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.

Pectin-derived oligosaccharides (POS) are emerging as promising functional prebiotics with growing industrial interest. This study reports a synergistic fungal pectinolytic biocatalytic system comprising endopolygalacturonase (EndoPG) and pectin methylesterase (PET11) from Aspergillus aculeatinus BCC 17849 for the controlled depolymerization of pomelo (Citrus maxima) albedo pectin. PET11-mediated demethylation increased substrate accessibility, thereby enhancing EndoPG-catalyzed hydrolysis and resulting in higher POS yields than those obtained with single-enzyme systems. The highest production of short-chain POS, comprising GalA, di-GalA, and tri-GalA (681 mg/g substrate), was achieved at an EndoPG:PET11 dosage ratio of 15:5. The resulting POS fraction significantly promoted the growth of five probiotic strains, including Lactobacilli and Bifidobacteria species, and enhanced probiotic adherence to intestinal epithelial cells. In particular, Lactobacillus acidophilus TBRC 5030 exhibited the highest adhesion level (35.24 ± 6.43%) in the presence of 2.0 mg/mL POS. Overall, this work demonstrated that enzyme-assisted demethylation coupled with targeted endo-hydrolysis enables effective tailoring of POS chain length, providing a promising biocatalytic strategy for pectin valorization into prebiotic ingredients.

The development of yeast cell factories for efficient xylose utilization and xylitol production is crucial for advancing sustainable biotechnological processes. Xylose, a major component of lignocellulosic biomass, presents challenges for microbial conversion due to its complex metabolic pathways. This study presents the genomic perspective and xylitol production capability of a novel xylose utilizing yeast Cyberlindnera fabianii TBRC 4498. Genome sequencing and functional annotation revealed key metabolic networks and genes involved in the xylose metabolism pathway, providing insights into the strain’s performance. The Cy. fabianii TBRC 4498 had excellent growth and xylose assimilation at a broad range of xylose concentrations from 40 to 140 g/L, with the highest growth rate at 80 g/L of xylose. The highest xylitol production yield (83.19 g/L) was detected from 120 g/L of xylose at 30 °C for 72 h, equivalent to 0.65 g xylitol/g xylose and 1.16 g/L/h productivity. Remarkably, Cy. fabianii TBRC 4498 produced high-purity xylitol, achieving over 95% homogeneity without forming undesirable byproducts, such as acid or ethanol. These results demonstrated the potential of Cy. fabianii TBRC 4498 as a whole-cell biocatalyst for xylitol production using high xylose concentrations, offering a promising microbial cell factory for large-scale xylitol production from lignocellulosic sugar.