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| The Effects of N-Terminal Amino Acids Truncation on Pullulanase Properties |
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Abstract Starch is one kind of abundant biomass resources which is widely used in lots of industries such as food, brewing and pharmaceutical industries. Pullulanase (pulA, EC 3.2.1.41) is one of the starch-debranching enzymes which catalyzes the hydrolysis of α-1,6 glycosidic bonds in starch, and improves the utilization of starch. Hence, it has a more profound reason to develop pullulanase in our own way. In the previous study, a pullulanase high-producing strain, Klebiella variicola strain 7, was obtained from the soil near a starch factory. The gene encoding pullulanase (GenBank No. KJ146839) from Klebiella variicola strain 7 was expression in Escherich coli BL21(DE3). In order to improve expression quantity, secretion efficiency and properties of pullulanase, Mutants of M1 and M2 were built using the method of genetic engineering of removing N- terminal amino acids of pullulanase to improve expression quantity, secretion efficiency and properties of pullulanase. The results showed that, the optimum temperature of both M1 and M2 were 45 ℃, and the optimum pH of M1 was 6.0, while the optimum pH of M2 was 5.6. The half-life and specific activity of M2 were 37 minutes and 582.204 U/mg, which were 6.17- and 1.6- fold that of M1. With pullulan as substrate, Kinetic studies showed that the Vmax, Kcat, Km and Kcat /Km of M2 was 0.001 3 μmoL/(mL·s)-1, 191.80 s-1, 0.30 mg/mL, 693.30, respectively. Compared with M1, the substance affinity increased, and the catalytic efficiency was 2- fold that of M1. This study provides a new way and thinking for improving the specific activity and half-life period of enzymes by N-terminal amino acids truncation.
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Received: 08 December 2015
Published: 20 May 2016
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