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 β-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 β-galactosidase in immobilized cells towards o-nitrophenyl β-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.

The transgalactosylase activity of β-galactosidases offers a convenient and promising strategy for conversion of lactose into high-value oligosaccharides, such as galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs). In this study, we cloned and biochemically characterized a novel C-terminally truncated β-galactosidase (PaBgal2A-D) from Paenibacillus antarcticus with high transglycosylation activity. PaBgal2A-D is a member of glycoside hydrolase (GH) family 2. The optimal pH and temperature of PaBgal2A-D were determined to be pH 6.5 and 50°C, respectively. It was relatively stable within pH 5.0-8.0 and up to 50°C. PaBgal2A-D showed high transglycosylation activity for GOS synthesis, and the maximum yield of 50.8% (wt/wt) was obtained in 2 h. Moreover, PaBgal2A-D could synthesize lacto-N-neotetraose (LNnT) using lactose and lacto-N-triose II (LNT2), with a conversion rate of 16.4%. This study demonstrated that PaBgal2A-D could be a promising tool to prepare GOS and LNnT.

A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917 bp, encoding 638 amino acids with a predicted molecular mass of 62.3 kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50 °C, respectively, and it was stable within pH 4.5‒7.0 and up to 45 °C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4 °C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products.

This study aimed to immobilize β-galactosidase (β-GAL) into enhanced polystyrene (PS) electrospun nanofiber membranes (ENMs) with functionalized graphene oxide (GO). Initially, GO sheets were functionalized by salinization with 3-aminopropyl triethoxysilane (APTES). Then the ENMs (PS, PS/GO, and PS/GO-APTES) were prepared and characterized. Then, the β-GAL was immobilized in the different ENMs to produce the β-GAL-bound nanocomposites (PS-GAL, PS/GO-GAL, and PS/GO-APTES-GAL). Immobilization of β-GAL into PS/GO-APTES significantly improved enzyme adsorption by up to 87 %. Also, PS/GO-APTES-GAL improved the enzyme activity, where the highest enzyme activity was obtained at enzyme concentrations of 4 mg/L, 50 °C, and pH 4.5. Likewise, the storage stability and reusability of immobilized β-GAL were improved. Furthermore, this process led to enhanced catalytic behavior and transgalactosylation efficiency, where GOS synthesis (72 %) and lactose conversion (81 %) increased significantly compared to the free enzyme. Overall, the immobilized β-GAL produced in this study showed potential as an effective biocatalyst in the food industry.

β-Galactosidase (β-gal), an enzyme related to cell wall degradation, plays an important role in regulating cell wall metabolism and reconstruction. However, activatable fluorescence probes for the detection and imaging of β-gal fluctuations in plants have been less exploited. Herein, we report an activatable fluorescent probe based on intramolecular charge transfer (ICT), benzothiazole coumarin-bearing β-galactoside (BC-βgal), to achieve distinct in situ imaging of β-gal in plant cells. It exhibits high sensitivity and selectivity to β-gal with a fast response (8 min). BC-βgal can be used to efficiently detect the alternations of intracellular β-gal levels in cabbage root cells with considerable imaging integrity and imaging contrast. Significantly, BC-βgal can assess β-gal activity in cabbage roots under heavy metal stress (Cd2+, Cu2+, and Pb2+), revealing that β-gal activity is negatively correlated with the severity of heavy metal stress. Our work thus facilitates the study of β-gal biological mechanisms.

β-Galactosidase (β-Gala) is an essential biomarker enzyme for early detection of breast tumors and cellular senescence. Creating an accurate way to monitor β-Gala activity is critical for biological research and early cancer detection. This work used fluorometric, colorimetric, and paper-based color sensing approaches to determine β-Gala activity effectively. Via the sensing performance, the catalytic activity of β-Gala resulted in silicon nanoparticles (SiNPs), fluorescent indicators obtained via a one-pot hydrothermal process. As a standard enzymatic hydrolysis product of the substrate, kaempferol 3-O-β-d-galactopyranoside (KOβDG) caused the fluorometric signal to be attenuated on kaempferol-silicon nanoparticles (K-SiNPs). The sensing methods demonstrated a satisfactory linear response in sensing β-Gala and a low detection limit. The findings showed the low limit of detection (LOD) as 0.00057 and 0.098 U/mL for fluorometric and colorimetric, respectively. The designed probe was then used to evaluate the catalytic activity of β-Gala in yogurt and human serum, with recoveries ranging from 98.33 to 107.9%. The designed sensing approach was also applied to biological sample analysis. In contrast, breast cancer cells (MCF-7) were used as a model to test the in vitro toxicity and molecular fluorescence imaging potential of K-SiNPs. Hence, our fluorescent K-SiNPs can be used in the clinic to diagnose breast cellular carcinoma, since they can accurately measure the presence of invasive ductal carcinoma in serologic tests.

Yogurt usually contains 5% to 7% sugar and 3% to 5% lactose. As β-galactosidases can hydrolyze lactose and improve sweetness, they have the potential to produce lactose-free (LF) and no-sugar-added (NSA) yogurt. In this study, the β-galactosidase AoBgal35A from Aspergillus oryzae was engineered by site-saturation mutagenesis. Results of 19 variants of T955 residue showed that the lactose hydrolysis rate of T955R-AoBgal35A was up to 90.7%, which is much higher than the 78.5% of the wild type. Moreover, the optimal pH of T955R-AoBgal35A was shifted from pH 4.5 to pH 5.5, and the optimal temperature decreased from 60°C to 50°C. The mutant T955R-AoBgal35A was successfully expressed in Komagataella pastoris, which produced extracellularly 4,528 U/mL of β-galactosidase activity. The mutant T955R-AoBgal35A was used to produce LF yogurt. The Streptococcus thermophilus count of LF yogurt increased from 7.9 to 9.5 log cfu/g, which is significantly higher than that of the control group (8.9 log cfu/g). The residual lactose content of LF yogurt was 0.13%, meeting the requirements of the national standard in China for the \"lactose-free\" label (<0.5%). Furthermore, sugar in yogurt was replaced by whey powder to produce LF-NSA yogurt. The optimal addition content of whey powder was 7.5%. The texture, water-holding capacity, and titratable acidity of LF and LF-NSA yogurt achieved good shelf life stability. Therefore, this study provides an insight for technological implications of β-galactosidases in the dairy industry.

A novel β-galactosidase (TsGal48) from Thermus scotoductus was cloned, and the enzyme was biochemically characterized. TsGal48 catalyzed the synthesis of lacto-N-neotetraose (LNnT) from lactose via the transglycosylation reaction with a maximal yield of 20%, which is the highest yield for the synthesis of LNnT so far. To further improve the yield of LNnT, TsGal48 was successfully engineered by directed evolution and site-saturation mutagenesis. A mutated β-galactosidase (mTsGal48) was selected and characterized. mTsGal48 produced LNnT with a yield of 27.7 g/L, which is 1.4-fold higher than that of TsGal48 (19.7 g/L). Then, a developed strategy for LNnT synthesis from chitin powder was provided in a 30 L bioreactor. The reaction process included chitin powder hydrolysis, lacto-N-triose II (LNT2) synthesis, and LNnT synthesis. The reaction time was reduced from 44 to 17 h in chitin powder hydrolysis and LNT2 synthesis. The content of LNnT was up to 25 g/L in the multienzyme system. The green and efficient route may be suitable for large-scale production of LNnT from chitin powder.

Colorectal cancer was one of the major malignant tumors threatening human health and β-Gal was recognized as a principal biomarker for primary colorectal cancer. Thus, designing specific and efficient quantitative detection methods for measuring β-Gal enzyme activity was of great clinical test significance. Herein, an ultrasensitive detection method based on Turn-on fluorescence probe (CS-βGal) was reported for visualizing the detection of exogenous and endogenous β-galactosidase enzyme activity. The test method possessed a series of excellent performances, such as a significant fluorescence enhancement (about 11.3-fold), high selectivity as well as superior sensitivity. Furthermore, under the optimal experimental conditions, a relatively low limit of detection down to 0.024 U/mL was achieved for fluorescence titration experiment. It was thanks to the better biocompatibility and low cytotoxicity, CS-βGal had been triumphantly employed to visual detect endogenous and exogenous β-Gal concentration variations in living cells with noteworthy anti-interference performance. More biologically significant was the fact that the application of CS-βGal in BALB/c nude mice was also achieved successfully for monitoring endogenous β-Gal enzyme activity.

β-galactosidase (β-gal) has high activity in various malignancies, which is suitable for targeted positron emission tomography (PET) imaging. Meanwhile, β-gal can successfully guide the formation of nanofibers, which enhances the intensity of imaging and extends the imaging time. Herein, we designed a β-galactosidase-guided self-assembled PET imaging probe [68Ga]Nap-NOTA-1Gal. We envisage that β-gal could recognize and cleave the target site, bringing about self-assembling to form nanofibers, thereby enhancing the PET imaging effect. The targeting specificity of [68Ga]Nap-NOTA-1Gal for detecting β-gal activity was examined using the control probe [68Ga]Nap-NOTA-1. Micro-PET imaging showed that tumor regions of [68Ga]Nap-NOTA-1Gal were visible after injection. And the tumor uptake of [68Ga]Nap-NOTA-1Gal was higher than [68Ga]Nap-NOTA-1 at all-time points. Our results demonstrated that the [68Ga]Nap-NOTA-1Gal can be used for the purpose of a new promising PET probe for helping diagnose cancer with high levels of β-gal activity.