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 (k_cat/K_m) 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.

Exploring new raw starch-hydrolyzing α-amylases and understanding their biochemical characteristics are important for the utilization of starch-rich materials in bio-industry. In this work, the biochemical characteristics of a novel raw starch-degrading α-amylase (HL11 Amy) from Roseateles terrae HL11 was firstly reported. Evolutionary analysis revealed that HL11Amy was classified into glycoside hydrolase family 13 subfamily 32 (GH13_32). It contains four protein domains consisting of domain A, domain B, domain C and carbohydrate-binding module 20 (CMB20). The enzyme optimally worked at 50 °C, pH 4.0 with a specific activity of 6270 U/mg protein and 1030 raw starch-degrading (RSD) U/mg protein against soluble starch. Remarkably, HL11Amy exhibited activity toward both raw and gelatinized forms of various substrates, with the highest catalytic efficiency (k_cat/K_m) on starch from rice, followed by potato and cassava, respectively. HL11Amy effectively hydrolyzed cassava pulp (CP) hydrolysis, with a reducing sugar yield of 736 and 183 mg/g starch from gelatinized and raw CP, equivalent to 72% and 18% conversion based on starch content in the substrate, respectively. These demonstrated that HL11Amy represents a promising raw starch-degrading enzyme with potential applications in starch modification and cassava pulp saccharification.

This study presents the first report on the integration of levan produced by Priestia koreensis HL12 into PVA/gelatin-based composite films and provides a comprehensive characterization of the resulting edible films. The properties of PVA/gelatin composite films were enhanced by incorporating levan at different ratios (10, 20, 30, 40, and 50 wt% relative to PVA). A small amount of glycerol was added as a plasticizer, and the three components were crosslinked using genipin. The PVA/gelatin/levan composite films were fabricated via the solvent casting method. The fabricated films exhibited smooth surface morphologies and a gradual color transition from blue to violet with increasing levan content. Scanning electron microscopy (SEM) was used to examine film morphology, while Fourier transform infrared spectroscopy (FTIR) confirmed crosslinking interactions between genipin and gelatin in the PVA/gelatin/levan component. Thermogravimetric analysis (TGA) was employed to evaluate thermal stability, and tensile testing was conducted to determine mechanical strength as a function of levan content. Among the formulations, PVA/GE/20%LV film exhibited enhanced thermal stability (from 321 to 314 °C) compared to PVA/GE/0%LV, the highest elongation at break (166%), and the lowest swelling index. These findings highlight the potential of levan from P. koreensis HL12 as a functional biopolymer, with the PVA/GE/20%LV film identified as the most suitable formulation for edible food packaging applications.