title: Enhancing the secretion efficiency and thermostability of a Bacillus deramificans pullulanase mutant (D437H/D503Y) by N-terminal domain truncation.
abstract: Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072-4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 10(8) and 449.3 × 10(8) U · mol(-1), respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China jingwu@jiangnan.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013696:Temperature; D013997:Time Factors
chemical_list: D050505:Mutant Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase