Pawar Sandip V, Hallam Steven J, Yadav Vikramaditya G
Department of Chemical & Biological Engineering, The University of British Columbia Vancouver BC Canada
University Institute of Pharmaceutical Sciences, Punjab University Chandigarh Punjab India.
RSC Adv. 2018 Jun 20;8(40):22490-22497. doi: 10.1039/c8ra02764a. eCollection 2018 Jun 19.
The profitability of next-generation biorefineries is acutely contingent on the discovery and utilization of biocatalysts that can valorize lignin. To this end, the metabolic catalogues of diverse microbiota have been mined previously using functional metagenomics in order to identify biocatalysts that can selectively degrade lignin into monoaromatic compounds. Herein, we have further improved the valorization factor of biorefining by deploying functional metagenomics toward the identification of a novel transaminase that can selectively functionalize lignin-derived monoaromatics to produce value-added feedstocks for pharmaceutical synthesis. We implemented a high-throughput colorimetric assay using -xylylenediamine as the amino donor and successfully identified a transaminase that utilizes the canonical cofactor, pyridoxal 5'-phosphate, to aminate as many as 14 monoaromatic aldehydes and ketones. We subsequently identified the optimal conditions for enzyme activity towards the most favoured amino acceptor, benzaldehyde, including temperature, pH and choice of co-solvent. We also evaluated the specificity of the enzyme towards a variety of amino donors, as well as the optimal concentration of the most favoured amino donor. Significantly, the novel enzyme is markedly smaller than typical transaminases, and it is stably expressed in without any modifications to its amino acid sequence. Finally, we developed and implemented a computational methodology to assess the activity of the novel transaminase. The methodology is generalizable for assessing any transaminase and facilitates screening of enzyme-substrate combinations in order to develop efficient biocatalytic routes to value-added amines. The computational pipeline is an ideal complement to metagenomics and opens new possibilities for biocatalyst discovery.
下一代生物精炼厂的盈利能力严重依赖于能够使木质素增值的生物催化剂的发现和利用。为此，此前已通过功能宏基因组学挖掘了多种微生物群的代谢目录，以识别能够将木质素选择性降解为单芳族化合物的生物催化剂。在此，我们通过运用功能宏基因组学来鉴定一种新型转氨酶，进一步提高了生物精炼的增值系数，这种转氨酶能够选择性地使木质素衍生的单芳族化合物功能化，从而生产用于药物合成的增值原料。我们使用 - 二甲苯二胺作为氨基供体实施了高通量比色测定，并成功鉴定出一种利用典型辅因子磷酸吡哆醛将多达14种单芳族醛和酮胺化的转氨酶。随后，我们确定了该酶针对最适宜的氨基受体苯甲醛的酶活性最佳条件，包括温度、pH值和共溶剂的选择。我们还评估了该酶对多种氨基供体的特异性，以及最适宜氨基供体的最佳浓度。值得注意的是，这种新型酶明显小于典型的转氨酶，并且在不改变其氨基酸序列的情况下在 中稳定表达。最后，我们开发并实施了一种计算方法来评估这种新型转氨酶的活性。该方法可普遍用于评估任何转氨酶，并有助于筛选酶 - 底物组合，以开发通向增值胺的高效生物催化路线。该计算流程是宏基因组学的理想补充，为生物催化剂的发现开辟了新的可能性。
