Β-galactosidase Production Research Articles

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 β-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 β-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.

In Vitro Characterization and Identification of Potential Probiotic Yeasts Isolated from Zaopocu, a Traditional Fermented Dregs Vinegar from Hainan Island.

Recently, there has been an increasing interest in researching fermented food-derived yeasts as probiotics because they offer a natural and diverse source of potential strains with unique functional properties and health benefits. In this study, 13 yeast strains isolated from Zaopocu (ZPC), a traditional fermented dregs vinegar on Hainan Island, China, were evaluated for their probiotic characteristics in vitro. Yeast identification was conducted through 5.8S-ITS region sequencing, revealing Kodamaea ohmeri as the predominantly isolated species (ZPC_Y3, Y5, Y6, Y11), followed by Pichia kudriavzevii (ZPC_Y2, Y13, Y14), Rhodotorula mucilaginosa (ZPC_Y9, Y10), Pichia fermentans (ZPC_Y8, Y12), Pichia kluyveri (ZPC_Y4), and Pichia occidentalis (ZPC_Y1). Except for ZPC_Y4, ZPC_Y8, and ZPC_Y12, all isolated yeasts exhibited stable growth at 37°C. The survival rates of all test strains exceeded 60% under challenging conditions at pH = 2 and 0.3% bile salt, along with strong antioxidant activity (> 5 6%), notable autoaggregation (> 70%), and varying levels of cell hydrophobicity with xylene (ranging from 35.32 ± 8.57% to 89.73 ± 4.84%). In addition, all isolates showed resistance to multiple antibiotics, along with antagonistic activity, and were deemed safe as none exhibited hemolytic, gelatinase, or DNase activities. Significantly, two P. kudriavzevii strains (ZPC_Y2, Y14) exhibited the production of catalase, lipase, and β-galactosidase, along with the capacity to synthesize gamma-aminobutyric acid (GABA). In summary, this preliminary study represents the first attempt to identify and characterize potential probiotic yeast strains isolated from Zaopocu, providing a theoretical basis for exploring their application in developing novel therapeutic probiotics.

Enhancing β-galactosidase production via GAL80 gene knockout in Kluyveromyces marxianus; an in-vitro and in-silico study on GAL/LAC system proteins

Lactose is a disaccharide composed of galactose and glucose which naturally exist only in the milk. β-galactosidase is one of the crucial industrial enzymes that hydrolyzes lactose. Kluyveromyces marxianus can grow on lactose as the only source of carbon and energy allowing this species to use whey as a cheap source. So far, no structural and comprehensive study has been done on its GAL/LAC system. Therefore, in this study, the proteins involved in the GAL/LAC system of K. marxianus were identified, and their secondary and tertiary structures and functions were determined for the first time. Also, the GAL80 inhibitory gene was deleted from the chromosome of this species. Its effect on β-Galactosidase enzyme production and its expression was investigated, as well as the expression of other genes involved in the production system of this enzyme. Lac9 and Lac12 proteins do not have beta sheets in their structures, in the Gal1, Gal7, and Gal80 proteins, the amount of helix is more than beta sheets, while in the Gal10 protein, the amount of beta sheets is more. Also, in the Lac12 protein, 46% of the total protein comprises transmembrane helices. Gal7 and Gal80 form homodimers in the cell. We showed that by deletion of the GAL80 gene from K. marxianus chromosome, the expression of genes involved in the GAL system, including LAC4 (β-galactosidase), LAC12 (permease), LAC9 (transcriptional activator), GAL1 (galactokinase), GAL7 (transferase), and GAL10 (epimerase), and the production of β-galactosidase, increased significantly in the culture medium containing different sugars.

Freshwater microalgae Nannochloropsis limnetica for the production of β-galactosidase from whey powder

This study investigated the first-ever reported use of freshwater Nannochloropsis for the bioremediation of dairy processing side streams and co-generation of valuable products, such as β-galactosidase enzyme. In this study, N. limnetica was found to grow rapidly on both autoclaved and non-autoclaved whey-powder media (referred to dairy processing by-product or DPBP) without the need of salinity adjustment or nutrient additions, achieving a biomass concentration of 1.05–1.36 g L−1 after 8 days. The species secreted extracellular β-galactosidase (up to 40.84 ± 0.23 U L−1) in order to hydrolyse lactose in DPBP media into monosaccharides prior to absorption into biomass, demonstrating a mixotrophic pathway for lactose assimilation. The species was highly effective as a bioremediation agent, being able to remove > 80% of total nitrogen and phosphate in the DPBP medium within two days across all cultures. Population analysis using flow cytometry and multi-channel/multi-staining methods revealed that the culture grown on non-autoclaved medium contained a high initial bacterial load, comprising both contaminating bacteria in the medium and phycosphere bacteria associated with the microalgae. In both autoclaved and non-autoclaved DPBP media, Nannochloropsis cells were able to establish a stable microalgae–bacteria interaction, suppressing bacterial takeover and emerging as dominant population (53–80% of total cells) in the cultures. The extent of microalgal dominance, however, was less prominent in the non-autoclaved media. High initial bacterial loads in these cultures had mixed effects on microalgal performance, promoting β-galactosidase synthesis on the one hand while competing for nutrients and retarding microalgal growth on the other. These results alluded to the need of effective pre-treatment step to manage bacterial population in microalgal cultures on DPBP. Overall, N. limnetica cultures displayed competitive β-galactosidase productivity and propensity for efficient nutrient removal on DPBP medium, demonstrating their promising nature for use in the valorisation of dairy side streams.

In vitro characterization of lactic acid bacteria from Indian fermented rice for probiotic applications

AimTo investigate probiotic attributes of lactic acid bacteria (LAB) isolated from under-explored Indian fermented rice for potential application in consumer health and functional product development. Methods and resultsThe isolates endured conditions resembling gastric stress, including pH (3), bile salt (4%), NaCl (6%), and phenol (0.6%). The selected isolates were identified as L. fermentum and L. plantarum strains by 16 S rRNA sequencing. In a simulated gastrointestinal tract (GIT), isolate SR1 outperformed other cultures in terms of viability. Auto-aggregation, and mucin adhesion abilities were tested for colonization potential. In which, compared to other isolates, MCR1 showed greater mucin adherence. SR1 and MCR1 showed higher percentages of auto-aggregation than GG. The auto-aggregation ranged from 9% to 88%., the levels significantly increase after 24 h incubation. The lower Salt aggregation Test (SAT) values indicated efficient hydrophobicity necessary for persistence in GIT and competitive inhibition to pathogens. All the isolates displayed considerably higher galactosidase activity than GG, isolate FCR2 expressed the highest, up to 613±0.92 Miller’s unit/ml activity. The probiotic candidates displayed broad spectrum antimicrobial activity against food-spoilage fungi and entero-pathogens. The study of safety aspects such as hemolytic, DNase activity, antibiotic susceptibility confirmed unavailability of any virulent factor. ConclusionThe isolates developed substantial galactosidase activity and significant broad-spectrum antibacterial activity. These characteristics make isolates an effective commercial option for β-galactosidase production in the food sector to create low-lactose or lactose-free products. Overall, the isolates' capacity for survival, colonization, and other useful traits may be of commercial relevance. Additionally, isolates can be researched for the production of antibacterial agents and in vivo studies for development of future functional food products. Moreover, these findings lay a robust groundwork for future exploration in probiotics, functional foods and nutraceuticals, while also shedding light on the mechanisms underlying the health benefits of the specified traditional food source. SignificanceThis investigation uncovered potential probiotic LAB of Indian fermented rice origin. By doing so, it expands our understanding of LAB diversity and presents promising candidates for applications in gut health improvement, functional food development, and exploration of natural antibacterial solutions.

Non-pathogenic Heyndrickxia coagulans (Bacillus coagulans) 29-2E inhibits the virulence of pathogenic Salmonella Typhimurium by quorum-sensing regulation

Bacteria produce and release small signal molecules, autoinducers, as an indicator of their cell density. The system, called a quorum-sensing (QS) system, is used to control not only virulence factors but also antibiotic production, sporulation, competence, and biofilm formation in bacteria. Different from antibiotics, QS inhibitors are expected to specifically repress the virulence factors in pathogenic bacteria without inhibiting growth or bactericidal effects. Therefore, since QS inhibitors have little risk of antibiotic-resistant bacteria emergence, they have been proposed as promising anti-bacterial agents. In the present study, we aimed to find new QS inhibitors that prohibit the signaling cascade of autoinducer 3 (AI-3) recognized by a QseCB two-component system that regulates some virulence factors of pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica subsp. enterica serovar Typhimurium. We have established the method for QS-inhibitor screening using a newly constructed plasmid pLES-AQSA. E.coli DH5α transformed with the pLES-AQSA can produce β-galactosidase that converts 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) into blue pigment (5-bromo-4-chloro-indoxyl) under the control of the QseCB system. By screening, Heyndrickxia coagulans (formerly Bacillus coagulans) 29-2E was found to produce an exopolysaccharide (EPS)-like water-soluble polymer that prohibits QseCB-mediated β-galactosidase production without antibacterial activities. Further, the simultaneous injection of the 29-2E strain significantly improves the survival rate of Salmonella Typhimurium-infected silkworm larvae (from 0% to 83.3%), suggesting that the substance may be a promising inhibitor against the virulence of pathogens without risk of the emergence of antibiotic-resistant bacteria.

Catechol-Siderophore Mimics Convey Nucleic Acid Therapeutics into Bacteria.

Antibacterial resistance is a major threat for human health. There is a need for new antibacterials to stay ahead of constantly-evolving resistant bacteria. Nucleic acid therapeutics hold promise as powerful antibiotics, but issues with their delivery hamper their applicability. Here, we exploit the siderophore-mediated iron uptake pathway to efficiently transport antisense oligomers into bacteria. We appended a synthetic siderophore to antisense oligomers targeting the essential acpP gene in Escherichia coli. Siderophore-conjugated PNA and PMO antisense oligomers displayed potent antibacterial properties. Conjugates bearing a minimal siderophore consisting of a mono-catechol group showed equally effective. Targeting the lacZ transcript resulted in dose-dependent decreased β-galactosidase production, demonstrating selective protein downregulation. Applying this concept to Acinetobacter baumannii also showed concentration-dependent growth inhibition. Whole-genome sequencing of resistant mutants and competition experiments with the endogenous siderophore verified selective uptake through the siderophore-mediated iron uptake pathway. Lastly, no toxicity towards mammalian cells was found. Collectively, we demonstrate for the first time that large nucleic acid therapeutics can be efficiently transported into bacteria using synthetic siderophore mimics.

What happens to Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with eukaryotic cells?

BackgroundThe impact of probiotic strains on host health is widely known. The available studies on the interaction between bacteria and the host are focused on the changes induced by bacteria in the host mainly. The studies determining the changes that occurred in the bacteria cells are in the minority. Within this paper, we determined what happens to the selected Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with the intestinal epithelial layer. For this purpose, we tested the bacteria cells' viability, redox activity, membrane potential and enzymatic activity in different environments, including CaCo-2/HT-29 co-culture, cell culture medium, presence of inflammatory inductor (TNF-α) and oxygen.ResultsWe indicated that the external milieu impacts the viability and vitality of bacteria. Bifidobacterium adolescentis decrease the size of the live population in the cell culture medium with and without TNF-α (p < 0.001 and p < 0.01 respectively). In contrast, Bifidobacterium longum ssp. longum significantly increased survivability in contact with the eukaryotic cells and cell culture medium (p < 0.001). Bifidobacterium adolescentis showed significant changes in membrane potential, which was decreased in the presence of eukaryotic cells (p < 0.01), eukaryotic cells in an inflammatory state (p < 0.01), cell culture medium (p < 0.01) and cell culture medium with TNF-α (p < 0.05). In contrast, Bifidobacterium longum ssp. longum did not modulate membrane potential. Instead, bacteria significantly decreased the redox activity in response to milieus such as eukaryotic cells presence, inflamed eukaryotic cells as well as the culture medium (p < 0.001). The redox activity was significantly different in the cells culture medium vs the presence of eukaryotic cells (p < 0.001). The ability to β-galactosidase production was different for selected strains: Bifidobacterium longum ssp. longum indicated 91.5% of positive cells, whereas Bifidobacterium adolescentis 4.34% only. Both strains significantly reduced the enzyme production in contact with the eukaryotic milieu but not in the cell culture media.ConclusionThe environmental-induced changes may shape the probiotic properties of bacterial strains. It seems that the knowledge of the sensitivity of bacteria to the external environment may help to select the most promising probiotic strains, reduce research costs, and contribute to greater reproducibility of the obtained probiotic effects.

Mechanism of lactose assimilation in microalgae for the bioremediation of dairy processing side-streams and co-production of valuable food products

Abstract This study investigated the mechanism of lactose assimilation in Nannochloropsis oceanica for dairy-wastewater bioremediation and co-production of valuable feed/food ingredients in a circular dairy system (β-galactosidase and omega-3 polyunsaturated fatty acids). Mixotrophic cultivation was found to be mandatory for lactose assimilation in N. oceanica, with biomass production in mixotrophic cultures reaching a fourfold increase over that under heterotrophic conditions. Under mixotrophic conditions, the microalgae were able to produce β-galactosidase enzyme to hydrolyse lactose, with maximum extracellular secretion recorded on day 8 of growth cycle at 41.47 ± 0.33 U gbiomass−1. No increase in the concentration of glucose or galactose was observed in the medium, confirming the ability of microalgae to indiscriminately absorb the resultant monosaccharides derived from lactose breakdown. Population analysis revealed that microalgae cells were able to maintain dominance in the mixotrophic culture, with bacteria accounting for &lt; 12% of biomass. On the other hand, under heterotrophic conditions, native bacteria took over the culture (occupying over 95% of total biomass). The bacteria, however, were also unable to effectively assimilate lactose, resulting in limited biomass increase and negligible production of extracellular β-galactosidase. Results from the study indicate that N. oceanica can be effectively applied for onsite dairy wastewater treatment under strict mixotrophic conditions. This is commercially disadvantageous as it rules out the possibility of deploying heterotrophic fermentation with low-cost bioreactors and smaller areal footprint.

Improvement of β-galactosidase production by solid-state fermentation using cauliflower (Brassica oleraceae var. botrytis) waste by Enterobacter aerogenes KCTC2190

In this present study, cauliflower wastes were employed as a substrate for better β-galactosidase production by Enterobacter aerogenes KCTC2190. Cauliflower leaf and stalk were used separately as substrates for solid state fermentation. 10% cauliflower stalk enhanced β-galactosidase production at the maximum level. Cultural parameters such as incubation period, temperature and pH were optimized. Maximum β-galactosidase was produced at 40ºC at pH 7 containing cauliflower stalk supplemented medium after 30 h of incubation.

β-galactosidase 생산 우수 유산균 선별 및 배지 최적화

Seven strains showing activity in MRS(De Man, Rogosa, and Sharpe) solid medium containing X-gal were selected from kimchi, yogurt, and earthworm feces. The strain isolated from yogurt showed the greatest activity.&#x0D; Enzyme activity was higher in cell suspensions than in cells or culture filtrates. The isolate was inferred to be a lactic acid bacterium based on Gram staining, biochemical tests, and the fact that it was isolated from commercial yogurt. The optimal concentrations of carbon, nitrogen, and minerals for cell growth and enzyme production were investigated. The results of this study are applicable to the production of β-galactosidase using lactic acid bacteria.

Lysobacter lactosilyticus sp. nov., a Novel β-Galactosidase Producing Bacterial Strain Isolated from Farmland Soil Applied with Amino Acid Fertilizer.

To isolate β-galactosidase producing bacterial resources, a novel Gram-stain-negative, strictly aerobic bacterial strain designated as A6T was obtained from a farmland soil sample. Cells of the strain were rod-shaped (0.4-0.7μm × 1.8-2.2μm) without flagella and motility. Strain A6T grew optimally at 30 °C, pH 7.0 with 0% (w/v) NaCl. Based on phylogenetic analysis, strain A6T clustered within the genus Lysobacter clade and branched with Lysobacter dokdonensis KCTC 12822T (99.5%, 16S rRNA gene sequence similarity) and Lysobacter caseinilyticus KACC 19816T (98.5%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain A6T and Lysobacter dokdonensis KCTC 12822T were 82.7% and 26.2%, and the values for strain A6T and KACC 19816T were 81.4% and 23.8%, respectively. Iso-C16:0, iso-C15:0, summed feature 9 (C17:1 iso ω9c and/or C16:0 10-methyl) and summed feature 3 (C16:1ω7c and/or C16:1 ω6c) were the major fatty acids, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine were the major polar lipids, and ubiquinone 8 (Q-8) was the major ubiquinone. The genomic DNA G+C content was 67.2mol%. Furthermore, under the condition of 30 °C, pH 7.0, 4% inoculation with 10.0gL-1 lactose, the β-galactosidase activity produced by strain A6T was highest, reaching 95.3 UmL-1, indicating that this strain could be applied as a potential strain for β-galactosidase production. Strain A6T represents a novel species of the genus Lysobacter, and Lysobacter lactosilyticus sp. nov. is proposed on the basis of phenotypic, genotypic, and chemotaxonomic analysis. The type strain is A6T (=KCTC 82184T=CGMCC 1.18582T).

Recombinant production in Escherichia coli of a β-galactosidase fused to a cellulose-binding domain using low-cost inducers in fed-batch cultivation

This study aimed to determine the effects of different feeding strategies in fed-batch cultivations on the production of β-galactosidase fused to a cellulose-binding domain (CBD) by Escherichia coli C41(DE3) using a bench bioreactor. The highest enzyme activities (∼17,000 U/L) were obtained in fed-batch cultivations controlled by DO-stat and induced with lactose (5 g/L) at 18 h of cultivation. Under these cultivation conditions, the maximum values of β-galactosidase yields per substrate, β-galactosidase yields per biomass, and QP were approximately 950 U/gglucose, 1100 U/gcells, and 540 U/L.h, respectively, using Terrific Broth as culture medium and concentrated feed solution (2 ×). The dairy coproducts were efficient in inducing the expression of the recombinant enzyme, having reached the highest values of β-galactosidase-CBD activity after 40 h of cultivation. The maximum enzyme activities obtained when using whey permeate or ricotta whey as inducers were 22,950 or 24,640 U/L, respectively. Using the optimal temperature of E. coli growth before induction and ricotta whey as an inducer increased enzyme productivity in 50%. Plasmid construction showed high stability (>94%) throughout E. coli cultivation. This study demonstrated the feasibility of using the DO-stat feeding strategy to produce β-galactosidase-CBD employing ricotta whey as an inducer of enzyme expression.

Isolation, production and optimisation of beta-galactosidase by utilizing yeasts isolated from selected dairy products

β-galactosidase is an enzyme that converts lactose into glucose and galactose. It alleviates the issue associated with intolerance to lactose and pollution caused as a result of milk by-products (whey). This study aimed to isolate and select yeast strains that can produce β-galactosidase from fresh milk, yoghurt and locally made cheese. A total of 115 yeasts were isolated from the samples, 9 yeast isolates had the ability to produce β-galactosidase and three (3) were selected for further analysis. β-galactosidase producers were screened using Ortho-nitrophenyl-β-D-galactopyranoside (ONPG) assay. Kluyveromyces marxianus strain SLDY – 005 produced the highest β-galactosidase. It showed maximum enzyme activity (277U/mL) at temperature 30oC, pH 5.5, after an incubation period of 36 h. Glucose had a decrease in β-galactosidase production while yeast extract and urea were considered appropriate nitrogen sources for the best enzyme synthesis. Crude β-galactosidase produced by Kluyveromyces marxianus SLDY – 005 was purified. The partially purified enzyme after dialysis showed a specific activity of 165.12 U/mL and had a purification fold of 6.02 and yield of 45.91 %. The purified enzyme had an optimum temperature of 40oC and a pH 6.0. Kluyveromyces marxianus strain isolated from local cheese is a potential candidate for the production of βgalactosidase and it could be used for combating the problem of lactose intolerance.

WITHDRAWN: Production of β-galactosidase from a Lactobacillus species

Production of β-galactosidase from a bacterial strain isolated from dairy wastewater was investigated. The isolate was screened for β-galactosidase producing ability and was confirmed as Lactobacilli acidophilus using biochemical tests and 16S rRNA gene sequencing technique. The optimum pH for β-galactosidase production was 6.0 with an activity of 121.70 μmol/min at 37°C. Lactobacilli acidophilus produced enzyme with the highest β-galactosidase activity (126.56 μmol/min) on the 12 th day of fermentation. The isolate was able to produce an enzyme with β-galactosidase activities of 121.93 and 112.21 μmol/min to utilize lactose and sugarcane bagasse, respectively more than glucose and a compendium of glucose and lactose. The best nitrogen source for β-galactosidase production was peptone with a β-galactosidase activity of 119.34 μmol/min, followed by a compendium of ammonium sulfate salt and beef extract which gave β-galactosidase activity of 118.91 μmol/min. These findings show that Lactobacillus acidophilus from dairy industrial wastewater can be an alternative source for β-galactosidase production using sugarcane bagasse which is a cheap readily available agro-waste. Also, the enzyme showed more preference for lactose suggesting its potential in the reduction of lactose level in dairy products.

Мolecular-genetic analysis of determinants encoding β-galactosidases of bacteria Bifidobacterium longum BIM B-813 D

The molecular-genetic analysis of the bacterial genome of the strain B. longum BIM B-813D distinguished by a high level of β-galactosidase production was performed. Genes Bgal_small_N, lacZ1, bgaB1, bgaB2 and bgaB3, and lacZ2, encoding the synthesis of β-galactosidases, were revealed in the deciphered genome. It was shown that the genes lacZ1, bgaB2, and bgaB3 characterized by an enhanced degree of similarity to the genes of closely related bifidobacterial species, presumably code for the enzymes catalyzing the specific reactions of hydrolysis and transglycosylation of carbohydrates. It was found that the enzymes BgaB1, BgaB2 and BgaB3 belong to the GH42 family of glycosyl hydrolases, whereas the enzymes LacZ1 and LacZ2 – to the GH2 family. The genome domains responsible for the synthesis of β-galactosidases in the strain B. longum BIM B-813D were studied in detail. A comparative analysis of the locus of lacZ1 in B. longum BIM B-813D and the similar genome fragment AS143_01230 from B. longum subsp. longum MC-42 detected the presence of the transposase gene ISL3 in the former strain. It was suggested that the insertion of the sequence of ISL3 in the lacZ1 locus resulted in the modified gene expression and the increased production of β-galactosidase in the strain B. longum BIM B-813D.

Moesziomyces spp. cultivation using cheese whey: new yeast extract-free media, β-galactosidase biosynthesis and mannosylerythritol lipids production.

Mannosylerythritol lipids (MELs) are biosurfactants with excellent biochemical properties and a wide range of potential applications. However, high production costs, low productivity and unsatisfactory scale-up production have hampered commercial adoption. Herein, we report for the first time the β-galactosidase production by Moesziomyces spp. from different sugars (D-galactose, D-glucose and D-lactose), with D-galactose being the best β-galactosidase inducer, with 11.2 and 63.1 IU/mgbiomass, for Moesziomyces aphidis 5535 T and Moesziomyces antarcticus 5048 T, respectively. The production of this enzyme allows to break down D-lactose and thus to produce MEL directly from D-lactose or cheese whey (a cheese industry by-product). Remarkably, when CW was used as sole media component (carbon and mineral source), in combination with waste frying oil, MEL productivities were very close (1.40 and 1.31 gMEL/L/day) to the ones obtained with optimized medium containing yeast extract (1.92 and 1.50 gMEL/gsusbtrate), both for M. antarcticus and M. aphidis. The low-cost, facile and efficient process which generates large amounts of MELs potentiates its industrialization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13399-022-02837-y.

Characterization of Induction and Targeting of Senescent Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) from older donors have limited potential for bone tissue formation compared with cells from younger donors, and cellular senescence has been postulated as an underlying cause. There is a critical need for methods to induce premature senescence to study this phenomenon efficiently and reproducibly. However, the field lacks consensus on the appropriate method to induce and characterize senescence. Moreover, we have a limited understanding of the effects of commonly used induction methods on senescent phenotype. To address this significant challenge, we assessed the effect of replicative, hydrogen peroxide, etoposide, and irradiation-induced senescence on human MSCs using a battery of senescent cell characteristics. All methods arrested proliferation and resulted in increased cell spreading compared with low passage controls. Etoposide and irradiation increased expression of senescence-related genes in MSCs at early time points, proinflammatory cytokine secretion, DNA damage, and production of senescence-associated β-galactosidase. We then evaluated the effect of fisetin, a flavonoid and candidate senolytic agent, to clear senescent cells and promote osteogenic differentiation of MSCs entrapped in gelatin methacryloyl (GelMA) hydrogels in vitro. When studying a mixture of nonsenescent and senescent MSCs, we did not observe decreases in senescent markers or increases in osteogenesis with fisetin treatment. However, the application of the same treatment toward a heterogeneous population of human bone marrow-derived cells entrapped in GelMA decreased senescent markers and increased osteogenesis after 14 days in culture. These results identify best practices for inducing prematurely senescent MSCs and motivate the need for further study of fisetin as a senolytic agent. Impact Statement The accumulation of senescent cells within the body has detrimental effects on tissue homeostasis. To study the role of senescent cells on tissue repair and regeneration, there is a need for effective means to induce premature cell senescence. Herein, we characterized the influence of common stressors to induce premature senescence in human mesenchymal stromal cells (MSCs). Irradiation of MSCs resulted in a phenotype most similar to quiescent, high-passage cells. These studies establish key biomarkers for evaluation when studying senescent cells in vitro.

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield. Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated. Aquatic habitats harbor arrays of microorganisms that can participate in demineralization, nutrient restoration, circulation and hence confer significant properties on the habitats and significantly influence microbial interactions and metabolism. In view of this, aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production. Four fungal isolates tagged SB01 and SC02 from stream identified as Aspergillus oryzae and Aspergillus niger and SC01 and SA01 from pond identified as A. oryzae and A. niger were studied. β-galactosidase gene was successfully amplified from the four isolates and their enzyme production levels were further studied. All the isolates showed considerably higher extracellular secretion than the intracellular synthesis on glucose-based medium. The production level was probed by xylose supplementation but surprisingly, the intracellular synthesis was higher than the extracellular secretion for all the isolates. In an attempt to boost the enzyme production, temperature was varied and 25 °C showed improved β-galactosidase synthesis and secretion than the 30 °C. This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production, and more importantly, revealed the intracellular accumulation of the enzyme as a bottleneck for industrial production.&#x0D; HIGHLIGHTS&#x0D; &#x0D; The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield&#x0D; Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated&#x0D; Aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production&#x0D; All the isolates showed considerable higher extracellular secretion than the intracellular synthesis on glucose-based medium, and vice-versa on xylose-supplemented medium, with the highest β-galactosidase synthesis and secretion efficiency occurring at 25 °C in both instances&#x0D; This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production&#x0D; &#x0D; GRAPHICAL ABSTRACT

Statistical mixture designs for media development with agro-industrial residues – Supporting the circular bioeconomy

Nutrient-rich side streams can be used in fermentation media to produce biomaterials, biofuels and other high-value products, contributing to the circular bioeconomy. However, the efficient valorization of side streams requires media optimized for the fermentation organism and the desired product. In an exemplary process, we investigated statistical mixture designs for the development of a medium containing food-grade agro-industrial residues (whey, molasses and cornsteep powder (CSP)). The yeast Kluyveromyces lactis was used to convert these residues into biomass and a model-protein (β-galactosidase). With the obtained response surface, the effects and interactions of the residues and mixtures thereof on growth rate, biomass and β-galactosidase production could be determined. Two-thirds CSP with one-third molasses produced the most biomass, whereas 88% CSP with 12% whey achieved the optimal β-galactosidase production rate. The side streams used fulfilled cell-specific requirements and the mixture design even led to the identification of synergistic effects between residues. The models were verified in confirmation runs, showing that statistical mixture designs are ideal for targeted and resource-efficient media development. Expanding the use of mixture designs to other side streams, biocatalysts, and products increases the attractiveness and efficiency of using agro-industrial residues in fermentation media and thus can be beneficial in supporting the circular bioeconomy.