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取结构-功能方法来提高酶作为食品和生物制品加工助剂的性能。在这方面，我们研究了脯氨酸特异性肽酶。脯氨酸具有连接到其氮原子的侧链，这使得其在结构上不同于其他氨基酸并且对蛋白水解具有抗性。当食物发酵时，含脯氨酸的肽（前肽）的积累发生，引起不期望的苦味。前肽的苦味可以通过释放脯氨酸来降低。已知并研究了至少9种脯氨酸特异性肽酶，其中最小单位的前肽被两种主要酶水解：Xaa-Pro二肽酶（X-Xase）和Pro-Xaa二肽酶（P-Xase）。X-Pases也被认为可以降解有机磷（OP;含有C-P（或S-P）键的化合物;用作杀虫剂和神经毒气剂）。在这项DG研究（2016-2021）中，这两种二肽酶被选为模型系统，用于研究其在食品和生物制品加工中应用的结构-功能关系。本论文的主要目标是：1）利用X射线晶体学技术对植物乳杆菌P-Xase的结构进行表征，这将是第一个X-Xase的结构模型; 2）基于植物乳杆菌P-Xase的晶体结构进行蛋白质工程改造; 3）利用突变的X-Xase和P-Xase对乳酸乳球菌进行代谢工程改造。博士学位学生（HQP-1）将在第一年和第二年从事P-Xase的X射线晶体学研究。将检查所揭示的结构模型以找到负责P-Xase的底物特异性和催化活性的残基。这些残基的作用将使用定点诱变进行检查和修饰。将通过X射线晶体学检查突变的酶，以确认结构和功能之间的关系。在第三年，HQP-1和第二个博士学位。学生（HQP-2），谁将在今年3年初招募，将合作开始这项研究的蛋白质工程，和HQP-2将继续到今年4。从第三年开始，与蛋白质工程工作平行，HQP-2将致力于L。通过克隆工程化的X-Xase（来自我以前的研究）和P-Xase（这项拟议的研究）。工程湖将在第4年和第5年检查乳酸菌菌株降低苦味和OP的能力。该研究项目将培养两名博士。学生在生物技术领域的多学科的方式，并将有助于在加拿大不断增长的生物技术产业。未来5年，脯氨酸特异性二肽酶的结构-功能关系信息将有助于生物产品的开发，如生物活性肽的生产。

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.