A glucose tolerant β-glucosidase from Thermomicrobium roseum that can hydrolyze biomass in seawater,Green Chemistry

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A glucose tolerant β-glucosidase from Thermomicrobium roseum that can hydrolyze biomass in seawater
Green Chemistry ( IF 9.3 ) Pub Date : 2021-08-13 , DOI: 10.1039/d1gc01357b
Sushant K. Sinha ₁ , Maithili Datta ₁ , Supratim Datta _{1,

2,

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Affiliation

β-Glucosidase (EC 3.2.1.21) plays an essential role in the hydrolysis of the β-1,4 linkage of cellobiose. Accumulated glucose during saccharification leads to the inhibition of the production of β-glucosidase, which causes an accumulation of cellobiose and inhibition of other cellulolytic enzymes. Thus, glucose tolerant and active β-glucosidase is required for the efficient saccharification of biomass. Freshwater is an essential ingredient of biotechnological processes and contributes to the environmental and economic cost of processes. Relatively few biocatalytic processes have been developed to utilize seawater, which is more abundant. Towards both the requirements, we set out to characterize a hyperthermophilic enzyme, B9L147, and evaluate its activity in seawater, and compared it with the industrial enzyme benchmarks. B9L147 from Thermomicrobium roseum was cloned and expressed in Escherichia coli. The overexpressed and purified wild-type showed a high specific activity of 280 ± 5.2 μmol min⁻¹ mg⁻¹ on pNPGlc when assayed at pH 7 and 84 °C. B9L147 retains at least 80% relative specific activity across a wide pH range from 5.5 to 10.0. The enzyme is glucose tolerant and remains fully active until 3 M glucose. The kinetic properties, stability, and glucose tolerance remain identical in seawater, unlike commercial enzymes. An engineered variant, V169C, showed a 15% enhanced specific activity and almost twice the half-life compared to the wild type (B9L147). Both B9L147 and V169C show very high synergistic activity when supplemented with commercial cellulases and enzymes cloned and overexpressed in our lab. To the best of our knowledge, B9L147 is the first β-glucosidase that can hydrolyze cellulose in seawater at elevated temperatures and thus may be of value for industrial applications. Our studies offer a framework for developing seawater tolerant in vitro saccharification systems for biomass hydrolysis towards the sustainable production of biofuels and chemicals from biomass.

中文翻译：

来自 Thermomicrobium roseum 的耐葡萄糖 β-葡萄糖苷酶，可水解海水中的生物质

β-葡萄糖苷酶 (EC 3.2.1.21) 在纤维二糖 β-1,4 键的水解中起重要作用。糖化过程中积累的葡萄糖会抑制 β-葡萄糖苷酶的产生，从而导致纤维二糖的积累和其他纤维素分解酶的抑制。因此，生物质的有效糖化需要葡萄糖耐受性和活性 β-葡萄糖苷酶。淡水是生物技术过程的重要组成部分，会增加过程的环境和经济成本。开发利用海水的生物催化工艺相对较少，而海水更丰富。针对这两个要求，我们着手表征一种超嗜热酶 B9L147，并评估其在海水中的活性，并将其与工业酶基准进行比较。B9L147 从Thermomicrobium roseum被克隆并在大肠杆菌中表达。过表达和纯化的野生型在p上显示出 280 ± 5.2 μmol min ^-1 mg ^-1的高比活性在 pH 7 和 84 °C 下测定时的 NPGlc。B9L147 在 5.5 至 10.0 的宽 pH 范围内保留了至少 80% 的相对比活性。该酶具有葡萄糖耐受性，并且在 3 M 葡萄糖之前保持完全活性。与商业酶不同，海水中的动力学特性、稳定性和葡萄糖耐受性保持相同。与野生型 (B9L147) 相比，工程变体 V169C 的比活性提高了 15%，半衰期几乎是其两倍。B9L147 和 V169C 在补充有我们实验室克隆和过表达的商业纤维素酶和酶时都显示出非常高的协同活性。据我们所知，B9L147 是第一种可以在高温下水解海水中纤维素的 β-葡萄糖苷酶，因此可能具有工业应用价值。我们的研究为开发耐海水性提供了一个框架用于生物质水解的体外糖化系统，以从生物质可持续生产生物燃料和化学品。

更新日期：2021-09-07

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