Disorder prediction-based construct optimization improves activity and catalytic efficiency of Bacillus naganoensis pullulanase

Abstract

Pullulanase is a well-known starch-debranching enzyme. However, the production level of pullulanase is yet low in both wide-type strains and heterologous expression systems. We predicted the disorder propensities of Bacillus naganoensis pullulanase (PUL) using the bioinformatics tool, Disorder Prediction Meta-Server. On the basis of disorder prediction, eight constructs, including PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78 and PULΔN106 by deleting the first 5, 22, 45, 64, 78 and 106 residues from the N-terminus, and PULΔC9 and PULΔC36 by deleting the last 9 and 36 residues from the C-terminus, were cloned into the recombinant expression vector pET-28a-PelB and auto-induced in Escherichia coli BL21 (DE3) cells. All constructs were evaluated in production level, specific activities and kinetic parameters. Both PULΔN5 and PULΔN106 gave higher production levels of protein than the wide type and displayed increased specific activities. Kinetic studies showed that substrate affinities of the mutants were improved in various degrees and the catalytic efficiency of PULΔN5, PULΔN45, PULΔN78, PULΔN106 and PULΔC9 were enhanced. However, the truncated mutations did not change the advantageous properties of the enzyme involving optimum temperature and pH for further application. Therefore, Disorder prediction-based truncation would be helpful to efficiently improve the enzyme activity and catalytic efficiency.

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

“…All assays were performed in triplicate. Double mutants exhibiting enzyme activity greater than that of WT pullulanase (cutoff 580 U·mL −1 ) were selected for protein purification to accurately measure the Michaelis–Menten parameters.…”

“…However, the catalytic efficiency of pullulanase needs to be further improved for industrial application. To date, engineering of pullulanase is mainly focused on the catalytic pocket or domain tailoring based on structure/sequence information …”

Understanding Thermostability Factors of Barley Limit Dextrinase by Molecular Dynamics Simulations

“…There are several works focused on improvement of the themostability of pullulanase derived from bacteria (Chen et al, 2015;Li et al, 2015;Chang et al, 2016;Wang et al, 2016). However, it is still lacking the understanding on the factor affecting the themostability of HvLD at the atomic level.…”

Efficient production of extracellular pullulanase in Bacillus subtilis ATCC6051 using the host strain construction and promoter optimization expression system

“…Pullulanase (PUL, EC 3.2.1.41) is a debranching enzyme in the α-amylase family GH13, capable of specifically hydrolyzing a-1,6-glycosidic linkages in pullulan, amylopectin, glycogen, and other related polysaccharides [ 35 ]. In food industries, there is a great commercial interest of using pullulanase, especially in the saccharification process together with saccharifying amylases for the production of high-glucose syrup and maltose [ 36 ]. Over the years, a large number of pullulanases have been discovered and identified from different microorganisms, and have been successfully heterologously expressed in Escherichia coli [ 37 , 38 ] and various Bacillus species [ 35 , 39 ].…”

“…Over the years, a large number of pullulanases have been discovered and identified from different microorganisms, and have been successfully heterologously expressed in Escherichia coli [ 37 , 38 ] and various Bacillus species [ 35 , 39 ]. However, only pullulanase derived from few strains such as Bacillus acidopullulyticus [ 40 ] and Bacillus naganoensis has great commercial value; and the production of pullulanase faces many difficulties, such as the expression levels of recombinant pullulanase were yet limit, low yields and low enzyme activity [ 36 ]. As shown in Table 1 , the protease-deficient B. subtilis host strains (WB600, WB800) have been explored for overexpressing B. naganoensis pullulanase, but the expression levels are poorer, and cannot satisfy industrial needs.…”