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

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 alpha-1,6 glycosidic linkage-rich substrates. The enzyme optimally works at 40 degrees C, pH 6.0, with the maximum specific activity of 181.14 +/- 3.55 U/mg protein and catalytic efficiency (k(cat)/K-m) of 49.39 mL/mg center dot s toward pullulan. In addition, HL12Pul worked in synergy with raw starch-degrading alpha-amylase, promoting raw cassava starch hydrolysis and increasing the sugar yield by 2.9-fold in comparison to the alpha-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.

Automated summary

In this study, the biochemical characteristics and potential applications of a new type I pullulanase (HL12Pul) were evaluated and reported for the first time. The enzyme shows specific hydrolysis activity towards alpha-1,6 glycosidic linkage-rich substrates and exhibits optimal activity at 40 degrees C, pH 6.0. HL12Pul also works synergistically with alpha-amylase, enhancing raw cassava starch hydrolysis and increasing the sugar yield. Additionally, HL12Pul effectively produces type III-resistant starch from cassava starch.