Chakrit Tachaapaikoon

BACKGROUND: Kestoses, short‐chain fructooligosaccharides (FOSs), are valued for their intestinal, immunomodulatory, sweetness, and prebiotic properties compared with longer‐chain FOSs. Although invertases are widely used for kestose production, most enzymes generate mixed FOS products. This study investigates a GH32 invertase from Saccharomyces cerevisiae (ScINV), heterologously expressed in Escherichia coli , with an emphasis on its product selectivity for kestose production. RESULTS: Under optimized conditions (600 g L ⁻¹ initial sucrose concentration, 1.0 μmol L ⁻¹ enzyme, pH 6.0, and 45 °C), ScINV produced 54.1 g L ⁻¹ kestoses after 24 h, corresponding to a yield of 9.0% and a purity of 7.5%. Purification by ligand‐exchange chromatography using XA2004‐30‐Ca resin increased kestose purity to 19.4% with complete recovery, whereas higher‐resolution fractionation achieved 100% purity with a reduced recovery of 14.9%. CONCLUSION: ScINV exhibits selective kestose production without formation of higher‐degree FOSs. Although partial peak overlap limits single‐step recovery, purification using XA2004‐30‐Ca resin provides a promising basis for scale‐up. Coupling this approach with continuous simulated moving bed technology may improve process efficiency and industrial feasibility. © 2026 Society of Chemical Industry.

Synthetic azo dyes are widely used in the textile industry; however, their use often poses environmental challenges. Here, we characterized the compost bacterium Bacillus subtilis strain CKCC for the decolorization of various azo dyes, including Congo Red, Reactive Black 5, Reactive Green 19, Reactive Red 120, and Reactive Blue 4. The application of strain CKCC exhibited high decolorization efficiency by utilizing various extracellular enzymes, including azoreductase and ligninolytic enzymes such as laccase, lignin peroxidase, and manganese peroxidase, which are essential for the decolorization of azo dyes. Fourier transform infrared spectroscopy (FTIR) analysis revealed structural changes during decolorization, consistent with the degradation of key functional groups. This transformation was attributed to the cleavage of azo linkages by azoreductase, with ligninolytic enzymes functioning on phenolic and aromatic moieties. While FTIR confirmed these structural changes, our findings only provided insights at the functional-group level, and the presence or absence of specific decolorized metabolites, such as aromatic amines, requires additional analytical techniques. In this study, the phytotoxic metabolites positively affected the germination and growth of Vigna radiata, confirming that decolorization using strain CKCC significantly reduced the toxic properties of the metabolites produced during dye decolorization. Hence, our isolated strain CKCC offers a potentially effective and environmentally sustainable method for treating azo-dye effluent in the textile industry.

Background: Lactulose is a rare disaccharide with applications in the pharmaceutical, food, and animal feed industries. It can be synthesized through a transgalactosylation reaction between fructose and lactose, mediated by β‐galactosidases. However, the need for high excess fructose to drive lactulose synthesis, along with relatively low yields and selectivity, has posed challenges for commercial production. This study extensively characterizes a novel lactulose‐producing glycoside hydrolase family 2 (GH2) β‐galactosidase from Caldicellulosiruptor saccharolyticus expressed in Escherichia coli (CsBgl2A).Results: The enzyme exhibited high activity across a broad pH and temperature range, maintaining more than 95% of its maximal activity between pH 6.0–9.0 and 40–55 °C. Under optimal conditions – 750 g L⁻¹ initial sugar concentration, a fructose‐to‐lactose molar ratio of 4, 6 μmol L⁻¹ enzyme, pH 7.0, and 55 °C – the reaction produced a maximum lactulose concentration of 81.9 g L⁻¹ at 3 h, with 32.5% lactulose yield, 10.9% lactulose‐to‐total sugars yield, and 61.6% lactose conversion. By 6 h, nearly all lactose had been hydrolyzed.Conclusion: CsBgl2A is among the few β‐galactosidases that achieve high lactulose yields with low fructose‐to‐lactose ratios. This enzyme is thus promising for producing low lactose dairy products and for cost‐effective lactulose synthesis. © 2025 Society of Chemical Industry.

BACKGROUND: Cellobiose 2‐epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3‐1 (IfCE) was successfully expressed in Escherichia coli and characterized.RESULTS: Unlike other CEs, the purified IfCE shows only epimerization activity toward β‐1,4‐glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, β‐1,4‐glycosidic linkages of trisaccharides and α‐1,4‐glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low‐calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL⁻¹ lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L⁻¹, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl⁻ and Na⁺ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen.CONCLUSION: The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry.

Recently, we reported the discovery of a novel endoglucanase of the glycoside hydrolase family 12 (GH12), designated IfCelS12A, from the haloalkaliphilic anaerobic bacterium Iocasia fonsfrigidae strain SP3-1, which was isolated from a hypersaline pond in the Samut Sakhon province of Thailand (ca. 2017). IfCelS12A exhibits high substrate specificity on carboxymethyl cellulose and amorphous cellulose but low substrate specificity on b-1,3;1,4-glucan. Unlike some endoglucanases of the GH12 family, IfCelS12A does not exhibit hydrolytic activity on crystalline cellulose (i.e., Avicel™). High-Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) analyses of products resulting from IfCelS12-mediated hydrolysis indicate mode of action for this enzyme. Notably, IfCelS12A preferentially hydrolyzes cellotetraoses, cellopentaoses, and cellohexaoses with negligible activity on cellobiose or cellotriose. Kinetic analysis with cellopentaose and barely b-d-glucan as cellulosic substrates were conducted. On cellopentaose, IfCelS12A demonstrates a 16-fold increase in activity (K _M = 0.27 mM; k _cat = 0.36 s⁻¹; k _cat /K _M = 1.34 mM⁻¹ s⁻¹) compared to the enzymatic hydrolysis of barley b-d-glucan (K _M : 0.04 mM, k _cat : 0.51 s⁻¹, k _cat /K _M = 0.08 mM⁻¹ s⁻¹). Moreover, IfCelS12A enzymatic efficacy is stable in hypersaline sodium chlorids (NaCl) solutions (up to 10% NaCl). Specifically, IfCel12A retains notable activity after 24 h at 2M NaCl (10% saline solution). IfCelS12A used as a cocktail component with other cellulolytic enzymes and in conjunction with mobile sequestration platform technology offers additional options for deconstruction of ionic liquid–pretreated cellulosic feedstock. Key points: • IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance • IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass • IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis

Ake-kavitch Siriatcharanon, Sawannee Sutheeworapong, Sirilak Baramee, Rattiya Waeonukul, Patthra Pason, Akihiko Kosugi, Ayaka Uke, Khanok Ratanakhanokchai, and Chakrit Tachaapaikoon. J. Microbiol. Biotechnol. 2024;34:457-66. https://doi.org/10.4014/jmb.2307.07004

Bacillus subtilis strain AP-1, which produces α-glucosidase with transglucosidase activity, was used to produce a series of long-chain isomaltooligosaccharides (IMOs) with degree of polymerization (DP) ranging from 2 to 14 by direct fermentation of maltose. A total IMOs yield of 36.33 g/L without contabacillusmination from glucose and maltose was achieved at 36 h of cultivation using 50 g/L of maltose, with a yield of 72.7%. IMOs were purified by size exclusion chromatography with a Superdex 30 Increase column. The molecular mass and DP of IMOs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Subsequently, linkages in produced oligosaccharides were verified by enzymatic hydrolysis with α-amylase and oligo-α-1,6-glucosidase. These IMOs showed prebiotic properties, namely tolerance to acidic conditions and digestive enzymes of the gastrointestinal tract, stimulation of probiotic bacteria growth to produce short-chain fatty acids and no stimulating effect on pathogenic bacteria growth. Moreover, these IMOs were not toxic to mammalian cells at up to 5 mg/mL, indicating their biocompatibility. Therefore, this research demonstrated a simple and economical method for producing IMOs with DP2–14 without additional operations; moreover, the excellent prebiotic properties of the IMOs offer great prospects for their application in functional foods.

D-Psicose is a rare, low-calorie sugar that is found in limited quantities in national products. Recently, D-psicose has gained considerable attention due to its potential applications in the food, nutraceutical, and pharmaceutical industries. In this study, a novel D-psicose 3-epimerase (a group of ketose 3-epimerase) from an extremely halophilic, anaerobic bacterium, Iocasia fonsfrigidae strain SP3-1 (IfDPEase), was cloned, expressed in Escherichia coli, and characterized. Unlike other ketose 3-epimerase members, IfDPEase shows reversible epimerization only for D-fructose and D-psicose at the C-3 position but not for D-tagatose, most likely because the Gly218 and Cys6 at the substrate-binding subsites of IfDPEase, which are involved in interactions at the O-1 and O-6 positions of D-fructose, respectively, differ from those of other 3-epimerases. Under optimum conditions (5 µM IfDPEase, 1 mM Mn²⁺, 50 °C, and pH 7.5), 36.1% of D-psicose was obtained from 10 mg/mL D-fructose. The IfDPEase is highly active against D-fructose under NaCl concentrations of up to 500 mM, possibly due to the excessive negative charges of acidic amino acid residues (aspartic and glutamic acids), which are localized on the surface of the halophilic enzyme. These negative charges may protect the enzyme from Na⁺ ions from the environment and result in the lowest pI value compared to those of other 3-epimerase members. Moreover, without adjusting any ingredients, IfDPEase could improve coconut water quality by converting D-fructose into D-psicose with a yield of 26.8%. Therefore, IfDPEase is an attractive alternative to enhancing the quality of fructose-containing foods.

The valorization of lignocellulosic feedstocks into biofuels and biochemicals has received much attention due to its environmental friendliness and sustainability. However, engineering an ideal microorganism that can both produce sufficient cellulases and ferment ethanol is highly challenging. In this study, we have tested seven different genes that are involved in glycosylphosphatidylinositol (GPI) biosynthesis and remodeling for the improvement of cellulase activity tethered on the S. cerevisiae cell surface. It was found that the overexpression of LAS21 can improve β-glucosidase activity by 48.8% compared to the original strain. Then, the three cellulase genes (cellobiohydrolase, endoglucanase, and β-glucosidase) and the LAS21 gene were co-introduced into a diploid thermotolerant S. cerevisiae strain by a multiple-round transformation approach, resulting in the cellulolytic ECBLCCE5 strain. Further optimization of the bioprocess parameters was found to enhance the ethanol yield of the ECBLCCE5 strain. Scaling up the valorization of pretreated sugarcane bagasses in a 1 L bioreactor resulted in a maximum ethanol concentration of 28.0 g/L (86.5% of theoretical yield). Our study provides a promising way to improve the economic viability of second-generation ethanol production. Moreover, the engineering of genes involved in GPI biosynthesis and remodeling can be applied to other yeast cell surface display applications.

Background: Cellulolytic, hemicellulolytic, and amylolytic (CHA) enzyme-producing halophiles are understudied. The recently defined taxon Iocasia fonsfrigidae consists of one well-described anaerobic bacterial strain: NS-1^T. Prior to characterization of strain NS-1^T, an isolate designated Halocella sp. SP3-1 was isolated and its genome was published. Based on physiological and genetic comparisons, it was suggested that Halocella sp. SP3-1 may be another isolate of I. fronsfrigidae. Despite being geographic variants of the same species, data indicate that strain SP3-1 exhibits genetic, genomic, and physiological characteristics that distinguish it from strain NS-1^T. In this study, we examine the halophilic and alkaliphilic nature of strain SP3-1 and the genetic substrates underlying phenotypic differences between strains SP3-1 and NS-1^T with focus on sugar metabolism and CHA enzyme expression. Methods: Standard methods in anaerobic cell culture were used to grow strains SP3-1 as well as other comparator species. Morphological characterization was done via electron microscopy and Schaeffer-Fulton staining. Data for sequence comparisons (e.g., 16S rRNA) were retrieved via BLAST and EzBioCloud. Alignments and phylogenetic trees were generated via CLUTAL_X and neighbor joining functions in MEGA (version 11). Genomes were assembled/annotated via the Prokka annotation pipeline. Clusters of Orthologous Groups (COGs) were defined by eegNOG 4.5. DNA-DNA hybridization calculations were performed by the ANI Calculator web service. Results: Cells of strain SP3-1 are rods. SP3-1 cells grow at NaCl concentrations of 5-30% (w/v). Optimal growth occurs at 37 °C, pH 8.0, and 20% NaCl (w/v). Although phylogenetic analysis based on 16S rRNA gene indicates that strain SP3-1 belongs to the genus Iocasia with 99.58% average nucleotide sequence identity to Iocasia fonsfrigida NS-1^T, strain SP3-1 is uniquely an extreme haloalkaliphile. Moreover, strain SP3-1 ferments D-glucose to acetate, butyrate, carbon dioxide, hydrogen, ethanol, and butanol and will grow on L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-raffinose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, xylan and phosphoric acid swollen cellulose (PASC). D-rhamnose, alginate, and lignin do not serve as suitable culture substrates for strain SP3-1. Thus, the carbon utilization profile of strain SP3-1 differs from that of I. fronsfrigidae strain NS-1^T. Differences between these two strains are also noted in their lipid composition. Genomic data reveal key differences between the genetic profiles of strain SP3-1 and NS-1^T that likely account for differences in morphology, sugar metabolism, and CHA-enzyme potential. Important to this study, I. fonsfrigidae SP3-1 produces and extracellularly secretes CHA enzymes at different levels and composition than type strain NS-1^T. The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery.