Enzyme Engineering

Enzymes are biocatalyst and regulate the chemical reactions occurring in living cell. They are essential for all life forms. The enzymes are also used widely in-vitro for a variety of purposes. They are extensively used in chemical, food, cosmetic and pharmaceutical industries because of their several unique advantages such as;

• ·         Non-toxic
• ·         Eco-friendly
• ·         Product purity
• ·         High specificity for substrate
• ·         Simpler chemical reactions
• ·         Lower energy requirement
• ·         Cost effectiveness

The industrial use of enzymes is increasing day by day as there is the need of cleaner and greener industrial practices. According to a research conducted by Business Communication Company (BCC) the enzyme market is estimated to increase up to $6.32 billion which was $5.01 billion in 2016¹.

Beside all their qualities there are some limitations such as optimal range of temperature and pH and lower stability the use of enzymes is narrowed in the industries. Therefore, some modifications are made to get the desired catalytic properties i.e., “Enzyme Engineering”. Except ribozymes all enzymes are protein in nature and hence are made up of amino acids. Modifying the amino acid sequence of an enzyme in order to enhance the quality and activity of an enzyme is known as enzyme engineering. It is usually applied to achieve thermal and pH stability, enhanced substrate specificity and better kinetic properties.

Methods of Enzyme Engineering

Mainly these approaches are applied for enzyme engineering i.e.,

• 1.      Rational Approach

• 2.      Directed evolution

• 3.      Semi-Rational Approach

• 4.      De-novo Designing

• 1.      Rational approach

It is the earlier and widely used approach in enzyme engineering. It is based on extensive understanding of the structure function dynamics of the enzyme and it involves making precise changes in the amino acid sequence of an enzyme. Then these precise changes are introduced by site-directed mutagenesis. Due to the increased beneficial mutations and the reduction of library size, it takes lesser time, cost and effort to screen the library. Multiple sequence alignment, detailed study of enzyme’s structure and different computational methods are used in this regard. Different bioinformatics tools are employed to study the three-dimensional structure of enzyme and to analyze the effect of any change in primary structure on its function. Molecular Dynamic (MD) simulations have brought a revolutionary change in this field, as it allows the scientist to study those dynamic molecular interactions which are involved in stability, solubility and function of the enzyme. The engineering of superoxide dismutase is one of the classic examples of rational designing. The coenzyme specificity of Thermus thermophilus isopropylmalate dehydrogenase was enhanced from a 100-fold preference for NAD to a 1000-fold preference for NADP. The engineered mutant was found to be twice as active as the wild type and resulted from substitution of only four amino acids ².  Apart from a number of successful attempts there are also some failures which are most probably the result of incomplete understanding of structure-function relationships.

• 2.      Directed evolution

Directed evolution also known as in-vitro evolution does not require structural data of enzyme. It is an empirical approach and mimics the process of natural evolution and harness the power of mutation. This technique employs a random process in which error-prone PCR or homologous recombination (DNA shuffling) is used to create a library of mutagenized genes. After that enzymes with improved catalytic properties are identified and their respective genes are amplified. The most critical step of directed evolution is to identify the best variant among several potential mutants and many methods are employed to assess the activity of enzyme and the selection is mostly based on the survival of cell after mutation. Direct evolution is the preferred strategy in enzyme engineering as it requires minimal data. The engineering of cytochrome P450 enzyme to alter its substrate specificity from long-chain fatty acids towards short alkanes³ and evolution of pyruvate aldolase⁴ are the examples of directed evolution.

• 3.      Semi-rational Approach

It is a hybrid of rational and directed evolution strategies. In this approach instead of larger libraries smaller and smarter libraries are designed and this approach requires the structural and functional data of the enzyme as well as also includes computational algorithms to predict the promising targets for modification. Machine-learning and quantum mechanics QM calculations are employed to study different features of the tailored enzyme. Semi-rational approach has increased the efficiency of enzyme engineering. The modification of human guanine deaminase to alter its specificity from ammelide to cytosine via semi-rational approach is an example⁵.

• 4.      De-novo Enzyme Design

Instead of remodeling an existing enzyme, the creation of biocatalysts from scratch is known as de-novo designing of enzyme. It mainly depends upon computational methods and involves in-silico designing of synthetic enzymes. It is the most recent and rapidly developing approach in enzyme engineering and has become the focus of biotechnologists around the globe. The recent de-novo designing of synthetic enzyme for stereoselective bimolecular Diels-Alder reaction⁶ is a breakthrough in enzyme engineering.

The developments in the field of enzyme engineering have enabled the researchers to manipulate and tailor the enzymes according to their current demands and needs. These modified enzymes with enhanced thermal and storage stability, better catalytic properties and altered substrate specificity have made their room in industry and medicine. It is also a step towards the establishment of ‘green’ industries and will surely bring a bright future for biotechnologists.

By: Mehwish Hamid

 

References:

1. Industrial enzymes market by type (carbohydrases, proteases, non-starch polysaccharides & others), application (food & beverage, cleaning agents, animal feed&others), brands&by region—global trends and forecasts to 2020. www.bccresearch.com. http://www.marketsandmarkets.com/Market-Reports/industrial-enzymesmarket-237327836.html.
2. Chen, R., Greer, A., & Dean, A. M. (1996). Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase. Proceedings of the National Academy of Sciences, 93(22), 12171-12176.
3. Fasan, R., Chen, M. M., Crook, N. C., & Arnold, F. H. (2007). Engineered alkane‐hydroxylating cytochrome P450BM3 exhibiting nativelike catalytic properties. Angewandte Chemie International Edition, 46(44), 8414-8418.
4. Cheriyan, M., Walters, M. J., Kang, B. D., Anzaldi, L. L., Toone, E. J., & Fierke, C. A. (2011). Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate. Bioorganic & medicinal chemistry, 19(21), 6447-6453.
5. Murphy, P. M., Bolduc, J. M., Gallaher, J. L., Stoddard, B. L., & Baker, D. (2009). Alteration of enzyme specificity by computational loop remodeling and design. Proceedings of the National Academy of Sciences, 106(23), 9215-9220.
6. Siegel, J. B., Zanghellini, A., Lovick, H. M., Kiss, G., Lambert, A. R., Clair, J. L. S., ... & Houk, K. N. (2010). Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction. Science, 329(5989), 309-313.
7. Ali, M., Ishqi, H. M., & Husain, Q. (2020). Enzyme engineering: Reshaping the biocatalytic functions. Biotechnology and Bioengineering, 117(6), 1877-1894.