Recombinant expression and characterization of a novel cold-adapted type I pullulanase for efficient amylopectin hydrolysis

Highlights

• PulPB1 can efficiently hydrolyze pullulan and amylopectin at ambient temperatures.

• PulPB1 showed great stability; its half-life at 50 °C was measured as 137 h.

• The N-terminal domain of PulPB1 significantly affected the enzymatic performances.

• PulPB1 exhibited huge potentialities in cold amylopectin hydrolysis.

Abstract

Cold-adapted pullulanase with high catalytic activity and stability is of special interest for its wide application in cold starch hydrolysis, but few pullulanases displaying excellent characteristics at ambient temperature and acidic pH have hitherto been reported. Here, a novel pullulanase from Bacillus methanolicus PB1 was successfully expressed in Escherichia coli BL21 (DE3) and determined to be a cold-adapted type I pullulanase (PulPB1) with maximum activity at 50 °C and pH 5.5. The recombinant PulPB1 showed great stability, its half-life at 50 °C was 137 h. PulPB1 can efficiently hydrolyze pullulan and amylopectin, with activities of 292 and 184 U/mg at 50 °C and pH 5.5, respectively. Moreover, the N-terminal domain of PulPB1 was found to significantly affect the enzymatic performance. Following truncation of the N-terminal domain, the activity towards pullulan decreased markedly from 292 to 141 U/mg and the half-life at 50 °C decreased from 137 to 10 h. Compared to the hydrolysis system with amyloglucosidase alone, the catalytic efficiency showed a 2.4-fold increase on combining PulPB1 with amyloglucosidase for amylopectin hydrolysis at 40 °C. This demonstrates that PulPB1 is promising for development as a superior candidate for cold amylopectin hydrolysis.

Introduction

Due to increasing concerns about energy and the environmental crisis, it is generally accepted that the gradual replacement of oil with renewable biomass resources is an effective way to develop a sustainable society (Cinelli et al., 2015; Hii et al., 2012). Therefore, efficient biotransformation of inexpensive and environmentally friendly biomass resources into industrial raw materials or biofuels has attracted a great deal of attention. Starch, one of the most abundant biopolymers on Earth, has been used as a major raw material for foods, chemicals, and biofuels (van der Maarel et al., 2002; Lu et al., 2018; Wang et al., 2017). Most starch contains 70–95 % amylopectin, which is composed of α-1,4-linked glucose chains ramified with α-1,6-linked branch points. Since most amylases specifically attack the α-1,4-glycosidic linkage, enzymes that cleave the α-1,6-glycosidic linkages are indispensable for effective and complete decomposition of starch into small-molecule sugars (Chang et al., 2016; Duan et al., 2013; Sun et al., 2010).

Pullulanase (EC 3.2.1.41), a well-known starch-debranching enzyme, has been widely applied to catalyze the hydrolysis of α-1,6-glycosidic linkages at branching points in pullulan, amylopectin, and related saccharides (Wang et al., 2019b). Based on substrate preference and reaction products, pullulanases are classified into five groups: type I pullulanases, amylopullulanases, neopullulanases, isopululanases and pullulan hydrolases type III (Møller et al., 2016; Vincent Lombard et al., 2014). Type I pullulanases specifically attack the α-1,6-glycosidic linkages due to the consensus sequence YNWGYDP at the catalytic domain (Hatada et al., 1996; Yamashita et al., 1997). Pullulanases are members of the glycoside hydrolase family 13 (GH13) (CAZy; http://www.cazy.org) and consist of a highly conserved catalytic domain, C-terminal domain and additional carbohydrate-binding modules (CBMs) at the N-terminus (Xu et al., 2014; Zeng et al., 2019a).

In the saccharification process, the addition of pullulanase can lower the dosage of glucoamylase, reduce the reaction time, and effectively improve the conversion rate (Duan et al., 2013; Lu et al., 2018). In recent years, a great deal of research has been focused on isolating thermoactive pullulanases from various microbial sources, such as Staphylothermus marinus (Li et al., 2013), Geobacillus kaustophilus DSM7263 (Li et al., 2018a), Bacillus sp. CICIM 263 (Li et al., 2012), and Thermotoga neapolitana (Chang et al., 2016; Kang et al., 2011). Further research has been focused on improving thermostability through site-directed mutagenesis or domain modification based on structure or sequence information (Chang et al., 2016; Chen et al., 2015, 2019; Duan and Wu, 2015a; Li et al., 2015; Wang et al., 2018b). All of these studies have been aimed at making pullulanases suitable for the current starch saccharification process, which is performed at least at 60 °C (Hii et al., 2012).

However, it should be noted that starch hydrolysis at high temperature is expected to be replaced by new technologies functioning at relatively moderate temperatures owing to its high energy consumption and complex process. In the past few years, cold starch hydrolysis, also known as “granular starch hydrolysis”, which is enabled by enzymes that synergistically hydrolyze starch at 35 °C and pH 5–6, has been an emerging technology (Lu et al., 2018). Since the granular starch hydrolysis process entails direct hydrolysis of the starch-based substrate, without high-temperature incubation, gelatinization, and subsequent cooling, the operational costs of industrial plants have been estimated to be 51 % lower (Cinelli et al., 2015; Lu et al., 2018). The best conversion rate for starch into glucose hitherto achieved is 98.6 % (Cinelli et al., 2015). In recent years, some cold-adapted α-amylases (Yang et al., 2017), that randomly attack the α-1,4-glycosidic linkages in a linear amylose chain, and raw-starch-digesting glucoamylases (Sun et al., 2007), capable of releasing glucose from the non-reducing ends of starch and related substrates, have been studied. Pullulanase plays a fundamental role as a debranching enzyme in the hydrolysis of starch. In the research of using corn starch as substrate to produce ethanol through deep enzymolysis and fermentation, the addition of pullulanase accelerates the corn starch hydrolysis at 55 °C and pH 5.5, leading to lower amounts of dextrins and concomitantly higher ethanol yield (Klosowski et al., 2010; Lu et al., 2018). Therefore, cold-adapted pullulanases showing high activity and stability at around 40 °C have huge potential for application in granular starch hydrolysis.

In the last few years, only a few cold-adapted pullulanases exhibiting maximum activity at around 40 °C have been reported (Elleuche et al., 2015; Lu et al., 2018; Rajaei et al., 2015; Wei et al., 2015). Pul703 isolated from Bacillus pseudofirmus 703 and Pul-SH3 from Exiguobacterium sp. SH3, with maximum activity at 45 °C and pH 7.0–8.0, are current candidates for cold starch hydrolysis (Lu et al., 2018; Rajaei et al., 2014). However, the exhibition of optimal activity in alkaline environments and low catalytic efficiency greatly restrict their application. Herein, we present an excellent type I pullulanase (PulPB1) isolated from Bacillus methanolicus PB1 with great activity and stability over a wide temperature range 30–50 °C at pH 5.5. PulPB1 can be used for efficient cold starch hydrolysis without adjusting the temperature or pH, indicating its great potential in the granular starch hydrolysis industry.