Biochemical characterization of mutants in the active site residues of the beta-galactosidase enzyme of Bacillus circulans ATCC 31382

The Bacillus circulans ATCC 31382 beta-galactosidase (BgaD) is a retaining-type glycosidase of glycoside hydrolase family 2 (GH2). Its commercial enzyme preparation, Biolacta N5, is used for commercial-scale production of galacto-oligosaccharides (GOS). The BgaD active site and catalytic amino acid residues have not been studied. Using bioinformatic routines we identified two putative catalytic glutamates and two highly conserved active site histidines. The site-directed mutants E447N, E532Q, and H345F, H379F had lost (almost) all catalytic activity. This confirmed their essential role in catalysis, as general acid/base catalyst (E447) and nucleophile (E532), and as transition state stabilizers (H345, H379), respectively. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.</p

Growth of the salt-tolerant yeast Zygosaccharomyces rouxii in microtiter plates:effects of NaCl, pH and temperature on growth and fusel alcohol production from branched-chain amino acids

Zygosaccharomyces rouxii, a salt-tolerant yeast isolated from the soy sauce process, produces fusel alcohols (isoamyl alcohol, active amyl alcohol and isobutyl alcohol) from branched-chain amino acids (leucine, isoleucine and valine, respectively) via the Ehrlich pathway. Using a high-throughput screening approach in microtiter plates, we have studied the effects of pH, temperature and salt concentration on growth of Z. rouxii and formation of fusel alcohols from branched-chain amino acids. Application of minor variations in pH (range 3-7) and NaCl concentrations (range 0-20%) per microtiter plate well allowed a rapid and detailed evaluation of fermentation conditions for optimal growth and metabolite production. Conditions yielding the highest cell densities were not optimal for fusel alcohol production. Maximal fusel alcohol production occurred at low pH (3.0-4.0) and low NaCl concentrations (0-4%) at 25°C. At pH 4.0-6.0 and 0-18% NaCl, considerable amounts of α-keto acids, the deaminated products from the branched-chain amino acids, accumulated extracellularly. The highest cell densities were obtained in plates incubated at 30°C. The results obtained under various incubation conditions with (deep-well) microtiter plates were validated in Erlenmeyer shake-flask cultures.

Potential Dental Biofilm Inhibitors: Dynamic Combinatorial Chemistry Affords Sugar-Based Molecules that Target Bacterial Glucosyltransferase

We applied dynamic combinatorial chemistry (DCC) to find novel ligands of the bacterial virulence factor glucosyltransferase (GTF) 180. GTFs are the major producers of extracellular polysaccharides, which are important factors in the initiation and development of cariogenic dental biofilms. Following a structure-based strategy, we designed a series of 36 glucose- and maltose-based acylhydrazones as substrate mimics. Synthesis of the required mono- and disaccharide-based aldehydes set the stage for DCC experiments. Analysis of the dynamic combinatorial libraries (DCLs) by UPLC-MS revealed major amplification of four compounds in the presence of GTF180. Moreover, we found that derivatives of the glucose-acceptor maltose at the C1-hydroxy group act as glucose-donors and are cleaved by GTF180. The synthesized hits display medium to low binding affinity (KD values of 0.4–10.0 mm) according to surface plasmon resonance. In addition, they were investigated for inhibitory activity in GTF-activity assays. The early-stage DCC study reveals that careful design of DCLs opens up easy access to a broad class of novel compounds that can be developed further as potential inhibitors

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates

Patient satisfaction in treatment of non-complex fractures and dislocations in general practice in the Netherlands:prospective cohort study protocol

INTRODUCTION: Diagnosis and treatment of fractures and dislocations are mostly performed in hospital settings. However, equal care for patients with non--complex fractures or dislocations ('minor trauma care') may be provided in general practice. While substitution of care from secondary to primary care settings is stimulated by governments and insurers, it is unknown what the effects are on patient satisfaction level. Therefore, our primary objective is to determine the effect of minor trauma care delivered in a general practice as compared with a hospital on patient satisfaction. Secondary objectives are to assess the effects on treatment outcomes, cost-effectiveness and time consumption. METHODS AND ANALYSIS: In a prospective cohort study, we will include 200 patients aged 12 and over with an X-ray confirmed diagnosis of a non--complex fracture or dislocation out of whom 100 treated in a general practice and 100 in a secondary care hospital, both located in the Netherlands. All treatment procedures and follow-up will be done in accordance to the hospital's standards of trauma care. Study assessments will be performed pre-treatment, and 1, 6 and 12 weeks after treatment. Data collected include demographics, patient satisfaction and patient-reported outcomes including physical functioning, complications, pain scores and treatment-related costs. The primary outcome patient satisfaction measured at 12 weeks will be compared between the settings and additionally multivariable regression will be performed to assess potential confounding effects of unbalanced prognostic factors. Treatment outcomes and time consumption will be analysed following the same approach while cost-effectiveness will be assessed using an incremental cost-effectiveness ratio. Subsequently, results will be discussed using focus groups consisting of patients (n=15) and healthcare providers. ETHICS AND DISSEMINATION: The Medical Ethics Committee from the University Medical Center Groningen reviewed this study protocol and granted exemption from ethical approval (METc UMCG 2017/277). Study results will be presented at (inter)national conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03506958; Pre-results

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus

Alginate (alginic acid) is a linear polysaccharide, wherein (1->4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1->4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.EU FP7BBIBlueBio Cofund consortiu

A phase 1b/2b multicenter study of oral panobinostat plus azacitidine in adults with MDS, CMML or AML with less than or equal to 30% blasts

Treatment with azacitidine (AZA), a demethylating agent, prolonged overall survival (OS) vs conventional care in patients with higher-risk myelodysplastic syndromes (MDS). As median survival with monotherapy is <2 years, novel agents are needed to improve outcomes. This phase 1b/2b trial (n=113) was designed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of panobinostat (PAN)+AZA (phase 1b) and evaluate the early efficacy and safety of PAN+AZA vs AZA monotherapy (phase 2b) in patients with higher-risk MDS, chronic myelomonocytic leukemia or oligoblastic acute myeloid leukemia with <30% blasts. The MTD was not reached; the RP2D was PAN 30 mg plus AZA 75 mg/m2. More patients receiving PAN+AZA achieved a composite complete response ([CR)+morphologic CR with incomplete blood count+bone marrow CR (27.5% (95% CI, 14.6–43.9%)) vs AZA (14.3% (5.4–28.5%)). However, no significant difference was observed in the 1-year OS rate (PAN+AZA, 60% (50–80%); AZA, 70% (50–80%)) or time to progression (PAN+AZA, 70% (40–90%); AZA, 70% (40–80%)). More grade 3/4 adverse events (97.4 vs 81.0%) and on-treatment deaths (13.2 vs 4.8%) occurred with PAN+AZA. Further dose or schedule optimization may improve the risk/benefit profile of this regimen

Proposed diagnostic criteria for classical CMML, CMML variants and pre-CMML conditions

Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by dysplasia, abnormal production and accumulation of monocytic cells and an elevated risk to transform into acute leukemia. Over the past two decades, our knowledge about the pathogenesis and molecular mechanisms in CMML has increased substantially. In parallel, better diagnostic criteria and therapeutic strategies have been developed. However, many questions remain regarding prognostication and optimal therapy. In addition, there is a need to define potential pre-phases of CMML and special CMML variants, and to separate these entities from each other and from conditions mimicking CMML. To address these unmet needs, an international consensus group met in a Working Conference in August 2018 and discussed open questions and issues around CMML, its variants, and pre-CMML conditions. The outcomes of this meeting are summarized herein and include diagnostic criteria and a proposed classification of pre-CMML conditions as well as refined minimal diagnostic criteria for classical CMML and special CMML variants, including oligomonocytic CMML and CMML associated with systemic mastocytosis. Moreover, we propose diagnostic standards and tools to delineate between normal, pre-CMML and CMML entities. These criteria and standards should facilitate diagnostic and prognostic evaluations in daily practice and clinical studies in applied hematology

The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity

Glucansucrase (mutant) enzymes from Lactobacillus reuteri 180 efficiently transglucosylate Stevia component rebaudioside A, resulting in a superior taste

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(β1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 β-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties