Electrochemical High-Throughput Discovery of Novel Lytic Polysaccharide Monooxygenase Enzymes

木质素和纤维素是由植物产生的碳水化合物分子，以赋予它们的细胞壁结构和保护免受降解。因此，木质素和纤维素都是高度稳定的非反应性分子，并且为了将它们化学降解成其较小的糖结构单元，需要苛刻的化学反应条件。Prozomix（http：//www.prozomix.com/）是一家英国生物技术公司，总部设在诺森伯兰郡，专门从事生物催化剂酶的生产和销售。目前，LPMO是Prozomix目录中唯一缺失的糖苷键裂解酶家族，因为（i）通过公司独特的专有内部宏基因组文库和序列搜索和快速商业蛋白质生产方面的专业知识，以前还没有快速LPMO活性筛选方法来支持LPMO酶的发现;和（ii）在LPMO生物化学中，活性位点配体在调节铜化学的能量学中的作用尚不清楚，因此还不可能将LPMO序列与底物反应性/特异性合理地联系起来。该项目结合了酶电化学（帕金），结构（戴维斯）和光谱学（沃尔顿）研究与工业酶的发现和商业化的互补专业知识（Prozomix）发现和表征新的溶解性多糖单加氧酶（LPMO）碳水化合物激活酶。在开创性的工作中，Davies和Walton（约克）证明了催化纤维素第一糖苷键裂解的溶解性多糖单加氧酶（LPMO）是铜酶。Parkin（约克）与Davies、Walton和来自巴西的合作者合作，展示了她的团队在蛋白质膜电化学方面的专业知识，使纯化的酶能够在低样品要求（mcg）、快速（约10 μ g）、低样品要求（mcg）和低样品要求（约10 μ g）的条件下筛选LPMO电子转移催化活性。该项目的目标将是：（i）从Prozomix宏基因组文库中鉴定（CaZy序列分析和AlphaFold 2）并克隆出新的细菌LPMO（ii）使用电化学快速筛选电子转移活性并鉴定推定酶中的氧化LPMO活性（iii）测试新LPMO的碳水化合物底物特异性，寻找潜在生物燃料兴趣的高度木质素和纤维素活性酶（iv）大规模生产多达5种具有新活性的先导酶，即，高温稳定性或不寻常的立体选择性（v）进一步从结构上表征前导酶，并使用分子生物学来探测结构-功能假设（vii）鉴定高稳定性，高活性的纤维素和/或木质素降解LPMO，Prozomix以生产用于规模化工业测试的酶学生将获得蛋白质纯化和分子生物学方面的专家培训，并学习纯化LPMO。还将在电化学和EPR方面提供专家支持。在实习期间，学生将参加Prozomix组织的适当培训课程，涵盖新型酶发现，开发和生产的各个方面。

Lignin and cellulose are carbohydrate molecules which are produced by plants to give their cell walls structure and protection from degradation. Both lignin and cellulose are therefore highly stable, unreactive molecules and in order to chemically degrade them into their smaller sugar building blocks harsh chemical reaction conditions are required. As a result of this, it is more cost effective to use enzyme cocktails to convert plant matter into biofuel, and biocatalysts which are useful in this process have a significant commercial value.Prozomix (http://www.prozomix.com/) is a British Biotech company based in Northumberland specializing in biocatalyst enzyme production and sales. Currently LPMOs are the only family of glycosidic bond cleavage enzymes missing from Prozomix's catalogue because (i) a rapid LPMO activity-screening method has not been previously available to support LPMO enzyme discovery via the companies unique, proprietary in-house metagenome library and expertise in sequence searches and rapid, commercial protein production; and (ii) in LPMO biochemistry, the role of active site ligands in tuning the energetics of the copper chemistry is not known so as-yet it is impossible to rationally relate LPMO sequence to substrate-reactivity/specificity. This project combines complementary expertise in enzyme electrochemistry (Parkin), structure (Davies) and spectroscopy (Walton) studies with industrial enzyme discovery and commercialization (Prozomix) to find and characterize new lytic polysaccharide monooxygenase (LPMO) carbohydrate-activating enzymes.In ground-breaking work, Davies and Walton (York) proved that the lytic polysaccharide monooxygenase (LPMO) enzymes which catalyze the first glycosidic bond cleavages of cellulose are copper enzymes. Parkin (York) has worked with Davies, Walton and collaborators from Brazil to show that her group's expertise in protein film electrochemistry enables purified enzymes to be screened for LPMO electron-transfer catalytic activity in a low-sample requiring (mcg), rapid (approx. 15 min) and high throughput, multiplexed manner.The project objectives will be to:(i) Identify (CaZy sequence analysis and AlphaFold2) and clone out new bacterial LPMOs from the Prozomix metagenome library(ii) Use electrochemistry to rapidly screen for electron-transfer activity and identify oxidative LPMO activity in the putative enzymes(iii) Test the carbohydrate substrate specificity of new LPMOs, searching for highly lignin and cellulose active enzymes of potential bio-fuel interest(iv) Large scale production of up to 5 lead enzymes with novel activity, i.e., high temperature stability or unusual stereo-selectivity(v) Further structurally characterize the lead enzymes and use molecular biology to probe structure-function hypotheses(vii) Identify high-stability, high-activity cellulose- and/or lignin-degrading LPMO, Prozomix to produce enzyme for scaled industrial testingThe student will be provided with expert training in protein purification and molecular biology and learn to purify an LPMO. Expert support will also be provided in electrochemistry and EPR. While on placement, the student will attend appropriate training sessions organised by Prozomix, covering all aspects of novel enzyme discovery, development and production.