Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, México.
PLoS One. 2024 Apr 18;19(4):e0301604. doi: 10.1371/journal.pone.0301604. eCollection 2024.
The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-β-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a β-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.
红鲍（Haliotis rufescens）是北美的最重要的水产养殖物种之一。它的肝胰腺富含纤维素酶和其他多糖降解酶，使其具有消化富含纤维素的大型藻类的显著能力；然而，其纤维素酶系统尚未得到充分探索。本文描述了一种来自红鲍的内源性三聚体糖基内切葡聚糖酶的一些功能和结构特性。通过 MALDI-TOF 质谱法测定，纯化后的酶的分子量为 23.4 kDa，在凝胶过滤色谱和同工酶中表现为三聚体。根据过碘酸-Schiff 试剂染色，在 SDS-PAGE 凝胶中检测糖基证实了鲍鱼肉酶是一种糖蛋白。水解纤维寡糖和对硝基苯-β-D-吡喃葡萄糖苷证实了其内切/外切活性。在 45°C 和 pH 6.0 下，对 0.5%（w/v）羧甲基纤维素的最大酶活为 53.9±1.0 U/mg。我们使用蛋白酶和质谱法阐明了鲍鱼肉酶的一级结构。基于这些结果，并使用生物信息学方法，我们鉴定了编码该酶的基因，并推导出其全长氨基酸序列；成熟蛋白由 177 个残基组成，理论分子量为 19.1 kDa，根据序列相似性，它被归类为糖苷水解酶家族 45 亚家族 B。AlphaFold 理论模型和与纤维五糖的对接模拟证实，鲍鱼肉酶是一种富含β-折叠的蛋白质，如圆二色性实验也观察到的，具有保守的催化残基：Asp26、Asn109 和 Asp134。有趣的是，AlphaFold-Multimer 分析表明鲍鱼肉酶为三聚体组装，这支持了我们的实验发现。这些酶的发现和特性可能有助于开发高效的纤维素生物转化工艺，用于生物燃料和可持续的生物产品。
