Muderspach Sebastian J, Fredslund Folmer, Volf Verena, Poulsen Jens-Christian Navarro, Blicher Thomas H, Clausen Mads Hartvig, Rasmussen Kim Krighaar, Krogh Kristian B R M, Jensen Kenneth, Lo Leggio Leila
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100, Copenhagen, Denmark.
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.
Biotechnol Biofuels. 2021 Sep 16;14(1):183. doi: 10.1186/s13068-021-02025-6.
Endo-β-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-β-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family's biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths.
A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (βα) barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π-π and cation-π interactions. The length of the substrate binding site-and thus produced oligosaccharide products-is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable.
We have characterized in detail the most thermophilic endo-β-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.
内切-β-1,4-半乳聚糖酶是糖苷水解酶（GH），属于GH53家族，该家族属于最大的GH家族——GH-A家族。GH广泛存在，参与众多生物学功能，在工业上也有广泛应用。内切-β-1,4-半乳聚糖酶尤其能水解果胶中的半乳聚糖和阿拉伯半乳聚糖，果胶是植物初生细胞壁的主要成分，在植物防御以及食品和其他行业中具有重要作用。在此，我们通过表征首个古生菌和嗜热GH53半乳聚糖酶来探索该家族的生物多样性，并将其用作构建具有不同产物长度的工程酶的支架。
在厌氧嗜热古菌聚集火球菌的基因组中鉴定出一个半乳聚糖酶基因，并对分离出的催化结构域进行表达和表征（IaGal）。IaGal呈现出GH-A家族酶典型的（βα）桶状结构，在第4个和第7个桶状链末端有催化羧酸盐。其至少95°C的最佳活性温度和超过100°C的熔点表明其具有极高的热稳定性，这对工业应用来说是非常有利的特性。如果酶消耗减少，重新添加酶的需求也会减少，从而降低成本。与其他已进行结构表征的成员相比，IaGal的主要稳定特征是π-π和阳离子-π相互作用。与先前表征的半乳聚糖酶相比，底物结合位点的长度——以及由此产生的寡糖产物——处于中间水平。受GH53家族结构多样性启发，合理设计了变体以缩短或延长底物结合凹槽，从而调节产物长度。缺失亚位点的变体产生的产物比IaGal短，这与启发设计的真菌半乳聚糖酶情况相同。设计了具有更长结合位点的IaGal变体，其产生的降解模式与预期不同，不过仍与野生型IaGal的降解模式不同。所有变体都保持了极高的稳定性。
我们详细表征了迄今为止已知的最嗜热的内切-β-1,4-半乳聚糖酶，并成功对其进行工程改造以改变降解谱，同时保持其许多理想的热稳定性。这是一项重要成果，因为寡糖产物长度对工业和天然GH来说都是一项重要特性。
