Biotechnology

Biography

Biography: Risa Yamashiro

Abstract

l-Asparaginase hydrolyzes l-asparagine into l-aspartate and ammonia, and is widely distributed in microorganisms, plants, and animals. l-Asparaginase has been applied in the food industry for reducing acrylamide, a carcinogenic compound in foods, which is mainly formed by a reaction between reducing sugars and asparagine at high temperature more than 120ºC. Although l-asparaginases from Escherichia coli and Erwinia chrysanthemi are commercially available, approved l-asparaginases for the application are few because of their lack of appropriate properties. Therefore, new l-asparaginases with better performance have been investigated worldwide.

In the present study, we focused on l-asparaginases from lactic acid bacteria, Lactobacillus delbrueckii and Lactobacillus sakei, designated as LdASNase and LsASNase, respectively. The expression plasmids were constructed by inserting LdASNase or LsASNase gene into pET-28a. Escherichia coli BL21 (DE3) transformed with the plasmid was cultured in LB medium in the presence of isopropyl β-D-thiogalactoside at 37ºC. The SDS-PAGE analysis showed that LdASNase was only expressed in insoluble form, whereas LsASNase was expressed in soluble and insoluble form. The soluble LsASNase was purified by Ni-affinity chromatography to electrophoretic homogeneity. The enzyme activity was measured by determining the amount of ammonia formed with glutamate dehydrogenase. The specific activity of purified LsASNase was calculated to be 1.32 U/mg. The optimum pH and reaction temperature were found to be 7-9 and 40ºC, respectively. LsASNase showed high substrate specificity towards l-asparagine compared with l-glutamine. In addition, LsASNase activity was inhibited by Zn²⁺ and Fe³⁺.