Improving and Controlling Natural Enzyme Reactions

From a random library of 60,000 clones, three improved variants of amylosucrase (two double mutants and one single mutant) were isolated that showed an improvement of their thermostability at 50 °C by 3.5- to 10-fold compared to the wild-type. The activity half-life of the best variant was 32 min at 50 °C, while the wild-type was completely inactivated at this temperature in less than 3 min. 

At 50°C, amylose synthesis using the improved mutant and high sucrose concentration (600 mM) allowed the production of amylose chains twice as long as those obtained by the parental enzyme at 30°C [125, 126], 

Most of the applications of a-glucans concern derived products of lower molecular weights, ranging from 1 to 70 kDa, which are currently obtained by acidic hydrolysis followed by solvent fractionation. Recently, engineered L. mesenteroides NRRL B-512F dextransucrases (DSR-S), truncated at the C-terminal domain, have made possible the direct synthesis of IMOs and dextrans of controlled molecular weight (10 and 40 kDa) in one pot (directly from sucrose) that are of interest for food, health, and fine chemical applications [129]. 

To reduce the extent of hydrolysis, several approaches can be attempted (1) continuous removal of the transglucosylation product by crystallization or selective adsorption onto different carriers (2) the presence of a suitable glycosyl acceptor that reacts as a nucleophile faster than water (3) the use of high concentration of acceptor (4) engineering the active site of the enzyme. 

In this regard, remarkable results were obtained for amylosucrase from 

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