Identification of Putative β-Galactosidase Genes in the Genome of Lactobacillus helveticus OSU-PECh-4A

The Lactobacillus helveticus OSU-PECh-4A strain, from the Ohio State University Parker Chair collection, produces exceptional β-galactosidase activity using acid whey as a culture medium, compared with a commercial broth. The strain has a genome sequence of 1,834,843 bp, and its GC content is 36.69%. Using InterProScan v5.50-84.0 software, four genes with putative β-galactosidase function were found.

Characterization of β-galactosidase and α-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

Purpose Gracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them. Methods G. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis. Result Formation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745). Conclusions G. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

Valorisation of whey for lactose recycling products production = Sūkalu valorizācija laktozes pārstrādes produktu ieguvei

The doctoral thesis “Valorisation of whey for lactose recycling products production” was developed from 2016 to 2021. Experiments were carried out in the research laboratories of the Faculty of Food Technology, Latvia University of Life Sciences and Technologies; Dairy Innovation Institute, California Polytechnic State University (USA); Institute of Microbiology and Biotechnology, Latvia University; Faculty of Chemistry, Latvia University; Institute of Solid State Physics, Latvia University and J.S. Hamilton Baltic Ltd. The aim of the doctoral thesis was to improve the lactose hydrolysis process for obtaining glucose-galactose and oligosaccharide syrups. The hypothesis of the doctoral thesis – the two-stage fermentation increases the sweetness of glucose-galactose syrup. The hypothesis of the doctoral thesis has been confirmed by the defended thesis: 1. The presence of cations affects the β-galactosidase activity in the sweet and acid whey permeate. 2. The chemical composition and quality of whey affect the physical properties of lactose. 3. Enzymatic reactions affect the functional and sensory properties of syrups. The research objects – sweet and acid whey permeates, glucose isomerase, commercial β-galactosidases and glucose-galactose syrup. The following tasks were set to achieve the aim of the doctoral thesis: 1. To evaluate the effect of cation concentration to ensure the β-galactosidase activity in substrate. 2. To investigate the physical properties of whey lactose in order to better understand its behaviour. 3. To study the changes of monosaccharide concentration in the lactose hydrolysis, varying with the solids concentration of the substrates and enzyme units. 4. To assess the possibilities of glucose isomerase to increase the sweetness of glucose-galactose syrup. 5. To evaluate the sensory properties of the developed syrups. The novelty of the doctoral thesis: 1. The study of the relationship of lactose hydrolysis process in the formation of galacto-oligosaccharides and lactulose. 2. The combination of β-galactosidase and glucose isomerase increases the sweetness of glucose-galactose syrup. The economic significance of the doctoral thesis: 1. The studies have shown the possibility to obtain syrup that can be used as sugar and sweeteners replacer in the food industry and to produce functional products. 2. A technological solution for hydrolysis of lactose is proposed, comprehensively evaluating the physical properties of lactose, fermentation parameters and whey composition. The doctoral thesis consists of three chapters: Chapter 1 describes the composition of whey and the possibilities of using it. An overview of the chemical and physical properties of lactose, lactose hydrolysis methods, the application of β-galactosidases and the properties of glucose-galactose syrup are provided. Chapter 2 summarises the materials and methods used in the thesis. Chapter 3 provides a summary of the results obtained in the study, the properties of commercial enzymes in different cation concentrations, the stability of enzymes in the gastrointestinal tract model, methods for the determination of lactose, the properties of dehydrated permeates are evaluated. The influence of factors on the hydrolysis of permeates and the profile of the obtained sugars was analysed. Possibilities for lactulose synthesis are considered. Sensory analysis of glucose-galactose syrups and syrups obtained in the two-stage fermentation are given. During the PhD studies the author had an internship at the Dairy Innovation Institute California Polytechnic State University (USA), where the experimental work was done. Internship was provided by the Baltic – American Freedom Foundation (BAFF) and the Council on International Education Exchange (CIEE). The study was partly financed by the LLU programme “Strengthening Research Capacity at the Latvia University of Agriculture” grant (Contract No. 3.2.-10/2017/LLU/27) “The optimization of bioprocesses for lactose recycling products”. The study was partly financed by the doctoral studies grant “Transition to the new doctoral funding model at the Latvia University of Life Sciences and Technologies” (Contract No. 3.2.-10/90). The thesis is written in English, it consists of 111 pages, 32 tables, 41 figures, 3 appendixes, and 233 bibliographic sources.

Phytochemical analysis, antioxidant and in vitro β-galactosidase inhibition activities of Juniperus phoenicea and Calicotome villosa methanolic extracts

Background Juniperus Phoenicea (JP) and Calicotome Villosa (CV) are used by Jordanian populations as herbal remedies in traditional medicine. Herein, the phytochemical contents of their methanolic extracts were analyzed and their antioxidant as well as in vitro anti- β-Galactosidase activities were evaluated; their effect on β-Galactosidase enzyme kinetics was evaluated and the thermodynamic of the enzyme was determined. Methods The antioxidant activity of JP and CV crude methanolic extracts was evaluated using 1,1-diphenyl,2-picrylhydrazyl (DPPH) free radical scavenging and ferric reducing antioxidant power (FRAP) assays; however, the effect of the plants’ crude extracts on β-Galactosidase activity and kinetics was evaluated in vitro. Moreover, total phenolic, flavonoids, and flavonols content in plants’ extracts were determined and expressed in Gallic acid equivalent (mg GAE/g dry extract) or rutin equivalent (mg RE/g dry extract). Results Phytochemical screening of the crude extracts of JP and CV leaves revealed the presence of phenols, alkaloids, flavonoids, terpenoids, anthraquinones, and glycosides. Flavonoids and flavonols contents were significantly higher in JP than in CV (p < 0.05). Furthermore, an analogous phenolic content was detected in both JP and CV methanolic extracts (103.6 vs 99.1 mg GAE/g extract). The ability of JP extract to scavenge DPPH radicals was significantly higher than that of CV extract with IC50 = 11.1 μg/ml and 15.6 μg/ml, respectively. However, their extracts revealed relatively similar antioxidant capacities in FRAP assay; their activity was concentration dependent. The JP extract inhibited β—galactosidase enzyme activity with a significant IC50 value compared to CV extract; they exhibited their inhibitory activities at IC50 values 65 µg/ml and 700 µg/ml, respectively. Rutin revealed anti-β-galactosidase activity at IC50 = 75 µg/ml. The mode of inhibition of β-galactosidase by JP, CV, and rutin was non-competitive, mixed, and competitive inhibition, respectively. Thermodynamic and enzyme inactivation kinetics revealed that β-galactosidase has a half-life time of 108 min at 55 °C, activation energy of 208.88 kJ mol−1 and the inactivation kinetics follows a first-order reaction with k-values 0.0023–0.0862 min−1 and positive entropy of inactivation (∆S°) values at various temperatures, indicating non-significant processes of aggregation. Conclusions The methanolic extracts of JP and CV possess anti-hyperglycemic and antioxidant activities with potential pharmaceutical applications.

Effect of N-acyl-homoserine lactones（AHL) on biogenic amine formation by Pantoea agglomerans

The effect of N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) on biogenic amine (BA) formation by Pantoea agglomerans was studied. Agrobacterium tumefaciens A136 and KYC55-based bioassays confirmed AHLs production by Pantoea agglomerans. The production ability of AHLs was quantified on the basis of β-galactosidase activity. The influence of temperature (10°C and 20°C) and pH (5.5 and 6.5) on β-galactosidase activity and BAs production by Pantoea agglomerans was determined. Acidification of the environment adversely affected the growth and β-galactosidase activity of Pantoea agglomerans, and AHLs production and BAs accumulation by Pantoea agglomerans was inhibited at low temperature. A significant correlation between β-galactosidase activity and BAs (putrescine, histamine, putrescine and tryptamine) was identified (P < 0.01). Based on the results of this study, the AHL-based QS system influences the concentrations and types of BAs produced by Pantoea agglomerans.

Intravenous delivery of adeno-associated viral gene therapy in feline GM1 gangliosidosis

GM1 gangliosidosis is a fatal neurodegenerative disease caused by a deficiency of lysosomal β-galactosidase. In its most severe form, GM1 gangliosidosis causes death by 4 years of age, and no effective treatments exist. Previous work has shown that injection of the brain parenchyma with an adeno-associated viral vector provides pronounced therapeutic benefit in a feline GM1 model. To develop a less invasive treatment for the brain and increase systemic biodistribution, intravenous injection of AAV9 was evaluated. AAV9 expressing feline β-galactosidase was intravenously administered at 1.5x1013 vector genomes/kilogram body weight to six GM1 cats at approximately 1 month of age. The animals were divided into two cohorts: 1) a long-term group, which was followed to humane endpoint, and 2) a short-term group, which was analyzed 16-weeks post treatment. Clinical assessments included neurological exams, cerebrospinal fluid and urine biomarkers, and 7-Telsa magnetic resonance imaging and spectroscopy. Postmortem analysis included β-galactosidase and virus distribution, histological analysis, and ganglioside content. Untreated GM1 animals survived 8.0 ± 0.6 months while intravenous treatment increased survival to an average of 3.5 years (n = 2) with substantial improvements in quality of life and neurologic function. Neurological abnormalities, which in untreated animals progress to the inability to stand and debilitating neurological disease by 8 months of age, were mild in all treated animals. Cerebrospinal fluid biomarkers were normalized, indicating decreased central nervous system cell damage in the treated animals. Urinary glycosaminoglycans decreased to normal levels in the long-term cohort. Magnetic resonance imaging and spectroscopy showed partial preservation of the brain in treated animals, which was supported by postmortem histological evaluation. β-galactosidase activity was increased throughout the central nervous system, reaching carrier levels in much of the cerebrum and normal levels in the cerebellum, spinal cord and cerebrospinal fluid. Ganglioside accumulation was significantly reduced by treatment. Peripheral tissues such as heart, skeletal muscle, and sciatic nerve also had normal β-galactosidase activity in treated GM1 cats. GM1 histopathology was largely corrected with treatment. There was no evidence of tumorigenesis or toxicity. Restoration of β-galactosidase activity in the central nervous system and peripheral organs by intravenous gene therapy led to profound increases in lifespan and quality of life in GM1 cats. This data supports the promise of intravenous gene therapy as a safe, effective treatment for GM1 gangliosidosis.