Discovery of Alkyl Triphenylphosphonium Pinostrobin Derivatives as Potent Anti-Breast Cancer Agents.

Pinostrobin demonstrated anticancer properties, but its hydrophobic feature led to a reduction in bioavailability. The mitochondria-targeted approach successfully synthesized eight new alkyl triphenylphosphonium pinostrobin derivatives (1-8) with good yield in this study. Seven compounds (1-3, 5-8) showed greater cytotoxic potency against the human MCF-7 breast cancer cell line than pinostrobin. Molecular docking studies were performed with two important targets in hormone-dependent anticancer strategies, estrogen receptor α (ERα) ligand binding domains, 3ERT (antagonist recognition and antiproliferative function), and 1GWR (agonist recognition and pro-proliferative function). In addition, the MD simulation study of the two most potent compounds (2 and 3) complexed with both ERα forms suggested that compounds 2 and 3 could serve as favourable antagonists. Furthermore, the in silico ADMET prediction indicated that compounds 2 and 3 could be potential drug candidates.

Effects of a low-iodine diet in post-thyroidectomy thyroid cancer patients undergoing I131 therapy at the Vietnam National Cancer Hospital.

Background: I131 therapy is regarded as an "internal surgery" (i.e., a non-invasive approach involving no incision or bleeding) that supports "external surgery" (i.e., using a scalpel) in completely eradicating the root cause of thyroid cancer. Limiting iodine intake is of paramount importance in I131 therapy. I131 therapy protocols recommend that patients follow a low-iodine diet, ideally with a maximum iodine intake of 50 µg/day for two weeks before the I131 therapy. Methods: A pre-post compassion uncontrolled clinic intervention study was conducted on a group of over 70 post-thyroidectomy thyroid cancer patients with indications for I131 therapy at the Vietnam National Cancer Hospital from December 2020 to December 2022. Aim: It aimed to assess the effects of a low-iodine diet on post-thyroidectomy thyroid cancer patients with indications for I131 therapy. Results: The study found that following the intervention, the percentage of participants at risk of mild to moderate malnutrition, as assessed by the PG-SGA tool, decreased to 4.3% from 40.0% before the intervention, with a statistically significant difference of p < 0.001. There was a considerable improvement in the low calcemia level among the study participants, with 35.7% of patients experiencing hypocalcemia prior to the intervention, which reduced to 17.1% after the intervention. This difference was statistically significant (p = 0.01). The study also revealed a urinary iodine level improvement among the study participants. Before the intervention, patients' average urinary iodine level was 14.9 ± 11.3 µg/dl. Following the intervention, it reduced to 12.7 ± 3.9 µg/dl, although this difference was not statistically significant (p = 0.29). Patients' quality of life after adhering to the low-iodine diet tended to decline; however, the change in scores before and after the intervention did not show a significant difference. Conclusion: Despite its negative impact on patients' quality of life, active nutrition counseling and intervention during the low-iodine diet contributed to the substantial improvement in the hypocalcemia level and the reduced urinary iodine level among patients, which in turn could enhance the efficacy of the subsequent I131 therapy.

Impact of the COVID-19 pandemic on provision of HIV/AIDS services for key populations.

The COVID-19 pandemic has aggravated the obstacles for HIV/AIDS programs in limited-resource countries like Vietnam to achieve the HIV/AIDS-related Sustainable Development target. The paper aims to evaluate the impact of the COVID-19 pandemic on the provision of HIV/AIDS services-a pathway to achieving universal health coverage for key populations (KPs). Employing mix-methods, we conducted a desk study, one focus group discussion, and ten in-depth interviews with participants from the Ministry of Health, Provincial Centres for Disease Control, and HIV/AIDS-related facilities. The results showed the reduced coverage of KPs with access to prevention (i.e., harm-reduction services, counselling), testing, and treatment services (i.e., antiretroviral therapy, isoniazid preventive therapy). It also showed the reduced coverage of quality essential services, mainly in skipping consultation and testing, delaying un-emergency services, and redirecting KPs to non-HIV-specialised facilities. There was a gap in providing support for mental health, violence/abuse, and reproductive health. Financial risk protection for KPs was reduced due to uncertain local budget allocation; decreasing their ability to pay for HIV/AIDS-related services and social health insurance premiums; and increased out-of-pocket payments to comply with the COVID-19 control measures. This paper provides recommendations for strategic planning to ensure universal health coverage for KPs in the post COVID-19 era, especially for limited-resource countries like Vietnam.

Co-production of gallic acid and a novel cell-associated tannase by a pigment-producing yeast, Sporidiobolus ruineniae A45.2.

BACKGROUND: Gallic acid has received a significant amount of interest for its biological properties. Thus, there have been recent attempts to apply this substance in various industries and in particular the feed industry. As opposed to yeasts, fungi and bacteria and their tannases have been well documented for their potential bioconversion and specifically for the biotransformation of tannic acid to gallic acid. In this research, Sporidiobolus ruineniae A45.2 is introduced as a newly pigment-producing and tannase-producing yeast that has gained great interest for its use as an additive in animal feed. However, there is a lack of information on the efficacy of gallic acid production from tannic acid and the relevant tannase properties. The objective of this research study is to optimize the medium composition and conditions for the co-production of gallic acid from tannic acid and tannase with a focus on developing an integrated production strategy for its application as a feed additive. RESULTS: Tannase produced by S. ruineniae A45.2 has been classified as a cell-associated tannase (CAT). Co-production of gallic acid obtained from tannic acid and CAT by S. ruineniae A45.2 was optimized using response surface methodology and then validated with the synthesis of 11.2 g/L gallic acid from 12.3 g/L tannic acid and the production of 31.1 mU/mL CAT after 48 h of cultivation in a 1-L stirred tank fermenter. Tannase was isolated from the cell wall, purified and characterized in comparison with its native form (CAT). The purified enzyme (PT) revealed the same range of pH and temperature optima (pH 7) as CAT but was distinctively less stable. Specifically, CAT was stable at up to 70 °C for 60 min, and active under its optimal conditions (40 °C) at up to 8 runs. CONCLUSION: Co-production of gallic acid and CAT is considered an integrated and green production strategy. S. ruineniae biomass could be promoted as an alternative source of carotenoids and tannase. Thus, the biomass, in combination with gallic acid that was formed in the fermentation medium, could be directly used as a feed additive. On the other hand, gallic acid could be isolated and purified for food and pharmaceutical applications. This paper is the first of its kind to report that the CAT obtained from yeast can be resistant to high temperatures of up to 70 °C.

Burden of injuries in Vietnam: emerging trends from a decade of economic achievement.

BACKGROUND: Vietnam has been one of the fastest-growing world economies in the past decade. The burden of injuries can be affected by economic growth given the increased exposure to causes of injury as well as decreased morbidity and mortality of those that experience injury. It is of interest to evaluate the trends in injury burden that occurred alongside Vietnam's economic growth in the past decade. METHODS: Results from Global Burden of Disease 2017 were obtained and reviewed. Estimates of incidence, cause-specific mortality, years lived with disability, years of life lost, disability-adjusted life years were analysed and reported for 30 causes of injury in Vietnam from 2007 to 2017. RESULTS: Between 2007 and 2017, the age-standardised incidence rate of all injuries increased by 14.6% (11.5%-18.2%), while the age-standardised mortality rate decreased by 11.6% (3.0%-20.2%). Interpersonal violence experienced the largest increase in age-standardised incidence (28.3% (17.6%-40.1%)), while exposure to forces of nature had the largest decrease in age-standardised mortality (47.1% (37.9%-54.6%)). The five leading causes of injury in both 2007 and 2017 were road injuries, falls, exposure to mechanical forces, interpersonal violence and other unintentional injuries, all of which increased in incidence from 2007 to 2017. Injury burden varied markedly by age and sex. CONCLUSIONS: The rapid expansions of economic growth in Vietnam as well as improvements in the Sociodemographic Index have occurred alongside dynamic patterns in injury burden. These results should be used to develop and implement prevention and treatment programme.

Cell Wall Anchoring of a Bacterial Chitosanase in Lactobacillus plantarum Using a Food-Grade Expression System and Two Versions of an LP TG Anchor.

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.

Expression and biochemical characterization of a new alkaline tannase from Lactobacillus pentosus.

Lactobacillus pentosus BA-7 and L. pentosus QA1-5 are tannin-tolerant lactic acid bacteria that were isolated from Miang, a traditional fermented tea-leaf found in northern Thailand and a tannin-rich substrate. Tannase encoding genes were isolated, cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant tannase was produced with production yields of 40 and 39 KU/L for LpTanBA-7 and LpTanQA1-5, respectively. Both revealed the same molecular weight of 50 kDa as estimated by SDS-PAGE and were optimally active under alkaline pH conditions LpTanQA1-5 revealed optimal temperatures in a range of 37-40 °C as is typically found in lactic acid bacteria, while LpTanBA-7 was active at higher temperatures with an optimum temperature range of 45-55 °C. LpTanBA-7 was found to be more stable within the same range of temperatures than LpTanQA1-5. Furthermore, it was active and stable toward various organic solvents and produced 50 mg/mL of gallic acid from 100 mg/mL tannic acid. Based on the results, LpTanBA-7 is considered a new alkali-moderately thermophilic tannase obtained from lactic acid bacterium that may be capable of a feasible production capacity of gallic acid and its esters. Furthermore, tannase that is active at high temperatures could also be used in tea products in order to develop a sweet aftertaste, as well as to improve levels of antioxidant activity.

Constitutive expression and cell-surface display of a bacterial ß-mannanase in Lactobacillus plantarum.

BACKGROUND: Lactic acid bacteria (LAB) are important microorganisms in the food and beverage industry. Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In this present study, we aimed at constitutive expression of a mannanase from Bacillus licheniformis DSM13, which was subsequently displayed on the cell surface of Lactobacillus plantarum WCFS1, for use as whole-cell biocatalyst in oligosaccharide production. RESULTS: Two strong constitutive promoters, Pgm and SlpA, from L. acidophilus NCFM and L. acidophilus ATCC4356, respectively, were used to replace the inducible promoter in the lactobacillal pSIP expression system for the construction of constitutive pSIP vectors. The mannanase-encoding gene (manB) was fused to the N-terminal lipoprotein anchor (Lp_1261) from L. plantarum and the resulting fusion protein was cloned into constitutive pSIP vectors and expressed in L. plantarum WCFS1. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The mannanase activity and the reusability of the constructed L. plantarum displaying cells were evaluated. The highest mannanase activities on the surface of L. plantarum cells obtained under the control of the Pgm and SlpA promoters were 1200 and 3500 U/g dry cell weight, respectively, which were 2.6- and 7.8-fold higher compared to the activity obtained from inducible pSIP anchoring vectors. Surface-displayed mannanase was shown to be able to degrade galactomannan into manno-oligosaccharides (MOS). CONCLUSION: This work demonstrated successful displaying of ManB on the cell surface of L. plantarum WCFS1 using constitutive promoter-based anchoring vectors for use in the production of manno-oligosaccharides, which are potentially prebiotic compounds with health-promoting effects. Our approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism with the use of strong constitutive promoters, which continuously drive synthesis of the recombinant protein without the need to add an inducer or change the growth conditions of the host strain, should result in the availability of safe, stable food-grade biocatalysts.

ß-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry.

ß-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant ß-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native ß-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant ß-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The Km and vmax values for lactose and o-nitrophenyl-ß-d-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and oNPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55 °C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of ß-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.

Fermentability of a Novel Galacto-Oligosaccharide Mixture by Lactobacillus spp. and Bifidobacterium spp.

This study aimed to investigate the specific growth stimulation of certain desired intestinal bacteria by a novel galacto-oligosaccharide mixture, which was produced with a ß-galactosidase from a potential probiotic Lactobacillus isolate that contained mainly oligosaccharides of ß-1,3 and ß-1,6 glycosidic linkages (termed Lb-GOS) using single-strain fermentations. The composition of this Lb-GOS mixture was 33.5% disaccharides, 60.5% trisaccharides, 4.8% tetrasaccharides, and 1.0% pentasaccharides with a negligible amount of monosaccharides, lactose, and lactobionic acid (0.3%). Eight Lactobacillus spp. strains and three Bifidobacterium spp. strains were used in single-strain fermentations to determine the fermentation activity scores of this Lb-GOS preparation compared to two commercially available prebiotic mixtures, 4'GOS-P and Vivinal GOS (V-GOS). The highest scores were obtained when L. reuteri Lb46 and the two Bifidobacterium strains, B. animalis subsp. lactis Bif1 and Bif3, were grown on these galacto-oligosaccharide mixtures. In addition, the Lb-GOS mixture was found to have higher fermentation activity scores; hence, it stimulated the growth of these probiotic strains more than 4'GOS-P and V-GOS, which may be attributed to the different glycosidic linkage types that are found in the Lb-GOS mixture compared to the other two commercial preparations. These findings suggested that the Lb-GOS mixture that is described in this work should be of interest for the formulations of new carbohydrate-based functional food ingredients.

Growth-Based Bacterial Viability Assay for Interference-Free and High-Throughput Toxicity Screening of Nanomaterials.

Current high-throughput approaches evaluating toxicity of chemical agents toward bacteria typically rely on optical assays, such as luminescence and absorbance, to probe the viability of the bacteria. However, when applied to toxicity induced by nanomaterials, scattering and absorbance from the nanomaterials act as interferences that complicate quantitative analysis. Herein, we describe a bacterial viability assay that is free of optical interference from nanomaterials and can be performed in a high-throughput format on 96-well plates. In this assay, bacteria were exposed to various materials and then diluted by a large factor into fresh growth medium. The large dilution ensured minimal optical interference from the nanomaterial when reading optical density, and the residue left from the exposure mixture after dilution was confirmed not to impact the bacterial growth profile. The fractions of viable cells after exposure were allowed to grow in fresh medium to generate measurable growth curves. Bacterial viability was then quantitatively correlated to the delay of bacterial growth compared to a reference regarded as 100% viable cells; data analysis was inspired by that in quantitative polymerase chain reactions, where the delay in the amplification curve is correlated to the starting amount of the template nucleic acid. Fast and robust data analysis was achieved by developing computer algorithms carried out using R. This method was tested on four bacterial strains, including both Gram-negative and Gram-positive bacteria, showing great potential for application to all culturable bacterial strains. With the increasing diversity of engineered nanomaterials being considered for large-scale use, this high-throughput screening method will facilitate rapid screening of nanomaterial toxicity and thus inform the risk assessment of nanoparticles in a timely fashion.

Secretory expression of ß-mannanase from Bacillus circulans NT 6.7 in Lactobacillus plantarum.

The ß-mannanase gene of Bacillus circulans NT 6.7 was successfully cloned in Lactobacillus plantarum WCFS1 using the pSIP403 expression vector and secreted to the supernatant rather than accumulated in the cells. The highest activity was achieved by controlling the pH at 6 during cultivation. Maximum mannanase activities detected in the supernatant and cell-free extract of 200 ml MRS broth were 8.2 and 0.86 U/ml, respectively. Enzyme activity in the supernatant increased to 27 U/ml by fermentation in a 5-L bioreactor with automatic pH control. The optimum temperature of recombinant ß-mannanase was 50 °C and stable between 30 and 50 °C. The optimum pH was 6 with stability in the range 5-7. Enzyme activity slightly increased with Co2+ but was strongly inhibited by EDTA. The enzyme exhibited high specificity to galactomannan substrates. The main products of copra meal and locust bean gum hydrolysis were manno-oligosaccharides. Therefore, recombinant ß-mannanase produced from a food grade host, L. plantarum WCFS1, showed potential for use in manno-oligosaccharides production and other food-related applications.

Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.

BACKGROUND: Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially food-grade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications. RESULTS: We studied overexpression of a chitosanase (CsnA) and a ß-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84% for ManB, and 79 kU/L and 56% for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host. CONCLUSIONS: ManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker.

Display of a ß-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

BACKGROUND: Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. RESULTS: ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. CONCLUSION: This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry.

Synthesis and cytotoxic evaluation of novel indenoisoquinoline-substituted triazole hybrids.

The synthesis of various substituted triazole-indenoisoquinoline hybrids was performed based on a CuI-catalyzed 1,3-cycloaddition between propargyl-substituted derivatives and the azide-containing indenoisoquinoline. Besides, a variety of N-(alkyl)propargylindenoisoquinolines was used as substrates for the construction of triazole-indenoisoquinoline-AZT conjugated via a click chemistry-mediated coupling with 3'-azido-3'-deoxythymidine (AZT). Thus, twenty three new indenoisoquinoline-substituted triazole hybrids were successfully prepared and evaluated as cytotoxic agents, revealing an interesting anticancer activity of four triazole linker-indenoisoquinoline-AZT hybrids in KB and HepG2 cancer cell lines.

Engineering a thermostable Halothermothrix orenii ß-glucosidase for improved galacto-oligosaccharide synthesis.

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining ß-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a ß-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k cat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k cat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using ß-galactosidase from Streptococcus thermophilus.

ß-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000 U of ß-galactosidase activity per liter of culture corresponding to approximately 170 mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205 g L-1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

Heterologous expression of a recombinant lactobacillal ß-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

BACKGROUND: Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric ß-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri ß-galactosidase. RESULTS: Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum ß-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), ß-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant ß-galactosidase. CONCLUSIONS: The over-expression of recombinant L. reuteri ß-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

Biochemical and structural characterization of a thermostable ß-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial ß-glucosidases (BGL) with significant ß-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific ß-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal ß-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are ß-D-Galp-(1→6)-D-Lac (6GALA) and ß-D-Galp-(1→3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

Fractionation of a galacto-oligosaccharides solution at low and high temperature using nanofiltration.

Like in many applications, solutions of high sugar content can cause serious problems due to microorganism contaminations. Hence, the main aim of this work was to study a nanofiltration process for GOS purification at 5 °C and 60 °C that may circumvent or reduce potential microbial growth. Process performances and rejection behaviors of monosaccharide as well as individual GOS components were compared. Operating at 5 °C is more advantageous especially with respect to the oligosaccharide (OS) recovery yield. Using a NF membrane (NP030) at 45 bar, a product purity of 85% (based on monosaccharide content) and an OS recovery yield of 82% could be achieved. However, a low average permeate flux of 3 L/m2 h had to be accepted. A diafiltration step improved product purity to 90% with 30% losses of OS. A qualitative theoretical discussion shows that a possible change of the pore radius distribution depending on temperature could play a role in solute rejection as well as selectivity.