Behind the Science: Engineering Enzymes

Behind the Science: Engineering Enzymes

Author: Cordula Buse, Rey-Ting Guo

Cordula Buse, Associate Editor for ChemBioEng Reviews, talks to Professor Rey-Ting Guo, Tianjin Institute of Industrial Biotechnology, China, about his recent article on enzyme design and engineering.

Professor Guo and his team work on the structural characterization and the engineering of industrial enzymes, especially phytases and xylanases. In their latest article, published in ChemBioEng Reviews as part of the special issue “Enzymes”, they discuss current findings in resolving the three-dimensional structure of phytases as well as different methods of protein design and engineering.

Professor Guo, your main research interests are structural biology and enzymology, especially phytases. Could you please briefly explain the focus of your research and why it is of current interest?

Phytase is one of the most important enzyme products today. The cereals used as animal feeds contain high amounts of phosphorus, which is stored as a form called phytate. Phytate is indigestible in monogastric animals such as pigs and chickens. Since phosphorus is essential for animal growth and health, supplementation of non-renewable inorganic phosphorus becomes a necessary strategy to compensate phosphorus insufficiency.

Excessive exogenous phosphorus is excreted in the animal manure and causes severe environmental pollution. Moreover, the indigestible phytate can form complexes with minerals and proteins and reduce the nutritional value of feed. Adding phytase, the phytate-degrading enzymes from microbes, to animal feeds to increase phosphorus availability and reduce inorganic phosphorus supplementation is vital for the modern animal farming industry. Thus, developing more powerful phytases is of enormous economic and environmental interest.

Since the first phytase product was launched in 1991, attempts to improve the enzyme have not ceased. Enzyme engineering modifies enzymes from the genetic level and changes the protein’s properties, i.e., towards higher stability and higher efficacy. Our work analyzes the protein’s three-dimensional structure to understand phytase at the most fundamental level. Our review article summarizes related works, which provide an important basis for further phytase engineering and for understanding the molecular mechanisms of other enzymes.

What is the broader impact of this paper for the scientific community?

Phytase is the most promising enzyme product nowadays, and brings remarkable benefits to economy and environment. The article summarizes the catalytic mechanism, classification, and protein structure of four different kinds of phytases, which provides not only a review of related studies but also an outlook for next-generation product development.

What was the inspiration behind your work?

My major at university was chemistry. I learned that chemical engineering is a powerful and fundamental technology which benefits every facet of our lives, yet could endanger the environment. This prompted me to search for more eco-friendly approaches. Hence, I turned to the application of biological catalysts, i.e., enzymes, as substitutes for chemicals in various applications.

How did you become interested in structural biology?

No enzyme can be comprehensively understood without its three-dimensional structure. When dealing with an enzyme with no information on protein folding and the residues that are crucial in the catalytic reaction, enzyme engineering would be performed like a blind man feeling an elephant. Hence, I learned to use NMR and X-ray crystallography to solve a protein’s three-dimensional structure, and utilized the obtained structure to conduct enzyme engineering.

NMR has an inherent limitation in determining the structure of proteins constituted by more than 100–200 amino acids, while X-ray crystallography can deal with proteins constituted by thousands of amino acids. Since most enzymes are large proteins, X-ray crystallography is a better approach for my research.

Which part of your work is most challenging?

Protein structure determination using X-ray crystallography involves protein expression, protein purification, crystallization, diffraction data collection, solving the phase, structure determination and refinement, and sometimes obtaining the structure in complex with ligands. Unpredictable problems could happen at every single step and bog down the entire process. A protein could be very easy to purify but very difficult to crystallize. A protein crystal could be very easy to grow but the diffraction resolution could be very difficult to improve. No one can cheer before the final protein structure is obtained.

Do you have a long-term vision for your research and what will be the next steps on your journey?

My team aims to study enzymes with unexplored function and structure. A very challenging, and very fascinating, target are the enzymes on the plasma membrane. Unlike most enzymes that exist in the cytoplasm or in secreted form, membrane enzymes are embedded in the cell membrane and perform their functions in a relatively hydrophobic environment. Very few of them are known to us, since these membrane proteins are very difficult to purify and even more difficult to crystallize. Thus, one of our long-term goals is to unveil these mysterious enzymes.

Which other topics does your research group investigate?

We work on enzymes which relate to biomass energy production, i.e., cellulases and hemicellulases, industrial applications, terpene synthesis, and the design of novel antibiotics.

Thank you for the interview!


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