Protein engineering thermostability

The application test of protein-engineered, thermostable glucose isomerase. The activity of immobilized enzyme is plotted as a function of time. The stability at 70 °C indicates how the enzymes will behave under industrial conditions. The variant Lys253Arg oi Actinoplanes missouriensis glucose isomerase has been shown to have (also under industrial conditions) a doubled half-life... 

Jensen LG, Olsen O, Kops O, Wolf N, Thomsen K, von Wettstein D. 1996. Transgenic Barley Expressing a Protein-Engineered, Thermostable (1,3-1,4)-B-Glucanase During Germination. Proceedings of the National Academy of Sciences USA. 93 3487-3491. 

I. Lasters, Enhancing the thermostability of glucose isomerase by protein engineering, Bio/Technology 1991, 9, 738-742. 

The by-products, such as isomaltose, have to be removed chromatographically, which is energy- and cost-intensive. Protein engineering focuses on increasing the glucose yield (affecting enzyme selectivity) and enzyme thermostability. 

T. J. Ahern, J. I. Casal, G. A. Petsko, and A. M. Klibanov, Control of oligomeric enzyme thermostability by protein engineering, Proc. Natl. Acad. Sci. USA 1987, 84, 675-679. 

Lehmann M, Wyss M. Engineering proteins for thermostability the use of sequence alignments versus rational design and directed evolution. Curr. Opin. Biotechnol. 2001 12 371-375. Berezovsky IN, Shakhnovich El. Physics and evolution of thermophilic adaptation. Proc. Natl. Acad. Sci. U.S.A. 2005 102 12742-12747. 

The examples above represent some of the most successful studies in protein engineering. They show that it is possible to enhance protein thermostability rationally, alter cofactor or substrate specificity, regiospecificity, and even change catalytic activity. Furtherm ore, the creation of enzymatic activity from a non-catalytic protein backbone, and the creation of a biocatalyst with an unprecedented catalytic activity not found in nature, have also been achieved. However, the examples published in the literature are probably only a tiny fraction of the many studies that have been, or are still, in progress awaiting positive results. 

Priolo N, Morcelle del Valle S, Arribere MC et al. (2000) Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits. J Protein Chem 19(l) 39-49 Quax W, Mrabet N, litten R et al. (1991) Enhancing the thermostability of glucose isomerase by protein engineering. Bio/Technology 9 738-742... 

Hartley B S, Hanlon N, Jackson R J, Rangrajan M (2000), Glucose isomer-ase insight into protein engineering for increased thermostability , Biochem. Biophys. Acta., 1543,294-335. 

Enzymes typically function at normal body temperature, 37°C. Their activities at higher temperatures are desirable for rate acceleration, but free enzymes denature at elevated temperatures. Upon intercalation in a-ZrRP (R = OH), both HRP and Hb showed peroxidase activities at over 85°C, observed for the first time, (258) while the free enzyme/protein deactivated rapidly at these temperatures with no activity. The maximum rate of the reaction (Vmax) increased 3.6-fold, while the concentration needed to achieve half the maximum rate (K ) decreased by 20% at these higher temperatures. Such high-temperature activities of enzymes/ proteins are unusual, and they indicate the promise of a-ZrRPs for enzyme stabilization in high-temperature applications. This strategy of enzyme stabilization in a-ZrRP may provide alternatives to thermophilic enzymes obtained from thermo-philes, and these may supplement thermostable enzymes obtained by protein engineering. 

Site-directed mutagenesis has been a valuable method for the engineering of many proteins, but a significant limitation on this technique is that it can be difficult to know what mutations should be made in order to obtain a desired functionality. For example, in order to increase the thermostability of a protein, it is not clear by looking at a 3-D structure which amino acid side chains will affect this trait. In addition, improvements made in the thermostability of the enzyme may adversely affect other properties of the protein, such as enzymatic activity. Therefore, there has been a good deal of interest in combinatorial methods for protein engineering, which can be used to sample a large area of the protein solution space, and thus rapidly identify proteins with desired functionalities. 

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