Induction of chronic asthma up regulated the transcription of senile factors in male rats.

BACKGROUND: The main characteristic of asthma is chronic inflammation. We examined cellular senescence by histology and molecular assay in the lungs of a rat model of asthma. This model comprises sensitization by several intraperitoneal injections of ovalbumin with aluminium hydroxide, followed by aerosol challenges every other day. RESULTS: Data showed that asthma induction caused histological changes including, hyperemia, interstitial pneumonia, fibrinogen clots, and accumulation of inflammatory cells in the pleura. There is an elevation of IL-1ß and NF-kB proteins in the asthmatic group (P < 0.001) compared to the control group. The expression of ß-galactosidase increased (P < 0.01), while the expression of Klotho and Sox2 genes was decreased in the lung tissue of the asthmatic group (P < 0.01). CONCLUSION: Taken together, these findings suggest that asthmatic conditions accelerated the cellular senescence in the lung tissue.

A Two-Photon-Active Zr-Based Metal-Organic Framework-Based Orthogonal Nanoprobe for Recognition of Cellular Senescence.

A luminescent nanoprobe capable of orthogonal sensing of two independent events is highly significant for unbiased disease-related detection such as the detection of senescent cells. Moreover, it is invaluable that the nanoprobe possesses a two-photon excitable characteristic that is highly suitable for imaging living cells and tissues. Herein, we present a two-photon-excitable multiluminescent orthogonal-sensing nanoprobe (OS nanoprobe) capable of detecting both pH elevation and ß-galactosidase (ß-gal) overexpression in senescent cells. In the design, Zr-based dual-emissive metal-organic frameworks prepared from two mixed amino linkers, referred to as NH2-MU, were used as the component for the ratiometric sensing of pH; additionally, fluorogenic resorufin-ß-d-galactopyranoside, linked to the NH2-MU framework, enables ß-gal detection. In the OS nanoprobe, the signals for pH and ß-gal sensing remain independent while maintaining high colocalization. The two-photon excitable organic linkers of NH2-MU impart the OS nanoprobe with a bioimaging capability, allowing for the differentiation of senescent human foreskin fibroblast (HFF) cells from younger HFF cells or LacZ positive cells with the 800 nm laser excitation. This study marks the first instance of achieving the multiplexed orthogonal fluorescent sensing of cellular senescence using a two-photon excitation strategy, suggesting the potential of using versatile metal-organic framework (MOFs)-based fluorophores to realize the orthogonal multiplexing of disease-related biomarkers through multiphoton excitation.

Recombinant production of Paenibacillus wynnii ß-galactosidase with Komagataella phaffii.

BACKGROUND: The ß-galactosidase from Paenibacillus wynnii (ß-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used ß-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, ß-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce ß-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the ß-gal-Pw in K. phaffii either extracellularly or intracellularly. RESULTS: Firstly, 11 different signal peptides were tested for extracellular production of ß-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of ß-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular ß-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric ß-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for ß-gal-Pw production compared to the AOX1 promoter. After partial purification, a ß-gal-Pw enzyme preparation with a total ß-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter). CONCLUSION: This study showed that the ß-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular ß-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

A Customizable Platform to Integrate CAR and Conditional Expression of Immunotherapeutics in T Cells.

The potential of chimeric antigen receptor (CAR)-based immunotherapy as a promising therapeutic approach is often hindered by the presence of highly immunosuppressive tumor microenvironments (TME). Combination therapies with either co-administration or built-in expression of additional TME-modulating therapeutic molecules to potentiate the functions of CAR-T cells can cause systemic toxicities due to the lack of control over the delivery of biologics. Here, we present a proof-of-concept engineered platform in human Jurkat T cells that combines CAR with a therapeutic gene circuit capable of sensing ß-galactosidase (a reported cancer-associated signal) and subsequently activate the production of customized therapeutic gene products. We have demonstrated the integration of the chemically induced proximity (CIP) and associated signal sensing technologies with CAR in this study. A ß-galactosidase-activatable prodrug was designed by conjugating a galactose moiety with a CIP inducer abscisic acid (ABA). We showed that Jurkat T cells engineered with CAR and the ABA-inducible genetic circuits can respond to recombinant ß-galactosidase to drive the production and secretion of various immunotherapeutics including an anti-cancer agent, an immunomodulatory cytokine, and immune checkpoint inhibitors. Our design is highly modular and could be adapted to sense different cancer-related signals to locally produce antitumor therapeutics that can potentially boost CAR-T efficacy and persistence.

Mechanism-Based Allylic Carbasugar Chlorides That Form Covalent Intermediates with α- and ß-Galactosidases.

Glycoside hydrolases have been implicated in a wide range of human conditions including lysosomal storage diseases. Consequently, many researchers have directed their efforts towards identifying new classes of glycoside hydrolase inhibitors, both synthetic and from natural sources. A large percentage of such inhibitors are reversible competitive inhibitors that bind in the active site often due to them possessing structural features, often a protonatable basic nitrogen atom, that mimic the enzymatic transition state. We report that mechanism-based small molecule galacto-like configured cyclohexenyl carbasugars form reversible covalent complexes with both α-galactosidase and ß-galactosidase. In addition, we show that the ß-galactosidase from Aspergillus oryzae reacts with three different carbasugar inhibitors, with three different second-order rate constants (kinact/Ki), to give the same enzyme-carbasugar covalent intermediate. The surprising observation that the α-galacto-configured inhibitor covalently labels the A. oryzae ß-galactosidase highlights the catalytic versatility of glycoside hydrolases. We expect that cyclohexenyl covalent inhibitors will become an important class of compounds in the chemical biologist's tool box.

Encapsulation of ß-Galactosidase into Polyallylamine/Polystyrene Sulphonate Polyelectrolyte Microcapsules.

More than half of the global population is unable to consume dairy products due to lactose intolerance (hypolactasia). Current enzyme replacement therapy methods are insufficiently effective as a therapeutic approach to treating lactose intolerance. The encapsulation of ß-galactosidase in polyelectrolyte microcapsules by using the layer-by-layer method could be a possible solution to this problem. In this study, adsorption and co-precipitation methods were employed for encapsulating ß-galactosidase in polyelectrolyte microcapsules composed of (polyallylamine /polystyrene sulphonate)3. As a result, the co-precipitation method was chosen for ß-galactosidase encapsulation. The adsorption method permits to encapsulate six times less enzyme compared with the co-precipitation method; the ß-galactosidase encapsulated via the co-precipitation method released no more than 20% of the initially encapsulated enzyme in pH 2 or 1 M NaCl solutions. In contrast, when using the sorption method, about 100% of the initially encapsulated enzyme was released from the microcapsules under the conditions described above. The co-precipitation method effectively prevents the complete loss of enzyme activity after 2 h of incubation in a solution with pH 2 while also alleviating the adverse effects of ionic strength. Consequently, the encapsulated form of ß-galactosidase shows promise as a potential therapeutic agent for enzyme replacement therapy in the treatment of hypolactasia.

Activity of exoglycosidases in blood, urine, cerebrospinal fluid, and vitreous humor in individuals who died from ethyl alcohol poisoning.

Lysosomal exoglycosidases, namely α-mannosidase (MAN), α-fucosidase (FUC), ß-galactosidase (GAL), and ß-glucuronidase (GLU), are of crucial importance in breaking down the oligosaccharide chains of multiple glycoconjugates. Those enzymes liberate monosaccharides from non-reducing ends of oligosaccharide chains. In this study, we have aimed to assess the potential utility of MAN, FUC, GAL, and GLU activities as indicators of ethanol abuse in individuals who died from ethanol intoxication, while also investigating the mechanisms underlying their deaths. The study group comprised 22 fatal ethanol-intoxicated individuals, while the control group included 30 deceased individuals whose body fluids showed no traces of alcohol. We measured the activities (pKat/mL) of MAN, FUC, GAL, and GLU in the supernatants of blood, urine, cerebrospinal fluid as well as vitreous humor. The results indicated significantly lower activities of MAN (p = 0.003), FUC (p = 0.008), GAL (p = 0.014), and GLU (p = 0.004) in the urine of individuals poisoned by ethanol as compared to the control group. Additionally, there was a significantly lower activity of MAN in the vitreous of those affected by ethyl alcohol poisoning (p = 0.016).

Filbertone Reduces Senescence in C2C12 Myotubes Treated with Doxorubicin or H2O2 through MuRF1 and Myogenin.

It has been demonstrated that filbertone, the principal flavor compound of hazelnuts, exhibits preventive effects against hypothalamic inflammation, obesity, neurodegenerative diseases, and muscle lipid accumulation. However, its influence on muscle aging has yet to be elucidated. The objective of this study was to investigate the effects of filbertone on muscle aging in C2C12 myotubes subjected to senescence induction by either doxorubicin or hydrogen peroxide. To ascertain the mechanisms by which filbertone exerts its effects, we conducted a series of experiments, including Western blot analysis, reverse transcription quantitative polymerase chain reaction (qRT-PCR), and senescence-associated ß-galactosidase (SA-ß-gal) staining. Filbertone was markedly observed to decrease not only the protein levels of p53 (p < 0.01) in senescence-induced skeletal muscle cells, but also the gene expression levels of p21 (p < 0.05), a direct target of p53. The expression of muscle-related genes, including myogenin and muscle RING-finger protein-1 (MuRF1), was found to be significantly enhanced in senescent muscle cells following treatment with filbertone (p < 0.05). In addition, the number of senescent skeletal muscle cells exhibiting ß-galactosidase activity was found to be markedly reduced in the presence of filbertone (p < 0.01). Collectively, these findings suggest that filbertone plays a pivotal role in the regulation of muscle aging.

Base editing of the GLB1 gene is therapeutic in GM1 gangliosidosis patient-derived cells.

GM1 gangliosidosis is an autosomal recessive neurodegenerative lysosomal storage disease caused by pathogenic variants in the GLB1 gene, limiting the production of active lysosomal ß-galactosidase. Phenotypic heterogeneity is due in part to variant type, location within GLB1, and the amount of residual enzyme activity; in the most severe form, death occurs in infancy. With no FDA approved therapeutics, development of efficacious strategies for the disease is pivotal. CRISPR/Cas based approaches have revolutionized precision medicine and have been indispensable to the development of treatments for several monogenic disorders with bespoke strategies central to current research pipelines. We used CRISPR/Cas-adenine base editing to correct the GLB1 c.380G>A (p.Cys127Tyr) variant in patient-derived dermal fibroblasts compound heterozygous with the GLB1 c.481T>G (p.Trp161Gly) pathogenic variant. Nucleofection of plasmids encoding the target sgRNA and ABEmax restored the canonical guanine (32.2 ± 2.2 % of the target allele) and synthesis of active ß-galactosidase. Analysis of cellular markers of pathology revealed normalization of both primary glycoconjugate storage and lysosomal pathology. Furthermore, analysis of off-target sites nominated by the in silico tools Cas-OFFinder and/or CRISTA revealed no significant editing or indels. This study supports the use of CRISPR/Cas-based approaches for the treatment of GM1 gangliosidosis, and provides foundational data for future translational studies.

Unveiling the genetic basis and metabolic rewiring behind the galactose-positive phenotype in a Streptococcus thermophilus mutant.

Streptococcus thermophilus (S. thermophilus) is a widely used starter culture in dairy fermentation, but most strains are galactose-negative and only metabolize glucose from lactose hydrolysis. In this study, we aimed to uncover the mechanisms underlying the acquisition of a stable galactose-positive (Gal+) phenotype in a mutant strain of S. thermophilus IMAU10636. By treating the wild-type strain with the mutagenic agent N-methyl-N-nitro-N-nitrosoguanidine, we successfully isolated a Gal+ mutant, S. thermophilus IMAU10636Y. Comparative enzyme activity assays revealed that the mutant exhibited higher ß-galactosidase and galactokinase activities, but lower glucokinase and pyruvate kinase activities compared to the wild-type. High-performance liquid chromatography analysis confirmed the mutant's enhanced ability to utilize lactose and galactose, leading to increased glucose secretion. Integrated genome and transcriptomics analyses provided deeper insights into the underlying genetic and metabolic mechanisms. We found that the metabolism regulatory network of the glycolysis / Leloir pathway was altered in the mutant, possibly due to the upregulation of the gene expression in the galR-galK intergenic region. This likely led to increased RNA polymerase binding and transcription of the gal operon, ultimately promoting the Gal+ phenotype. Additionally, we identified a mutation in the scrR gene, encoding a LacI family transcriptional repressor, which also contributed to the Gal+ phenotype. These findings offer new perspectives on the metabolic rewiring and regulatory mechanisms that enable S. thermophilus to acquire the ability to metabolize galactose. This knowledge can inform strategies for engineering and selecting Gal+ strains with desirable fermentation characteristics for dairy applications.

Correlations between in vitro gastrointestinal digestion of ß-galactosidase/carboxymethylchitosan-silica dosage powder and its physicochemical properties.

Oral administration of ß-galactosidase, which alleviate lactose intolerance symptoms, is challenging due to its instability throughout the gastrointestinal tract. The objective of this work was to make correlations between the in-vitro digestion and chemical characteristics of a ß-galactosidase/carboxymethylchitosan-silica biocatalyst powder. This was obtained by a one-pot silica gel route assisted by carboxymethyl chitosan, using maltose as lyoprotectant. The chemical characterization allowed to understand as was modulated the calcium incorporation, through electrostatic interactions and as maltose protects the enzyme from agglomeration, by vitrification and formation of hydrogen bonds. The formulated biocatalyst could be a complement of silicon and calcium, in turn, it preserves 96 % and 63 % of the enzymatic activity compared with the biocatalyst control (without simulated digestion), in the gastric and intestinal phases, respectively. This activity was even greater than that observed in the commercial products evaluated in these phases. Likewise, the biocatalyst obtained retained its activity after 12 months of storage at 25 °C and it did not present cytotoxicity in cells derived from human colon epithelial mucosa (NCM460) under the conditions and concentrations evaluated. These results make this biocatalyst in an excellent candidate for release of this enzyme. Therefore, it could be useful for lactose-intolerant people.

ß-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by ß-Galactosidase (ß-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with ß-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed ß-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.

2-Bromopalmitate treatment attenuates senescence phenotype in human adult cells - possible role of palmitoylation.

Cells may undergo senescence in response to DNA damage, which is associated with cell cycle arrest, altered gene expression and altered cell morphology. Protein palmitoylation is one of the mechanisms by which the DNA damage response is regulated. Therefore, we hypothesized that protein palmitoylation played a role in regulation of the senescent phenotype. Here, we showed that treatment of senescent human vascular smooth muscle cells (VSMCs) with 2-bromopalmitate (2-BP), an inhibitor of protein acyltransferases, is associated with changes in different aspects of the senescent phenotype, including the resumption of cell proliferation, a decrease in DNA damage markers and the downregulation of senescence-associated ß-galactosidase activity. The effects were dose dependent and associated with significantly decreased total protein palmitoylation level. We also showed that the senescence-modifying properties of 2-BP were at least partially mediated by the downregulation of elements of DNA damage-related molecular pathways, such as phosphorylated p53. Our data suggest that cell senescence may be regulated by palmitoylation, which provides a new perspective on the role of this posttranslational modification in age-related diseases.

Digital Ion Trap Isolation and Mass Analysis of Macromolecular Analytes with Multiply Charged Ion Attachment.

Multiply charged ions produced by electrospray ionization (ESI) of heterogeneous mixtures of macromolecular analytes under native conditions are typically confined to relatively narrow ranges of mass-to-charge (m/z) ratio, often with extensive overlap. This scenario makes charge and mass assignments extremely challenging, particularly when individual charge states are unresolved. An ion/ion reaction strategy involving multiply charged ion attachment (MIA) to the mixture components in a narrow range of m/z can facilitate charge and mass assignment. In MIA operation, multiply charged reagent ions are attached to the analyte ions of opposite polarity to provide large m/z displacements resulting from both large changes in mass and charge. However, charge reduction of the high m/z ions initially generated under native ESI conditions requires the ability to isolate high m/z ions and to analyze even higher m/z product ions. Digital ion trap (DIT) operation offers means for both high m/z ion isolation and high m/z mass analysis, in addition to providing conditions for the reaction of oppositely charged ions. The feasibility of conducting MIA experiments in a DIT that takes advantage of high m/z ion operation is demonstrated here using a tandem 2D-3D DIT instrument. Proof-of-concept MIA experiments with cations derived from ß-galactosidase using the 20- charge state of human serum immunoglobulin G (IgG, ∼149 kDa) as the reagent anion are described. MIA experiments involving mixtures of ions derived from the E. coli. ribosome are also described. For example, three components in a mixture of 70S particles (>2.2 MDa) were resolved and assigned with masses and charges following an MIA experiment involving the 20- charge state of human serum IgG.

Improved self-cleaving precipitation tags for efficient column free bioseparations.

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

Membrane separation assisted colorimetric/fluorescent detection of ß-galactosidase-positive bacteria in milk and milk powder based on the oxidase-like activity of CoOOH nanosheets.

Species-specific enzymes provide a substantial boost to the precision and selectivity of identifying dairy products contaminated with foodborne pathogens, due to their specificity for target organisms. In this study, we developed cobalt oxyhydroxide nanosheets (CoOOH NSs) for a dual-mode biosensor capable of detecting ß-galactosidase (ß-Gal)-positive bacteria in milk and milk powder. The sensor exploits the oxidase-mimicking activity of CoOOH NSs, where ß-Gal converts the substrate ß-D-galactopyranoside to p-aminophenol, reducing CoOOH NSs to Co2+ and inhibiting the formation of the blue product from 3,3',5,5'-tetramethylben-zidine. Sensitivity was enhanced through membrane filtration and ß-Gal induction by isopropyl ß-D-thiogalactoside. The assay achieved a detection limit of 5 cfu mL-1 and demonstrated recoveries (90.7 % to 103 %) and relative standard deviations <5.7 % in milk and milk powder samples. These findings underscore the potential of the sensor for detecting ß-Gal-positive bacteria in dairy products.

Enhancement of ß-galactosidase catalytic activity and stability through covalent immobilization onto alginate/tea waste beads and evaluating its impact on the quality of some dairy products.

The current study aimed to evaluate the hydrolysis of whole fat milk (WFM) and sweet whey (SW) using ß-galactosidase (ß-gal) after covalent immobilization onto activated alginate/tea waste (Alg/TW) beads as a novel carrier. The optimum temperature for free and Alg/TW/ß-gal was 40 °C and the ideal pH was 7.0. However, Alg/TW/ß-gal displayed better stabilities at high temperatures and a wide pH range. Additionally, the value of Km and Vmax for Alg/TW/ß-gal was higher than the free enzyme. The Alg/TW/ß-gal showed better residual activity (78.6 %) after 90 storage days at 4 °C. The reusability of Alg/TW/ß-gal was very good as it conserved its full activity after 15 consecutive cycles and conserved 93 % of its initial activity after 10 cycles with ONPG (O-nitrophenyl-ß-D-galactopyranoside) and lactose as a substrate, respectively. The impact of Alg/TW/ß-gal on WFM and SW using HPLC analysis revealed a remarkable decrease in lactose concentration and increase of glucose and galactose concentrations. The SW exhibited higher degree of lactose hydrolysis (97.3 %) compared to WFM (62.4 %). Besides, SW had a prominent increase in total phenolic content (96.8 mg/L) compared to WFM (54.3 mg/L). The antioxidant activity had increased after enzyme treatment in both WFM and SW. The GC-MS analysis for volatile compounds identified twenty-five flavour constituents. Finally, Alg/TW/ß-gal has a potential application for obtaining healthy, acceptable, and commercial dairy products of low lactose.

A concise synthetic approach for isoiminosugars.

Isoiminosugars are highly biological active substances. Herein, we report a concise synthetic approach for this class of compounds. The key step relies on a stereospecific 1,2-hydride shift in O-2 tosylated glycopyranosides leading to C-2 branched glycofuranosides. This approach enables a 4-step synthesis of powerful ß-galactosidase inhibitor 4-epi-isofagomine starting from a simple d-glucopyranoside.

Synthesis of C-glycoside analogues of isopropyl ß-D-1-thiogalactopyranoside (IPTG) and 1-ß-D-galactopyranosyl-2-methylpropane. Conformational analysis and evaluation as inhibitors of the lac repressor in E. coli and as galactosidase inhibitors.

Isopropyl 1-thio-ß-D-galactopyranoside (IPTG, 1) is used widely as an inducer of protein expression in E. coli and 1-ß-D-galactopyranosyl-2-methylpropane (2), a C-glycoside analogue of 1, has also been identified as an inducer. Here, synthesis and study of mimetics of 1 and 2, 1-ß-D-galactopyranosyl-2-methylpropan-1-ols and two cyclic acetals derivatives, that constrain the presentation of the iPr group in various geometries is described. Conformational analysis of C-glycosides in protic solvent is performed using (i) Desmond metadynamics simulations (OPLS4) and (ii) use of 3JHH values obtained by 1H-NMR spectroscopy. 1-ß-D-Galactopyranosyl-2-methylpropane (2) is an effective protein expression inducer when compared to the new mimetics, which were less effective or did not induce expression. 1-ß-D-Galactopyranosyl-2-methylpropane (2) led to significantly reduced proteolysis during protein expression, compared to IPTG suggesting that recombinant protein purification will be easier to achieve with 2, yielding proteins with higher quality and activity. IPTG reduced bacterial growth to a greater degree than 2 compared to the control. IPTG's isopropyl group was observed by molecular dynamics (MD) simulations to be flexible in the binding pocket, deviating from its crystal structure binding mode, without impacting other interactions. The MD simulations predicted that 1-ß-D-galactopyranosyl-2-methylpropane (2) was more likely than IPTG to bind the repressor with a conformation favoured in protic solvent, while maintaining interactions observed for IPTG. MD simulations predicted that isobutanol derivatives may disrupt interactions associated with IPTG's binding mode. The compounds were also evaluated as inhibitors of galactosidases, with 2 being the more potent inhibitor of the E. coli ß-galactosidase. The constrained cyclic acetals showed similar inhibition constants to IPTG indicating E. coli ß-galactosidase can recognize galactopyranoses with varying presentation of the iPr group.

Non-carrier immobilization of yeast cells by genipin crosslinking for the synthesis of prebiotic galactooligosaccharides from plant-derived galactose.

Galactooligosaccharides (GOS), as mimics of human milk oligosaccharides, are important prebiotics for modulating the ecological balance of intestinal microbiota. A novel carrier-free cell immobilization method was established using genipin to cross-link Kluyveromyces lactis CGMCC 2.1494, which produced ß-galactosidase, an enzyme essential for GOS synthesis. The resulting immobilized cells were characterized as stable by thermogravimetric analysis and confirmed to be crosslinked through scanning electron microscopy analysis (SEM) and Fourier transform infrared spectroscopy (FTIR). The Km and Vmax values of ß-galactosidase in immobilized cells towards o-nitrophenyl ß-D-galactoside were determined to be 3.446 mM and 2210 µmol min-1 g-1, respectively. The enzyme in the immobilized showed higher thermal and organic solvent tolerance compared to that in free cells. The immobilized cells were subsequently employed for GOS synthesis using plant-derived galactose as the substrate. The synthetic reaction conditions were optimized through both single-factor experiments and response surface methodology, resulting in a high yield of 49.1 %. Moreover, the immobilized cells showed good reusability and could be reused for at least 20 batches of GOS synthesis, with the enzyme activity remaining above 70 % at 35 °C.