In-Depth Characterization of Debranching Type I Pullulanase from Priestia koreensis HL12 as Potential Biocatalyst for Starch Saccharification and Modification

Abstract

Pullulanase is an effective starch debranching enzyme widely used in starch saccharification and modification. In this work, the biochemical characteristics and potential application of a new type I pullulanase from Priestia koreensis HL12 (HL12Pul) were evaluated and reported for the first time. Through in-depth evolutionary analysis, HL12Pul was classified as type I pullulanase belonging to glycoside hydrolase family 13, subfamily 14 (GH13_14). HL12Pul comprises multi-domains architecture, including two carbohydrate-binding domains, CBM68 and CBM48, at the N-terminus, the TIM barrel structure of glycoside hydrolase family 13 (GH13) and C-domain. Based on sequence analysis and experimental cleavage profile, HL12Pul specifically hydrolyzes only α-1,6 glycosidic linkage-rich substrates. The enzyme optimally works at 40 °C, pH 6.0, with the maximum specific activity of 181.14 ± 3.55 U/mg protein and catalytic efficiency (kcat/Km) of 49.39 mL/mg·s toward pullulan. In addition, HL12Pul worked in synergy with raw starch-degrading α-amylase, promoting raw cassava starch hydrolysis and increasing the sugar yield by 2.9-fold in comparison to the α-amylase alone in a short reaction time. Furthermore, HL12Pul effectively produces type III-resistant starch (RSIII) from cassava starch with a production yield of 70%. These indicate that HL12Pul has the potential as a biocatalyst for starch saccharification and modification.

A molecular study on recombinant pullulanase type I from Metabacillus indicus

“…In this context, the Pull_Met displayed an optimal pH of 6.0 which was well- compatible with the reported range of the optimal pH for pullulanases type I. Meanwhile, pullulanase type I from Lactococcus lactis (Waśko et al, 2011 ), Bacillus methanolicus PB1 (Zhang et al 2020 ), Priestia koreensis HL12 (Prongjit et al 2022 ), Bacillus megaterium Y103 (Wu et al 2022 ), and Anaerobranca gottschalkii (Bertoldo et al 2004 ), exhibited an optimal pH of 4.5, 5.5, 6.0, 6.5, and 8, respectively. The slight acidic to near neutral pH optima for Pull_Met could be explained by the perception that the enzyme is most likely secreted internally in the cytoplasm, which displays a low pH compared to the pH in the external environment (Krulwich et al 1997 ).…”

“…The conserved motif of seven amino acids YNWGYNP was reported to predominate in all pullulanases pullulanase type I that only could attack α-1,6 glycosidic bonds of pullulan (Erden-Karaoğlan et al, 2019 , Iqrar et al 2020 , Prongjit et al 2022 ), this region would appear to be involved in substrate binding or catalytic activity (Bertoldo et al 1999 , Yamashita et al, 1997 ). Reportedly, this conserved motif does not exist in pullulanases type II that could attack both α-1,4 glycosidic bonds and α-1,6 glycosidic bonds in pullulan.…”

“…SH3 (Rajaei et al 2015 )d methanolicus PB1 (Zheng et al 2021 ) exhibited an optimal temperature at 35, 35, 45, and 50 o C, respectively. Likewise Pull_Met, pullulanase type I from a hot-spring metagenome (Thakur et al 2021 ), B. subtilis BK07 (Erden-Karaoğlan et al, 2019), and B. subtilis PY22 (Erden-Karaoğlan et al, 2019) and Priestia koreensis HL12 (Prongjit et al 2022 ) showed an optimal temperature at 40 o C. Conversely, there is a plethora of reports addressing thermostable pullulanase type I with an optimal temperature above 50 o C. The optimal temperature of thermostable pullulanases type I from F. pennavorans Ven5 (Koch et al 1997 ), Anaerobranca gottschalkii (Bertoldo et al 2004 ), Bacillus thermoleovorans US105 (Messaoud et al 2002 ), Thermotoga maritima (Kriegshäuser et al, 2000 ) showed an optimal temperature at 65, 70, 75, and 90 o C, respectively. Pull_Met, a cold-adapted pullulanase type I, had a lower optimal temperature (40 o C) than other described cold-adapted pullulanases (45–50 o C) (e.g., pul-SH3 and pulPB1).…”

“…Typically, raw substrate hydrolysis requires the synergistic interaction of enzyme composites to achieve complete saccharification (conversion yield ≥ 80%), such as cooperation of α-amylase and pullulanase in starch hydrolysis (Pan and Lee 2005 ; Prongjit et al 2022 ).…”

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes

“…Besides, starch can be also applied to produce cyclodextrin and oligosaccharides through the action of cyclodextrin glycosyltransferase (EC 2.4.1.19) and maltooligosaccharide-forming amylases (EC 3.2.1.-), respectively. − Although various amylolytic enzymes have been used in starch processing, most of them are very expensive or inefficient in starch conversion . The majority of α-amylase, β-amylase, and α-glucosidase enzymes can hydrolyze α-1,4-glucosidic linkages in starch but have low activities for α-1,6-linkages, , resulting in a low overall conversion rate of starch material and large amounts of byproducts. , Therefore, eliminating α-1,6 branches by debranching enzymes (DBEs) during starch degradation is a critical and well-established approach to facilitate starch hydrolysis and improve production efficiency …”