨胀纤维素））表现出非常高的酶活性。该酶耐受 2 M 氯化钠，在 10 天后保留 74%的 CMC 比活，在报道的 GH9 内切葡聚糖酶中最高。全长 H1AD14 是一种进行性内切葡聚糖酶，能够有效地糖化甘蔗渣。CBM 的缺失降低了催化活性和进行性。研究结果增加了对 GH9 内切葡聚糖酶的了解，并为从地衣芽孢杆菌中鉴定和工程化其他酶提供了可能性，以开发用于提高生物质解构的纤维素酶混合物。关键点：• H1AD14 是一种来自地衣芽孢杆菌的高活性和进行性 GH9 内切葡聚糖酶。• H1AD14 具有热稳定性和很长的半衰期。• H1AD14 显示出比商业内切葡聚糖酶更高的糖化效率。
