Protein structure-based enhancement of enzyme performance for food and bioproduct applications using X-ray crystallography, protein modification and metabolic engineering methods

我的DG研究计划的总体目标是采用结构-功能方法来提高酶作为食品和生物制品加工助剂的性能。在这种情况下，我们已经研究了特定于脯氨酸的多肽酶。脯氨酸有一个侧链连接到它的氮原子上，这使它在结构上与其他氨基酸不同，并抵抗蛋白质降解。当食物发酵时，含有脯氨酸的多肽(Pro-多肽)的积累会引起不想要的苦味。前肽的苦味可以通过释放脯氨酸来减少。目前已知和研究的Pro多肽酶至少有9种，其中最小单位的Pro被两种主要的酶：Xaa-Pro二肽酶(X-Pase)和Pro-Xaa二肽酶(P-Xase)所降解。X-Pase还能降解有机磷(OP；含有C-P键(或S-P键；用作杀虫剂和神经毒剂)的化合物)。在这项DG研究中(2016-2021年)，这两种二肽酶被选为研究它们在食品和生物制品加工中应用的结构-功能关系的模型系统。在这个拟议的计划中，我的目标是实现以下三个目标：1)利用X射线结晶学表征植物乳杆菌P-Xase的结构，这将是X-Pase的第一个结构模型；2)根据其晶体结构对P-Xase进行蛋白质工程；3)将X-Pase和P-Xase突变的代谢工程乳酸菌。一名博士生(HQP-1)将在一年级和二年级从事P-Xase的X射线结晶学研究。将检查揭示的结构模型，以找出与底物专一性和催化活性有关的残基。这些残基的作用将通过定点突变进行检查和修饰。突变的酶将通过X射线结晶学进行检查，以确认结构和功能之间的关系。在第三年，HQP-1和第二个博士生(HQP-2)将合作开始这项蛋白质工程的研究，HQP-2将继续这项研究到第四年。从第三年开始，与蛋白质工程工作平行，HQP-2将通过克隆工程X-Pase(我之前的研究)和P-Xase(建议的研究)来研究乳酸乳杆菌的代谢操作。经过改造的乳酸乳杆菌菌株将在4年级和5年级接受测试，以了解它们减少苦味和OP的能力。该研究计划将以多学科的方式培训两名生物技术领域的博士生，并将为加拿大不断发展的生物技术产业做出贡献。在未来五年规划中，研究蛋白质结构与功能的关系有助于生物制品的开发，如生物活性多肽的生产。

The overarching goal of my DG research program is to take a structure-function approach to enhance the performance of enzymes as processing aids for food and bioproduct processing. We have worked on proline-specific peptidases in this context. Proline has a side chain attached to its nitrogen atom that makes it structurally distinct from the other amino acids and resistant to proteolysis. When foods are fermented, an accumulation of proline-containing peptides (Pro-peptides) occurs to elicit undesirable bitter tastes. Bitterness of Pro-peptides can be reduced through liberation of proline. At least 9 proline-specific peptidases are known and studied, with the smallest units of Pro-peptides being hydrolyzed by two principle enzymes: Xaa-Pro dipeptidase (X-Pase) and Pro-Xaa dipeptidase (P-Xase). X-Pases are also known to degrade organophosphorus (OP; compounds containing C-P (or S-P) bonds; used as pesticides and nerve gas agents). For this DG research (2016-2021), these two dipeptidases were chosen as model systems for investigating their structure-function relationships for applications in food and bioproduct processing. In this proposed plan, I aim to achieve the following three objectives: 1) To characterize the structure of Lactobacillus plantarum P-Xase using X-ray crystallography that will be the first structure model of X-Pases, 2) to protein engineer P-Xase based on its crystal structure, and 3) to metabolic engineer Lactococcus lactis with mutant X-Pase and P-Xase. A Ph.D. student (HQP-1) will work on the X-ray crystallography of P-Xase in year 1 and 2. The revealed structural models will be examined to find the residues responsible for substrate specificity and catalytic activity of P-Xase. Roles of these residues will be examined and modified using site-directed mutagenesis. The mutated enzymes will be examined by X-ray crystallography to confirm the relationship between structure and function. In year 3, HQP-1 and the second Ph.D. student (HQP-2), who will be recruited at the beginning of year 3, will collaboratively start this investigation of protein engineering, and HQP-2 will continue it into year 4. Starting in year 3, parallel to the protein engineering work, HQP-2 will work on the metabolic manipulation of L. lactis through cloning of engineered X-Pase (from my previous study) and P-Xase (this proposed study). Engineered L. lactis strains will be examined for their ability to reduce bitterness and OP in year 4 and 5. The research program will train two Ph.D. students in biotechnology fields in a multidisciplinary manner and will contribute to growing biotechnology industries in Canada. Beyond 5-year plan, the structure-function relationship information of proline-specific dipeptidases can contribute to the development of bioproducts, such as bioactive peptide production.