Tailor-made β-glucosidase with increased activity at lower temperature without loss of stability and glucose tolerance,Green Chemistry

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Tailor-made β-glucosidase with increased activity at lower temperature without loss of stability and glucose tolerance
Green Chemistry ( IF 9.3 ) Pub Date : 2020-03-11 , DOI: 10.1039/c9gc04166d
Florian Lenz _{1,

2,

3,

4} , Paul Zurek _{1,

2,

3,

4} , Martina Umlauf _{1,

2,

3,

4} , Iasson E. P. Tozakidis _{1,

2,

3,

4} , Joachim Jose _{1,

2,

3,

4}

Affiliation

The hydrolysis of cellobiose by β-glucosidases represents the crucial step in the degradation of lignocellulosic biomass and is therefore a major target for improvement by enzyme engineering. BglA from Caldicellulosiruptor saccharolyticus (CsBglA) is among the β-glucosidases with highest known activity and thermostability, however with a temperature optimum of 70–80 °C. To improve the enzyme's activity at moderate temperatures, a semi-rational approach based on homology modeling, conservation scoring and subsequent site-saturation mutagenesis was combined with random mutagenesis, resulting in the variant CsBglA-LYTH with 150% improvement of k_cat/K_M at 55 °C, a specific activity of 504 U mg⁻¹ (CsBglA: 359 U mg⁻¹) and a K_M-value of 37.1 mM (CsBglA: 61.2 mM). The temperature optimum of the enzyme was altered from 80 °C to 65 °C while retaining stability at the temperature of 55 °C as applied for cellulose hydrolysis. To our knowledge, this makes CsBglA-LYTH the bacterial β-glucosidase with highest activity and stability.

中文翻译：

量身定制的β-葡萄糖苷酶，在较低的温度下具有增强的活性，而不会失去稳定性和葡萄糖耐量

β-葡萄糖苷酶水解纤维二糖代表了木质纤维素生物质降解的关键步骤，因此是通过酶工程改进的主要目标。糖化卡尔维分解酶（Cs BglA）的BglA是已知活性和热稳定性最高的β-葡萄糖苷酶，但最适温度为70–80°C。为了提高酶在中等温度下的活性，将基于同源性建模，保守评分和随后的位点饱和诱变的半定量方法与随机诱变相结合，产生了变异Cs BglA-LYTH，k _cat / K改善了150％_中号在55℃下，比活度为504U mg ^-1（Cs BglA：359U mg ^-1），K _M值为37.1mM （Cs BglA：61.2mM）。将酶的最佳温度从80°C更改为65°C，同时保持在55°C的温度下的稳定性（用于纤维素水解）。据我们所知，这使Cs BglA-LYTH成为具有最高活性和稳定性的细菌β-葡萄糖苷酶。

更新日期：2020-04-24

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