ß-Galactosidase-activated near-infrared AIEgen for ovarian cancer imaging in vivo.

Near-infrared (NIR) aggregation induced-emission luminogens (AIEgens) circumvent the noisome aggregation-caused quenching (ACQ) effect in physiological milieu, thus holding high promise for real-time and sensitive imaging of biomarkers in vivo. ß-Galactosidase (ß-Gal) is a biomarker for primary ovarian carcinoma, but current AIEgens for ß-Gal sensing display emissions in the visible region and have not been applied in vivo. We herein propose an NIR AIEgen QM-TPA-Gal and applied it for imaging ß-Gal activity in vitro and in ovarian tumor model. After being internalized by ovarian cancer cells (e.g., SKOV3), the hydrophilic nonfluorescent QM-TPA-Gal undergoes hydrolyzation by ß-Gal to yield hydrophobic QM-TPA-OH, which subsequently aggregates into nanoparticles to turn NIR fluorescence "on" through the AIE mechanism. In vitro experimental results indicate that QM-TPA-Gal has a sensitive and selective response to ß-Gal with a limit of detection (LOD) of 0.21 U/mL. Molecular docking simulation confirms that QM-TPA-Gal has a good binding ability with ß-Gal to allow efficient hydrolysis. Furthermore, QM-TPA-Gal is successfully applied for ß-Gal imaging in SKOV3 cell and SKOV3-bearing living mouse models. It is anticipated that QM-TPA-Gal could be applied for early diagnosis of ovarian cancers or other ß-Gal-associated diseases in near future.

Covalent immobilization of ß-galactosidase using a novel carrier alginate/tea waste: statistical optimization of beads modification and reusability.

ß-galactosidase has been immobilized onto novel alginate/tea waste gel beads (Alg/TW) via covalent binding. Alg/TW beads were subjected to chemical modification through amination with polyethyleneimine (PEI) followed by activation with glutaraldehyde (GA). Chemical modification parameters including PEI concentration, PEI pH, and GA concentration were statistically optimized using Response Surface methodology (RSM) based on Box-Behnken Design (BBD). Analysis of variance (ANOVA) results confirmed the great significance of the model that had F value of 37.26 and P value < 0.05. Furthermore, the R2 value (0.9882), Adjusted R2 value (0.9617), and predicted R2 value (0.8130) referred to the high correlation between predicted and experimental values, demonstrating the fitness of the model. In addition, the coefficient of variation (CV) value was 2.90 that pointed to the accuracy of the experiments. The highest immobilization yield (IY) of ß-galactosidase (75.1%) was given under optimized conditions of PEI concentration (4%), PEI pH (9.5), and GA concentration (2.5%). Alg/TW beads were characterized by FT-IR, TGA, and SEM techniques at each step of immobilization process. Moreover, the immobilized ß-galactosidase revealed a very good reusability as it could be reused for 15 and 20 consecutive cycles keeping 99.7 and 72.1% of its initial activity, respectively. In conclusion, the environmental waste (tea waste) can be used in modern technological industries such as the food and pharmaceutical industry.

Amorphous-crystalline phase transition and intrinsic magnetic property of nickel organic framework for easy immobilization and recycling of ß-Galactosidase.

This work describes the synthesis of fibrous nickel-based metal organic framework (Ni-ZIF) via simple solvothermal method. The material formed was calcinated at 400, 600, 800 °C to improve its surface area, porosity and enzyme binding capacity. Changes in X-ray diffraction pattern after calcination revealed the Ni-ZIF transitioned from amorphous to crystalline structure. The surface area, pore volume and pore size for Ni-ZIF@600 were found to be 312.15 m2/g, 0.88 cm3/g and 10.28 nm, with an enzyme loading capacity of 593.85 mg/g after 30 h The free (ß-Gal-LEH) and immobilized ß-Galactosidase were stable at pH 7.5, temperature 50 °C, and yielded 70.70 and 63.95 mM glucose after milk lactose hydrolysis, respectively. The Ni-ZIF@600@ß-Gal-LEH exhibited high enzyme retention capacity, maintaining 59.44 % of its original activity after 6-cycles. The enhanced magnetic property, enzyme binding capacity and easy recoverability of the calcinated Ni-ZIF could guarantee its industrial significance as immobilization module for enzyme-mediated catalysis.

Magnetic core-shell cellulose system for the oriented immobilization of a recombinant ß-galactosidase with a protein tag.

The objective of this study was to immobilize a recombinant ß-galactosidase (Gal) tagged with a cellulose-binding domain (CBD) onto a magnetic core-shell (CS) cellulose system. After 30 min of reaction, 4 U/capsule were immobilized (CS@Gal), resulting in levels of yield and efficiency exceeding 80 %. The optimal temperature for ß-galactosidase-CBD activity increased from 40 to 50 °C following oriented immobilization. The inhibitory effect of galactose decreased in the enzyme reactions catalyzed by CS@Gal, and Mg2+ increased the immobilized enzyme activity by 40 % in the magnetic CS cellulose system. The relative enzyme activity of the CS@Gal was 20 % higher than that of the soluble enzyme activity after 20 min at 50 °C. The CS support and CS@Gal capsules exhibited an average size of 8 ± 1 mm, with the structure of the shell (alginate-pectin-cellulose) enveloping and isolating the magnetic core. The immobilized ß-galactosidase-CBD within the magnetic CS cellulose system retained ∼80 % of its capacity to hydrolyze lactose from skim milk after 10 reuse cycles. This study unveils a novel and promising support for the oriented immobilization of recombinant ß-galactosidase using a magnetic CS system and a CBD tag. This support facilitates ß-galactosidase reuse and efficient separation, consequently enhancing the catalytic properties of the enzyme.

Lactose hydrolysis in packed-and fluidized-bed reactors using a recombinant ß-galactosidase immobilized on magnetic core-shell capsules.

The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core-shell enzymatic system featuring ß-galactosidase fused to a Cellulose Binding Domain (CBD) tag (ß-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing ß-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability.

Biochemical Insights into a Novel Family 2 Glycoside Hydrolase with Both ß-1,3-Galactosidase and ß-1,4-Galactosidase Activity from the Arctic.

A novel GH2 (glycoside hydrolase family 2) ß-galactosidase from Marinomonas sp. BSi20584 was successfully expressed in E. coli with a stable soluble form. The recombinant enzyme (rMaBGA) was purified to electrophoretic homogeneity and characterized extensively. The specific activity of purified rMaBGA was determined as 96.827 U mg-1 at 30 °C using ONPG (o-nitrophenyl-ß-D-galactopyranoside) as a substrate. The optimum pH and temperature of rMaBGA was measured as 7.0 and 50 °C, respectively. The activity of rMaBGA was significantly enhanced by some divalent cations including Zn2+, Mg2+ and Ni2+, but inhibited by EDTA, suggesting that some divalent cations might play important roles in the catalytic process of rMaBGA. Although the enzyme was derived from a cold-adapted strain, it still showed considerable stability against various physical and chemical elements. Moreover, rMaBGA exhibited activity both toward Galß-(1,3)-GlcNAc and Galß-(1,4)-GlcNAc, which is a relatively rare occurrence in GH2 ß-galactosidase. The results showed that two domains in the C-terminal region might be contributed to the ß-1,3-galactosidase activity of rMaBGA. On account of its fine features, this enzyme is a promising candidate for the industrial application of ß-galactosidase.

Measuring ß-galactosidase activity in opaque dairy solutions under optimum conditions for galactooligosaccharide synthesis by isothermal titration calorimetry.

The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate (thermal power) from enzyme-catalyzed reactions as a function of time. This study used ITC to measure ß-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with 2 ß-galactosidase (Enzyme Commission number 3.2.1.23) isozymes derived from Aspergillus oryzae and Kluyveromyces lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the 2 homologues differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties.

Enzymatic synthesis of prebiotic galactooligosaccharides from galactose derived from gum arabic.

A novel enzymatic process was established for galactooligosaccharides (GOS) synthesis by using plant-derived galactose as substrate, without producing any byproducts. The galactose was prepared from the acid hydrolysate of gum arabic. The yeast Kluyveromyces lactis producing ß-galactosidase capable of catalyzing GOS synthesis from galactose was screened out. The synthesis conditions using the yeast cells as enzyme source were optimized by both single-factor experiment and response surface methodology, with the highest GOS yield reached 45%. The composition of reaction mixture contained only GOS and unreacted galactose, which could be easily separated by the cation exchange resin column. The structures of major GOS products were identified as Gal-ß-D-(1 â†’ 6)-Gal, Gal-ß-D-(1 â†’ 3)-Gal, and Gal-ß-D-(1 â†’ 6)-Gal-ß-D-(1 â†’ 6)-Gal by MS and NMR spectra. Moreover, the ß-galactosidase-containing cells can be recycled for at least 30 batches of GOS synthesis at 35 °C, with the enzyme activity remaining above 60%.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

GH2 family ß-galactosidases evolution using degenerate oligonucleotide gene shuffling.

OBJECTIVES: To improve the biochemical characteristics of the GH2 family ß-galactosidases using a family shuffling method based on degenerate oligonucleotide gene shuffling. RESULTS: Four ß-galactosidase genes from the genus Alteromonas were divided into 14 gene segments, and each included the homologous sequence in the adjacent segments. The gene segments were regenerated into complete ß-galactosidase genes and amplified by PCR. The obtained chimeric genes were cloned into a plasmid and screened for ß-galactosidase activity. Approximately 320 positive clones were observed on the screening plate, of which nine sequenced genes were chimera. Additionally, the M22 and M250 mutants were expressed, purified, and characterized. The optimal temperature and substrate specificity of the recombinant M22 and M250 were consistent with those of the wild-type enzymes. The catalytic efficiency of recombinant M22 enzyme was higher than that of the wild-type enzymes, and the recombinant M250 displayed weak transglycosylation activity. CONCLUSIONS: The chimeric genes of GH2 ß-galactosidase were obtained using a controlled family shuffling that will provide an enzyme evolutionary method to obtain the ß-galactosidases with excellent characteristics for laboratory and industrial purposes.

The Glycoside Hydrolase Family 35 ß-galactosidase from Trichoderma reesei debranches xyloglucan oligosaccharides from tamarind and jatobá.

Trichoderma reesei (anamorph Hypocrea jecorina) produces an extracellular beta-galactosidase from Glycoside Hydrolase Family 35 (TrBga1). Hydrolysis of xyloglucan oligosaccharides (XGOs) by TrBga1 has been studied by hydrolysis profile analysis of both tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seed storage xyloglucans using PACE and MALDI-ToF-MS for separation, quantification and identification of the hydrolysis products. The TrBga1 substrate preference for galactosylated oligosaccharides from both the XXXG- and XXXXG-series of jatobá xyloglucan showed that the doubly galactosylated oligosaccharides were the first to be hydrolyzed. Furthermore, the TrBga1 showed more efficient hydrolysis against non-reducing end dexylosylated oligosaccharides (GLXG/GXLG and GLLG). This preference may play a key role in xyloglucan degradation, since galactosyl removal alleviates steric hindrance for other enzymes in the xyloglucanolytic complex resulting in complete xyloglucan mobilization. Indeed, mixtures of TrBga1 with the α-xylosidase from Escherichia coli (YicI), which shows a preference towards non-galactosylated xyloglucan oligosaccharides, reveals efficient depolymerization when either enzyme is applied first. This understanding of the synergistic depolymerization contributes to the knowledge of plant cell wall structure, and reveals possible evolutionary mechanisms directing the preferences of debranching enzymes acting on xyloglucan oligosaccharides.

Chemosensor with Ultra-High Fluorescence Enhancement for Assisting in Diagnosis and Resection of Ovarian Cancer.

Fluorescence imaging-guided diagnostics is one of the most promising approaches for facile detection of tumors in situ owing to its simple operation and non-invasiveness. As a crucial biomarker for primary ovarian cancers, ß-galactosidase (ß-gal) has been demonstrated to be the significant molecular target for visualization of ovarian tumors. Herein, a membrane-permeable fluorescent chemosensor (namely, LAN-ßgal) was synthesized for ß-gal-specific detection using the d-galactose residue as a specific recognition unit and LAN-OH (ΦF = 0.47) as a fluorophore. After ß-gal was digested, the fluorescence of the initially quenched LAN-ßgal (ΦF < 0.001) was enhanced by up to more than 2000-fold, which exceeded the fluorescence enhancement of other previously reported probes. We also demonstrated that the chemosensor LAN-ßgal could visualize endogenous ß-gal and distinguish ovarian cancer cells from normal ovarian cells. Further, the chemosensor LAN-ßgal was successfully applied to visualize the back tumor-bearing mouse model and peritoneal metastatic ovarian cancer model in vivo. More importantly, through in situ spraying, the proposed chemosensor was successfully employed to assist in the surgical resection of ovarian cancer tumors due to its high tumor-to-normal (T/N) tissue fluorescence ratio of 218. To the best of our knowledge, this is the highest T/N tissue fluorescence ratio ever reported. We believe that the LAN-ßgal chemosensor can be utilized as a new tool for the clinical diagnosis and treatment of ovarian cancer.

Gene Editing Corrects In Vitro a G > A GLB1 Transition from a GM1 Gangliosidosis Patient.

Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced ß-galactosidase (ß-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of ß-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on ß-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.

Evaluation of Prebiotic Properties of Galactooligosaccharides Produced by Transgalactosylation Using Partially Purified ß-Galactosidase from Enterobacter aerogenes KCTC2190.

Transgalactosylation reaction is the penultimate step in the production of galactooligosaccharides (GOSs) which has prominent applications in the treatment of disorders. In the present study, partially purified ß-galactosidase from Enterobacter aerogenes KCTC2190 was used for the synthesis of prebiotic GOSs. GOSs were produced using lactose as substrate. Structural elucidation of collected fractions of GOSs by liquid chromatography electrospray ionization mass spectrometry exhibited the appearance of major peaks of produced GOSs at m/z 241.20, 481.39, 365.11, 527.17, and 701.51 respectively. GOSs facilitated the growth of potential probiotic strains (Lactobacillus delbrueckii ssp. helveticus, Bifidobacterium bifidum, and Lactiplantibacillus plantarum) and liberated propionate and butyrate as principal short-chain fatty acids which established its prebiotic potency. Synbiotic combinations exhibited good antioxidant activities. Synbiotic combinations also exhibited antimicrobial activities against pathogenic microorganisms namely Staphylococcus aureus and Escherichia coli. Synbiotic combinations of GOSs and the respective probiotic microorganisms were able to decrease viable human bone cancer cells (MG-63).

Cloning, Expression, Purification and Characterization of the ß-galactosidase PoßGal35A from Penicillium oxalicum.

Galactosidases are industrially important enzymes that hydrolyze galactosidic bonds in carbohydrates. Identifying new galactosidases with distinct functional characteristics is of paramount importance. In this study, we report the finding of a novel ß-galactosidase PoßGal35A from the fungus Penicillium oxalicum. PoßGal35A belongs to the glycoside hydrolase family 35 (GH35), functions optimally at 70 °C and pH 5.0, and exhibits a specific high activity (191 ± 6.2 U/mg) towards pNPßgal. Ca2+, Fe3+and Ba2+ ions enhance the activity of the enzyme, whereas Cu2+ and Hg2+ significantly reduce it. This enzyme releases galactose from ß-1,3-galactan, ß-1,4-galactan, ß-1,6-galactan, as well as arabinogalactan from larchwood (LWAG). In addition, PoßGal35A acts synergistically with arabinosidase to degrade LWAG. These results suggest that PoßGal35A is a high activity exo-ß-1,3/4/6-galactanase that can be used to establish glycan blocks in glycoconjugates, and thus provides a new tool for biotechnological applications.

A GH42 ß-galactosidase from Bifidobacterium thermophilum: Biochemical characterization and synthesis of galactosyl glycerol by reverse hydrolysis.

In this study, a ß-galactosidase gene (btgal42) was first cloned from Bifidobacterium thermophilum and successfully expressed in Escherichia coli. The molecular weight of the purified BtGal42 was estimated to be 78 kDa by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 225 kDa by the size-exclusion chromatography, indicating that the native BtGal42 was a homotrimer. BtGal42 belonged to the glycosidase hydrolase family 42, exhibiting the maximum activity at pH of 7 and at temperature of 50°C. The enzyme displayed a strictly specific activity toward substrates with ß-galactosyl linkages at the nonreducing ends, of which the activity on 4-nitrophenyl-ß-d-galactopyranoside was the highest, followed by 2-nitrophenyl-ß-d-galactopyranoside and lactose. Among the tested metals and reagents, BtGal42 showed tolerance in the presence of various organic solvents. Importantly, BtGal42 exhibited a high reverse hydrolysis activity when using galactose as the donor and the di-alcohol ethylene glycol and the trialcohol glycerol as the acceptors. Under unoptimized reaction conditions, the galactosyl glycerol yield reached 62.2 g/L (galactose conversion rate, 41.2%). This study might provide a feasible method for the biosynthesis of galactosyl glycerol from low-cost glycerol and galactose, which was associated with high conversion efficiency and few byproducts.

Immobilization of Kluyveromyces lactis ß-Galactosidase on Meso-macroporous Silica: Use of Infrared Spectroscopy to Rationalize the Catalytic Efficiency.

Enzyme immobilization on adequate carriers is a challenging strategy. Understanding the enzyme-carrier interactions and their effects on enzyme conformation and bioactivity is critical. In this study, a meso-macropores silica (MMS) was used to immobilize ß-galactosidase from the yeast Kluyveromyces lactis (ß-gal-KL) by physical adsorption. The bioactivity of the immobilized ß-gal-KL was altered, evidenced by the increased Km , decreased Vmax and kcat , and increased activity at alkaline values. By performing infrared spectroscopy analysis and subsequent secondary structure assessment from the amide I band, the immobilized ß-gal-KL suffered a ß-sheet (∼31-35 %) to α-helix (∼15-19 %) transition with increased turns (∼21-22 %) with respect to the free ß-gal-KL having ∼12 % α-helix, ∼42 % ß-sheet, and ∼17 % turns. These findings led us to correlate the observed bioactivity performance to structural alterations to a non-native conformation. The presented line of thought can lead to a better understanding of the reasons causing bioactivity alterations upon enzyme immobilization.

Identification, Characterization, and Expression of a ß-Galactosidase from Arion Species (Mollusca).

ß-Galactosidases (ß-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing ß-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a ß-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a putative ß-galactosidase from Arion vulgaris (A0A0B7AQJ9) in Sf9 cells. The entire gene codes for a protein consisting of 661 amino acids, comprising a putative signal peptide and an active domain. Specificity studies show exo- and endo-cleavage activity for galactose ß1,4-linkages. Both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, display similar biochemical parameters. Both ß-galactosidases are most active in acidic environments ranging from pH 3.5 to 4.5, and do not depend on metal ions. The ideal reaction temperature is 50 °C. Long-term storage is possible up to +4 °C for the A. vulgaris enzyme, and up to +20 °C for the A. lusitanicus enzyme. This is the first report of the expression and characterization of a mollusk exoglycosidase.

Optimal ß-galactosidases for producing high-titer 3,6-anhydro-L-galactose from red-algal agarobiose.

3,6-Anhydro-L-galactose (L-AHG) is a monomeric sugar in agarose derived from red macroalgae. Owing to its various physiological activities such as anti-inflammation, moisturizing, skin whitening, anti-colon cancer, and anti-cariogenicity, L-AHG is a potential functional ingredient. In our previous study, a simple and efficient two-step L-AHG production process was designed for high-titer L-AHG production, where a single enzyme was used after the liquefaction of agarose by acid prehydrolysis. However, the enzyme used did not completely hydrolyze agarobiose (AB). Therefore, in this study, for the efficient hydrolysis of AB and the high-titer production of L-AHG, various ß-galactosidases belonging to glycoside hydrolase families 1, 2, 35, and 42 were compared by testing their substrate specificities and kinetic parameters. Among the five ß-galactosidases, Bga42A, originating from Bifidobacterium longum ssp. infantis ATCC 15,697, showed the highest substrate specificity. Consequently, the two-step process utilizing Bga42A as a single enzyme resulted in a high-titer production of L-AHG at 85.9 g/L, demonstrating the feasibility of producing L-AHG from agarose. KEY POINTS: • L-AHG derived from red macroalgae has various physiological activities. • Various ß-galactosidases were evaluated to efficiently hydrolyze agarobiose. • Bga42A showed the highest substrate specificity against agarobiose. • The highest amount of L-AHG with 85.9 g/L was simply produced.

Enhanced thermal stability of the ß-galactosidase BgaB from Bacillus circulans by cyclization mediated via SpyTag/SpyCatcher interaction and its use in galacto-oligosaccharides synthesis.

Cyclization of proteins using SpyTag/SpyCatcher is a novel approach to increase their thermal stability. In this paper, we test this approach on two ß-galactosidases from Bacillus circulans, BgaB and BgaC, and find that BgaB was stabilized while BgaC was not. Wild-type BgaB precipitated completely upon heating above 70 °C, but after SpyRing cyclization, it remained soluble after heating to 90 °C. Similarly, wild-type BgaB retained only 50 % activity after heating at 60 °C for 10 min, but this increased to 80 % after SpyRing cyclization. In contrast, cyclization decreased the stability of BgaC. After SpyRing cyclization, BgaC only retained 2 % activity after 20-min incubation at 55 °C, whereas the wild-type BgaC retained 25 % activity. One reason for the different effect of cyclization may the shorter distance between the N- and C-termini in BgaB (20.2 Å) as compared to BgaC (43.7 Å). The intrinsic fluorescence and circular dichroism spectra suggested that SpyRing cyclization of BgaB did not significantly change its conformation or secondary structure. SpyRing cyclized BgaB yielded similar amounts and compositions of galacto-oligosaccharides using a high initial lactose concentration (40 %, w/v), but a slightly higher amount at low initial lactose concentration (5 %, w/v) suggesting increased transgalactosylation activity.