High cell density sequential batch fermentation for enhanced propionic acid production from glucose and glycerol/glucose mixture using Acidipropionibacterium acidipropionici.

BACKGROUND: Propionic acid fermentation from renewable feedstock suffers from low volumetric productivity and final product concentration, which limits the industrial feasibility of the microbial route. High cell density fermentation techniques overcome these limitations. Here, propionic acid (PA) production from glucose and a crude glycerol/glucose mixture was evaluated using Acidipropionibacterium acidipropionici, in high cell density (HCD) batch fermentations with cell recycle. The agro-industrial by-product, heat-treated potato juice, was used as N-source. RESULTS: Using 40 g/L glucose for nine consecutive batches yielded an average of 18.76 ± 1.34 g/L of PA per batch (0.59 gPA/gGlu) at a maximum rate of 1.15 gPA/L.h, and a maximum biomass of 39.89 gCDW/L. Succinic acid (SA) and acetic acid (AA) were obtained as major by-products and the mass ratio of PA:SA:AA was 100:23:25. When a crude glycerol/glucose mixture (60 g/L:30 g/L) was used for 6 consecutive batches with cell recycle, an average of 35.36 ± 2.17 g/L of PA was obtained per batch (0.51 gPA/gC-source) at a maximum rate of 0.35 g/L.h, and reaching a maximum biomass concentration of 12.66 gCDW/L. The PA:SA:AA mass ratio was 100:29:3. Further addition of 0.75 mg/L biotin as a supplement to the culture medium enhanced the cell growth reaching 21.89 gCDW/L, and PA productivity to 0.48 g/L.h, but also doubled AA concentration. CONCLUSION: This is the highest reported productivity from glycerol/glucose co-fermentation where majority of the culture medium components comprised industrial by-products (crude glycerol and HTPJ). HCD batch fermentations with cell recycling are promising approaches towards industrialization of the bioprocess.

Membrane-based continuous fermentation with cell recycling for propionic acid production from glycerol by Acidipropionibacterium acidipropionici.

BACKGROUND: Microbial production of propionic acid (PA) from renewable resources is limited by the slow growth of the producer bacteria and product-mediated inhibition. The present study evaluates high cell density continuous PA fermentation from glycerol (Gly) using Acidipropionibacterium acidipropionici DSM 4900 in a membrane-based cell recycling system. A ceramic tubular membrane filter of 0.22 µm pore size was used as the filtering device for cell recycling. The continuous fermentations were run sequentially at dilution rates of 0.05 and 0.025 1/h using varying glycerol concentrations and two different yeast extract concentrations. RESULTS: PA volumetric productivity of 0.98 g/L.h with a product yield of 0.38 gPA/gGly was obtained with 51.40 g/L glycerol at a yeast extract concentration of 10 g/L. Increasing the glycerol and yeast extract concentrations to 64.50 g/L and 20 g/L, respectively, increased in PA productivity, product yield, and concentration to 1.82 g/L.h, 0.79 gPA/gGly, and 38.37 g/L, respectively. However, lowering the dilution rate to 0.025 1/h reduced the production efficiency. The cell density increased from 5.80 to 91.83 gCDW/L throughout the operation, which lasted for a period of 5 months. A tolerant variant of A. acidipropoinici exhibiting growth at a PA concentration of 20 g/L was isolated at the end of the experiment. CONCLUSIONS: Applying the current approach for PA fermentation can overcome several limitations for process industrialization.

Carboligation of 5-(hydroxymethyl)furfural via whole-cell catalysis to form C12 furan derivatives and their use for hydrazone formation.

BACKGROUND: Biobased 5-(hydroxymethyl)furfural (5-HMF) is an important platform that offers numerous possibilities for upgrading to a range of chemical, material and fuel products. One reaction of special interest is the carboligation of 5-HMF into C12 compounds, including 5,5'-bis(hydroxymethyl)furoin (DHMF) and its subsequent oxidation to 5,5'-bis(hydroxymethyl)furil (BHMF), due to their potential applications as building blocks for polymers and hydrocarbon fuels. OBJECTIVES: This study was aimed at evaluating the use of whole cells of Escherichia coli carrying recombinant Pseudomonas fluorescens benzaldehyde lyase as biocatalysts for 5-HMF carboligation, recovery of the C12 derivatives DHMF and BHMF, and testing the reactivity of the carbonyl groups for hydrazone formation for potential use as cross-linking agents in surface coatings. The effects of different parameters on the reaction were investigated to find the conditions for achieving high product yield and productivity. RESULTS: The reaction with 5 g/L 5-HMF using 2 gCDW/L recombinant cells in 10% dimethyl carbonate, pH 8.0 at 30 °C resulted in DHMF yield of 81.7% (0.41 mol/mol) at 1 h, and BHMF yield of 96.7% (0.49 mol/mol) at 72 h reaction time. Fed-batch biotransformation generated a maximum DHMF concentration of 53.0 g/L (or 26.5 g DHMF/g cell catalyst) with productivity of 10.6 g/L.h, after five feeds of 20 g/L 5-HMF. Both DHMF and BHMF reacted with adipic acid dihydrazide to form hydrazone that was confirmed by Fourier-transform infrared spectroscopy and 1H NMR. CONCLUSION: The study demonstrates the potential application of recombinant E. coli cells for cost-effective production of commercially relevant products.

Usage Pattern of Fixed-dose Combinations at ICMR Network of Rational Use of Medicine Centres across India: Recommendations for Policymakers and Prescribers.

AIM: Irrational use of medicines is a global problem. In India, one contributing factor is the availability of a large number of fixed-dose combinations (FDCs). To improve rational use and to strengthen policies, it is important to assess the usage patterns and rationality of FDCs. METHODS: This study was conducted as part of a 1-year prospective cross-sectional analysis of prescriptions in the outpatient clinics of broad specialities from 13 tertiary care hospitals across India. Five most commonly prescribed FDCs in each center were analyzed. In addition, all the prescribed FDCs were classified as per the Kokate Committee classification and it was noted whether any of the FDCs were irrational or banned as per the reference lists released by regulatory authorities. RESULTS: A total of 4,838 prescriptions were analyzed. Of these, 2,093 (43.3%) prescriptions had at least one FDC. These 2,093 prescriptions had 366 different FDCs. Of the 366 FDCs, 241 were rational; 10 were irrational; 14 required further data generation; and the remaining 96 FDCs could not be categorized into any of the above. Vitamins and minerals/supplements, antibacterial for systemic use, and drugs for gastroesophageal reflux disease (GERD) and peptic ulcer were the most used FDCs. CONCLUSION: Based on the finding that some prescriptions contained irrational FDCs, it is recommended that a rigorous, regular, and uniform method of evaluation be implemented to approve/ban FDCs and that prescribers be periodically notified about the status of the bans.

A facile process for adipic acid production in high yield by oxidation of 1,6-hexanediol using the resting cells of Gluconobacter oxydans.

BACKGROUND: Adipic acid (AA) is one of the most important industrial chemicals used mainly for the production of Nylon 6,6 but also for making polyurethanes, plasticizers, and unsaturated polyester resins, and more recently as a component in the biodegradable polyester poly(butylene adipate terephthalate) (PBAT). The main route for AA production utilizes benzene as feedstock and generates copious amounts of the greenhouse gas NO2. Hence, alternative clean production routes for AA from renewable bio-based feedstock are drawing increasing attention. We have earlier reported the potential of Gluconobacter oxydans cells to oxidize 1,6-hexanediol, a potentially biobased diol to AA. RESULTS: The present report involves a study on the effect of different parameters on the microbial transformation of 1,6-hexanediol to adipic acid, and subsequently testing the process on a larger lab scale for achieving maximal conversion and yield. Comparison of three wild-type strains of G. oxydans DSM50049, DSM2003, and DSM2343 for the whole-cell biotransformation of 10 g/L 1,6-hexanediol to adipic acid in batch mode at pH 7 and 30 °C led to the selection of G. oxydans DSM50049, which showed 100% conversion of the substrate with over 99% yield of adipic acid in 30 h. An increase in the concentrations of the substrate decreased the degree of conversion, while the product up to 25 g/L in batch and 40 g/L in fed-batch showed no inhibition on the conversion. Moreover, controlling the pH of the reaction at 5-5.5 was required for the cascade oxidation reactions to work. Cell recycling for the biotransformation resulted in a significant decrease in activity during the third cycle. Meanwhile, the fed-batch mode of transformation by intermittent addition of 1,6-hexanediol (30 g in total) in 1 L scale resulted in complete conversion with over 99% yield of adipic acid (approximately 37 g/L). The product was recovered in a pure form using downstream steps without the use of any solvent. CONCLUSION: A facile, efficient microbial process for oxidation of 1,6-hexanediol to adipic acid, having potential for scale up was demonstrated. The entire process is performed in aqueous medium at ambient temperatures with minimal greenhouse gas emissions. The enzymes involved in catalyzing the oxidation steps are currently being identified.

A multicentre point prevalence survey (PPS) of antimicrobial use amongst admitted patients in tertiary care centres in India.

OBJECTIVES: Data from point prevalence surveys (PPSs) in India are scarce. Conducting PPSs is especially challenging in the absence of electronic medical records, a lack of dedicated resources and a high patient load in resource-poor settings. This multicentre survey was conducted to provide background data for planning and strengthening antimicrobial stewardship programmes across the country. METHODS: This inpatient PPS was conducted over 2 weeks in May 2019 simultaneously across five study centres in India. Data about patient characteristics, indications for antimicrobials use and details of each antimicrobial prescribed including supportive investigation reports were collected in predesigned forms. RESULTS: A total of 3473 admitted patients in wards and ICUs were covered across five study centres. Of these, 1747 (50.3%) patients were on antimicrobials, with 46.9% patients being on two or more antimicrobials. Out of the total antimicrobials prescribed, 40.2% of the antimicrobials were prescribed for community-acquired infection requiring hospitalization followed by surgical prophylaxis (32.6%). Third-generation cephalosporins and drugs from the 'Watch' category were prescribed most commonly. Only 22.8% of the antimicrobials were based on microbiology reports. CONCLUSIONS: The survey demonstrated a high use of antimicrobials in admitted patients with a considerable proportion of drugs from the 'Watch' category. The targets for interventions that emerged from the survey were: improving surgical prophylaxis, decreasing double anaerobic cover, initiating culture of sending cultures and de-escalation with targeted therapy.

Safety of hydroxychloroquine in healthcare workers for COVID-19 prophylaxis.

BACKGROUND & OBJECTIVES: Hydroxychloroquine (HCQ), reported to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication in in vitro studies, has been recommended for prophylaxis of COVID-19 in healthcare workers (HCWs). The objective of this study was to assess short-term adverse events (AEs) of HCQ in HCWs. METHODS: This cross-sectional study among consenting HCWs taking prophylaxis and working in hospitals with COVID-19 patients used online forms to collect details of HCWs, comorbidities, prophylactic drugs used and AEs after the first dose of HCQ. Verification of dose and AEs was done by personal contact. Multivariate logistic regression analysis was done to determine the effect of age, gender and dose of HCQ on AE. RESULTS: Of the 1303 HCWs included, 98.4 per cent (n=1282) took HCQ and 66 per cent (n=861) took 800 mg as first day's dose. Among the 19.9 per cent (n=259) reporting AEs, 1.5 per cent (n=20) took treatment for AE, none were hospitalized and three discontinued HCQ. Gastrointestinal AEs were the most common (172, 13.2%), with less in older [odds ratio (OR) 0.56, 95% confidence interval (CI) 0.35-0.89], with more in females (OR 2.46, 95% CI 1.78-3.38) and in those taking a total dose of 800 mg on day one compared to a lower dose. Hypoglycaemia (1.1%, n=14), cardiovascular events (0.7%, n=9) and other AEs were minimal. INTERPRETATION & CONCLUSIONS: HCQ prophylaxis first dose was well tolerated among HCWs as evidenced by a low discontinuation. For adverse effects, a small number required treatment, and none required hospitalization. The study had limitations of convenience sampling and lack of laboratory and electrocardiography confirmation of AEs.

Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes.

Mycobacteriophages possess different sets of lytic enzymes for disruption of the complex cell envelope of the mycobacteria host cells and release of the viral progeny. Lysin B (LysB) enzymes are mycolylarabinogalactan esterases that cleave the ester bond between the arabinogalactan and mycolic acids in the mycolylarabinogalactan-peptidoglycan (mAGP) complex in the cell envelope of mycobacteria. In the present study, four LysB enzymes were produced recombinantly and characterized with respect to their enzymatic and antibacterial activities. Examination of the kinetic parameters for the hydrolysis of para-nitrophenyl ester substrates, shows LysB-His6 enzymes to be active against a range of substrates (C4-C16), with a catalytic preference towards p-nitrophenyl laurate (C12). With p-nitrophenyl butyrate as substrate, LysB-His6 enzymes showed highest activity at 37 °C. LysB-His6 enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca2+ and Mn2+ enhanced the enzymatic activity of LysB-His6 enzymes, while transition metal ions like Zn2+ and Cu2+ inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His6 enzymes against mAGP complex was confirmed by LC-MS. LysB-His6 enzymes showed marginal antibacterial activity when tested alone against Mycobacterium smegmatis, however a synergetic activity was noticed when combined with outer membrane permealizers. These results confirm that LysB enzymes are lipolytic enzymes with potential application as antimycobacterials.

Stress induced biofilm formation in Propionibacterium acidipropionici and use in propionic acid production.

Propionibacterium acidipropionici produces propionic acid from different sugars and glycerol; the production can be improved by high cell density fermentations using immobilized cells that help to overcome the limitations of the non-productive lag phase and product inhibition. In this study, the use of stress factors to induce P. acidipropionici to form biofilm and its use as an immobilization procedure in fermentations in bioreactors for producing propionic acid was investigated. Citric acid and sodium chloride increased exopolysaccharide production, biofilm forming capacity index and trehalose production. Analysis of the expression of trehalose synthesis-related genes otsA and treY by RT-qPCR showed significantly increased expression of only treY during log phase with citric acid, while FISH analysis showed expression of treY and luxS under the influence of both stress factors. The stress factors were then used for development of microbial biofilms as immobilization procedure on Poraver® and AnoxKaldnes® carriers in recycle batch reactors for propionic acid production from 20 g/L glycerol. Highest productivities of 0.7 and 0.78 g/L/h were obtained in Poraver® reactors, and 0.39 and 0.43 g/L/h in AnoxKaldnes® reactors with citric acid and NaCl, respectively.

Immobilization to Positively Charged Cellulose Nanocrystals Enhances the Antibacterial Activity and Stability of Hen Egg White and T4 Lysozyme.

Antibacterial bionanostructures were produced from cellulose nanocrystals (CNC) with immobilized lysozyme from hen egg white (HEW) and T4 bacteriophage, respectively. The nanocrystals were prepared from microcrystalline cellulose by ammonium persulfate oxidation with a yield of 68% and having an average size of 250 nm and low polydispersity index. HEW lysozyme (HEWL) and T4 lysozyme (T4L) were immobilized to CNC by different mechanisms including adsorption and covalent coupling to carbodiimide-activated carboxylate groups and to glutaraldehyde-activated aminated CNC (Am-CNC), respectively. The effect of immobilization on the enzymatic activity (both lytic and hydrolytic) and antibacterial activity of the lysozymes was studied using different methods. Am-CNC-lysozyme conjugates retained the highest lytic activity, 86.3% and 78.3% for HEWL and T4L, respectively. They also showed enhanced bactericidal activity with high potency against Gram-positive as well as Gram-negative bacteria in a relatively shorter time as compared to the free enzymes and resulted in extensive cellular damage, as shown by transmission electron microscopy. The enhanced antibacterial activity was correlated with the increase in zeta potential of Am-CNC-lysozyme conjugates. The immobilized lysozyme preparations further exhibited enhanced storage stability at 4 and 22 °C.

Antimicrobial Protein Candidates from the Thermophilic Geobacillus sp. Strain ZGt-1: Production, Proteomics, and Bioinformatics Analysis.

A thermophilic bacterial strain, Geobacillus sp. ZGt-1, isolated from Zara hot spring in Jordan, was capable of inhibiting the growth of the thermophilic G. stearothermophilus and the mesophilic Bacillus subtilis and Salmonella typhimurium on a solid cultivation medium. Antibacterial activity was not observed when ZGt-1 was cultivated in a liquid medium; however, immobilization of the cells in agar beads that were subjected to sequential batch cultivation in the liquid medium at 60 °C showed increasing antibacterial activity up to 14 cycles. The antibacterial activity was lost on protease treatment of the culture supernatant. Concentration of the protein fraction by ammonium sulphate precipitation followed by denaturing polyacrylamide gel electrophoresis separation and analysis of the gel for antibacterial activity against G. stearothermophilus showed a distinct inhibition zone in 15-20 kDa range, suggesting that the active molecule(s) are resistant to denaturation by SDS. Mass spectrometric analysis of the protein bands around the active region resulted in identification of 22 proteins with molecular weight in the range of interest, three of which were new and are here proposed as potential antimicrobial protein candidates by in silico analysis of their amino acid sequences. Mass spectrometric analysis also indicated the presence of partial sequences of antimicrobial enzymes, amidase, and dd-carboxypeptidase.

Bio-based 3-hydroxypropionic- and acrylic acid production from biodiesel glycerol via integrated microbial and chemical catalysis.

BACKGROUND: 3-Hydroxypropionic acid (3HP) and acrylic acid (AA) are industrially important platform- and secondary chemical, respectively. Their production from renewable resources by environment-friendly processes is desirable. In the present study, both chemicals were almost quantitatively produced from biodiesel-derived glycerol by an integrated process involving microbial and chemical catalysis. RESULTS: Glycerol was initially converted in a fed-batch mode of operation to equimolar quantities of 3HP and 1,3-propanediol (1,3PDO) under anaerobic conditions using resting cells of Lactobacillus reuteri as a biocatalyst. The feeding rate of glycerol was controlled at 62.5 mg/g(CDW).h which is half the maximum metabolic flux of glycerol to 3HP and 1,3PDO through the L. reuteri propanediol-utilization (pdu) pathway to prevent accumulation of the inhibitory intermediate, 3-hydroxypronionaldehyde (3HPA). Subsequently, the cell-free supernatant containing the mixture of 3HP and 1,3PDO was subjected to selective oxidation under aerobic conditions using resting cells of Gluconobacter oxydans where 1,3PDO was quantitatively converted to 3HP in a batch system. The optimum conditions for the bioconversion were 10 g/L substrate and 5.2 g/L cell dry weight. Higher substrate concentrations led to enzyme inhibition and incomplete conversion. The resulting solution of 3HP was dehydrated to AA over titanium dioxide (TiO2) at 230 °C with a yield of >95%. CONCLUSIONS: The present study represents the first report on an integrated process for production of acrylic acid at high purity and -yield from glycerol through 3HP as intermediate without any purification step. The proposed process could have potential for industrial production of 3HP and AA after further optimization. Graphical abstract Integrated three-step process for conversion of biodiesel glycerol to 3-hydroxypropionic acid (3HP) and acrylic acid (AA). Glycerol was initially converted to equimolar quantities of 3HP and 1,3-propanediol (1,3PDO) using resting cells of Lactobacillus reuteri. Subsequently, the cell-free supernatant containing the mixture of 3HP and 1,3PDO was subjected to selective oxidation using resting cells of Gluconobacter oxydans where 1,3PDO was quantitatively converted to 3HP. The resulting solution of 3HP was dehydrated to AA over titanium dioxide (TiO2) at 230 °C.

Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol.

BACKGROUND: Lactobacillus reuteri converts glycerol to 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO) via 3-hydroxypropionaldehyde (3HPA) as an intermediate using enzymes encoded in its propanediol-utilization (pdu) operon. Since 3HP, 1,3PDO and 3HPA are important building blocks for the bio-based chemical industry, L. reuteri can be an attractive candidate for their production. However, little is known about the kinetics of glycerol utilization in the Pdu pathway in L. reuteri. In this study, the metabolic fluxes through the Pdu pathway were determined as a first step towards optimizing the production of 3HPA, and co-production of 3HP and 1,3PDO from glycerol. Resting cells of wild-type (DSM 20016) and recombinant (RPRB3007, with overexpressed pdu operon) strains were used as biocatalysts. RESULTS: The conversion rate of glycerol to 3HPA by the resting cells of L. reuteri was evaluated by in situ complexation of the aldehyde with carbohydrazide to avoid the aldehyde-mediated inactivation of glycerol dehydratase. Under operational conditions, the specific 3HPA production rate of the RPRB3007 strain was 1.9 times higher than that of the wild-type strain (1718.2 versus 889.0 mg/gCDW.h, respectively). Flux analysis of glycerol conversion to 1,3PDO and 3HP in the cells using multi-step variable-volume fed-batch operation showed that the maximum specific production rates of 3HP and 1,3PDO were 110.8 and 93.7 mg/gCDW.h, respectively, for the wild-type strain, and 179.2 and 151.4 mg/gCDW.h, respectively, for the RPRB3007 strain. The cumulative molar yield of the two compounds was ~1 mol/mol glycerol and their molar ratio was ~1 mol3HP/mol1,3PDO. A balance of redox equivalents between the glycerol oxidative and reductive pathway branches led to equimolar amounts of the two products. CONCLUSIONS: Metabolic flux analysis was a useful approach for finding conditions for maximal conversion of glycerol to 3HPA, 3HP and 1,3PDO. Improved specific production rates were obtained with resting cells of the engineered RPRB3007 strain, highlighting the potential of metabolic engineering to render an industrially sound strain. This is the first report on the production of 3HP and 1,3PDO as sole products using the wild-type or mutant L. reuteri strains, and has laid ground for further work on improving the productivity of the biotransformation process using resting cells.

Interaction of iron deficiency anemia and hemoglobinopathies among college students and pregnant women: a multi center evaluation in India.

Although iron deficiency anemia is very common in India, systematic large studies on the prevalence and hematological consequences of iron deficiency among carriers of ß-thalassemia (ß-thal) and other hemoglobinopathies are lacking. A multi center project was undertaken to screen college/university students and pregnant women for iron deficiency anemia and various hemoglobinopathies. Fifty-six thousand, seven hundred and seventy-two subjects from six states, Maharashtra, Gujarat, Karnataka, West Bengal, Assam and Punjab, were studied. Iron deficiency anemia was evaluated by measuring zinc protoporphyrin (ZPP) and hemoglobin (Hb) levels, while ß-thal and other hemoglobinopathies were detected by measuring the red cell indices and by Hb analysis using high performance liquid chromatography (HPLC). College boys (2.2%), college girls (14.3%) and antenatal women (27.0%) without any hemoglobinopathies had iron deficiency anemia. Among the ß-thal carriers, the prevalence of iron deficiency anemia was 17.3% in college boys, 38.1% in college girls and 55.9% in pregnant women, while in the Hb E [ß26(B8)Glu→Lys; HBB: c.79G>A] carriers, it was 7.3% in college boys, 25.4% in college girls and 78.0% in antenatal women. In individuals with Hb E disease, the prevalence of iron deficiency anemia varied from 31.2-77.3% in the three groups. A significant reduction in Hb levels was seen when iron deficiency anemia was associated with hemoglobinopathies. However, the Hb A2 levels in ß-thal carriers were not greatly reduced in the presence of iron deficiency anemia.

Amino acid oxidation of Candida antarctica lipase B studied by molecular dynamics simulations and site-directed mutagenesis.

Molecular dynamics simulations have been performed on lipase B from Candida antarctica (CalB) in its native form and with one or two oxidized residues, either methionine oxidized to methionine sulfoxide, tryptophan oxidized to 5-hydroxytryptophan, or cystine oxidized to a pair of cysteic acid residues. We have analyzed how these oxidations affect the general structure of the protein as well as the local structure around the oxidized amino acid and the active site. The results indicate that the methionine and tryptophan oxidations led to rather restricted changes in the structure, whereas the oxidation of cystines, which also caused cleavage of the cystine S-S linkage, gave rise to larger changes in the protein structure. Only two oxidized residues caused significant changes in the structure of the active site, viz., those of the Cys-22/64 and Cys-216/258 pairs. Site-directed mutagenesis studies were also performed. Two variants showed a behavior similar to that of native CalB (M83I and M129L), whereas W155Q and M72S had severely decreased specific activity. M83I had a slightly higher thermostability than native CalB. No significant increase in stability toward hydrogen peroxide was observed. The same mutants were also studied by molecular dynamics. Even though no significant increase in stability toward hydrogen peroxide was observed, the results from simulations and site-directed mutagenesis give some clues about the direction of further work on stabilization.

Improved production of 3-hydroxypropionaldehyde by complex formation with bisulfite during biotransformation of glycerol.

3-Hydroxypropionaldehyde (3HPA) is an important specialty chemical which can be produced from glycerol using resting cells of Lactobacillus reuteri. This biocatalytic route, however, suffers from substrate- and product-mediated loss of enzyme activity within 2 h of biotransformation. In order to overcome the inhibitory effects of 3HPA, complex formation with sodium bisulfite was investigated, optimized and applied for in situ capture of the aldehyde during biotransformation of glycerol in a fed-batch process. As a result, the activity of the cells was maintained for at least 18 h. The 3HPA produced per gram cell dry weight was increased 5.7 times compared to the batch production process, and 2.2 times compared to fed-batch process without in situ complex formation. This approach may have potential for production and in situ removal of 3HPA after further process development.

Ectoine-mediated protection of enzyme from the effect of pH and temperature stress: a study using Bacillus halodurans xylanase as a model.

Compatible solutes are small, soluble organic compounds that have the ability to stabilise proteins against various stress conditions. In this study, the protective effect of ectoines against pH stress is examined using a recombinant xylanase from Bacillus halodurans as a model. Ectoines improved the enzyme stability at low (4.5 and 5.0) and high pH (11 and 12); stabilisation effect of hydroxyectoine was superior to that of ectoine and trehalose. In the presence of hydroxyectoine, residual activity (after 10 h heating at 50 °C) increased from about 45 to 86 % at pH 5 and from 33 to 89 % at pH 12. When the xylanase was incubated at 65 °C for 5 h with 50 mM hydroxyectoine at pH 10, about 40 % of the original activity was retained while no residual activity was detected in the absence of additives or in the presence of ectoine or trehalose. The xylanase activity was slightly stimulated in the presence of 25 mM ectoines and then gradually decreased with increase in ectoines concentration. The thermal unfolding of the enzyme in the presence of the compatible solutes showed a modest increase in denaturation temperature but a larger increase in calorimetric enthalpy.

Inbreeding as a cause for deafness: Dadhkai study.

BACKGROUND: We report on the higher prevalence of deaf-mutes from a village in Jammu and Kashmir State of India. MATERIALS AND METHODS: A cross-sectional study among 79 deaf mutes using pedigree analysis, audiometry, imaging and molecular analysis. RESULTS: A high rate of hereditary deafness with 79 individuals diagnosed to be suffering from non-syndrome deafness in a total population of 2452 individuals residing in the village. INTERPRETATION: Flourishing of intermarriages led to a population with high prevalence of deafness.