Advances in the application of yeast surface display technology / 生物工程学报

Yeast surface display (YSD) is a technology that fuses the exogenous target protein gene sequence with a specific vector gene sequence, followed by introduction into yeast cells. Subsequently, the target protein is expressed and localized on the yeast cell surface by using the intracellular protein transport mechanism of yeast cells, whereas the most widely used YSD system is the α-agglutinin expression system. Yeast cells possess the eukaryotic post-translational modification mechanism, which helps the target protein fold correctly. This mechanism could be used to display various eukaryotic proteins, including antibodies, receptors, enzymes, and antigenic peptides. YSD has become a powerful protein engineering tool in biotechnology and biomedicine, and has been used to improve a broad range of protein properties including affinity, specificity, enzymatic function, and stability. This review summarized recent advances in the application of YSD technology from the aspects of library construction and screening, antibody engineering, protein engineering, enzyme engineering and vaccine development.

Teaching reform and practice of "food enzymology and enzyme engineering" based on cutting-edge researches / 生物工程学报

Food enzymology and enzyme engineering is an important professional course of food science. The course includes the basic theory of enzymology, enzyme engineering technology and the application of enzymes in food industry. Considering the knowledge gap between the teaching contents and the cutting-edge researches, the team constantly adjusted and optimized the course contents to enable students to keep up with state-of-the-art progress by carefully mining the cutting-edge researches. Taking cutting-edge researches as the breakthrough point, we explored the problem-based learning (PBL) teaching model under the guidance of outcome-based education (OBE) concept, and highlighted the importance of the teacher-student and student-student interactions to improve students' enthusiasm and participation. A diversified assessment system was established to evaluate the performance of students in the learning process. The teaching reform consolidated the basic knowledge and expanded the academic frontiers, and fostered students' ability in analyzing problems, designing solutions and achieving team communication. The course may give new insights into the teaching reform of food enzymology and enzyme engineering and other related courses.

Advances in poly(ethylene terephthalate) hydrolases / 生物工程学报

Plastics have brought invaluable convenience to human life since it was firstly synthesized in the last century. However, the stable polymer structure of plastics led to the continuous accumulation of plastic wastes, which poses serious threats to the ecological environment and human health. Poly(ethylene terephthalate) (PET) is the most widely produced polyester plastics. Recent researches on PET hydrolases have shown great potential of enzymatic degradation and recycling of plastics. Meanwhile, the biodegradation pathway of PET has become a reference model for the biodegradation of other plastics. This review summarizes the sources of PET hydrolases and their degradation capacity, degradation mechanism of PET by the most representative PET hydrolase-IsPETase, and recently reported highly efficient degrading enzymes through enzyme engineering. The advances of PET hydrolases may facilitate the research on the degradation mechanism of PET and further exploration and engineering of efficient PET degradation enzymes.

Substitutability of metal-binding sites in an alcohol dehydrogenase / 生物工程学报

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

Exploration and practice in ideological education in the course of "Protein and Enzyme Engineering" / 生物工程学报

Protein and Enzyme Engineering is the core and required course for colleague students majored in biotechnology, which plays an important role in the professional training system. In accordance with the "Guidelines for the Development of Ideological Education in Higher Education Institutions" issued by the Ministry of Education, we explored the combination of course teaching with ideological education by considering the features of the biotechnology major and the course and setting up rational teaching objectives. This paper described the strategy, design, implementation and evaluation approaches that were used in the course of "Protein and Enzyme Engineering" to achieve a good integration. The practice starts from story-telling, discussion of life, case study, hot issues discussion, literature discussion and presentations. The scientific spirits, civic character, global vision, eco-civilization and legal consciousness, as well as their native land emotion and cultural confidence, were boosted. The natural integration of the ideological education into the whole process of this course helped to better achieve the goal of curriculum education while promoting teaching excellence.

Oligomerization triggered by foldon to enhance the catalytic efficiency of feruloyl esterase / 生物工程学报

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.

Computational chemistry approaches in studies on industrial enzymes / 生物工程学报

We review major computational chemistry techniques applied in industrial enzyme studies, especially approaches intended for guiding enzyme engineering. These include molecular mechanics force field and molecular dynamics simulation, quantum mechanical and combined quantum mechanical/molecular mechanical approaches, electrostatic continuum models, molecular docking, etc. These approaches are essentially introduced from the following two angles for viewing: one is about the methods themselves, including the basic concepts, the primary computational results, and potential advantages and limitations; the other is about obtaining valuable information from the respective calculations to guide the design of mutants and mutant libraries.

Preface for special issue on enzyme engineering (2018) / 生物工程学报

Enzyme engineering combines enzymology and engineering, and is one of the major fields of modern biotechnology. To promote enzyme engineering research in China, we present in this special issue with reviews and original articles focusing on recent relevant advances reported by Chinese scientists.

Advances in study of plant type Ⅲ polyketide synthases / 中国中药杂志

Polyketides are a large class of natural products with notable structural diversity and different biological activities. They have essential pharmacological value for human health. In plants, the enzymes responsible for the formation of phenolic metabolites backbone structures are collectively known as type Ⅲ polyketide synthases (PKSs), which are the key enzymes for the polyketides biosynthesis. The PKSs catalyze a series of condensation reactions of two-carbon acetate units with an acyl starter. A brief overview of this group of enzymes, including their reaction mechanisms, function modification, expression regulation, molecular evolution, and recent interesting findings are presented here.

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

Enzymes have been long used in man-made biochemical processes, from brewing and fermentation to current industrial production of fine chemicals. The ever-growing demand for enzymes in increasingly specific applications requires tailoring naturally occurring enzymes to the non-natural conditions found in industrial processes. Relationships between enzyme sequence, structure and activity are far from understood, thus hindering the capacity to design tailored biocatalysts. In the field of protein engineering, directed enzyme evolution is a powerful algorithm to generate and identify novel and improved enzymes through iterative rounds of mutagenesis and screening applying a specific evolutive pressure. In practice, critical checkpoints in directed evolution are: selection of the starting point, generation of the mutant library, development of the screening assay and analysis of the output of the screening campaign. Each step in directed evolution can be performed using conceptually and technically different approaches, all having inherent advantages and challenges. In this article, we present and discuss in a general overview, challenges of designing and performing a directed enzyme evolution campaign, current advances in methods, as well as highlighting some examples of its applications in industrially relevant enzymes.