Microbial β-Glucosidases: screening, characterization, cloning and applications

Cellulose is the most abundant biomaterial in the biosphere and the major component of plant biomass. Cellulase is an enzymatic system required for conversion of renewable cellulose biomass into free sugar for subsequent use in different applications. Cellulase system mainly consists of three individual enzymes namely: endoglucanase, exoglucanase and β-glucosidases. β-Glucosidases are ubiquitous enzymes found in all living organisms with great biological significance. β-Glucosidases have also tremendous biotechnological applications such as biofuel production, beverage industry, food industry, cassava detoxification and oligosaccharides synthesis. Microbial β-glucosidases are preferred for industrial uses because of robust activity and novel properties exhibited by them. This review aims at describing the various biochemical methods used for screening and evaluating β-glucosidases activity from microbial sources. Subsequently, it generally highlights techniques used for purification of β-glucosidases. It then elaborates various biochemical and molecular properties of this valuable enzyme such as pH and temperature optima, glucose tolerance, substrate specificity, molecular weight, and multiplicity. Furthermore, it describes molecular cloning and expression of bacterial, fungal and metagenomic β-glucosidases. Finally, it highlights the potential biotechnological applications of β-glucosidases

Effects of detergents on activity, thermostability and aggregation of immobilized lipases

Predmetom tejto diplomovej práce bolo štúdium vplyvu detergentov na aktivitu, termostabilitu a agregáciu voľnej a imobilizovanej formy komerčného preparátu lipázy izolovanej z mikroskopickej huby Rhizopus arrhizus. Teoretická časť obsahuje ucelenú rešerš popisujúcu štruktúru, mechanizmus účinku a priemyselný význam spomínanej hydrolázy spolu s popisom chemických účinkov detergentov, pričom dôraz bol kladený predovšetkým na skupinu neionogénnych detergentov s názvom tweeny. V experimentálnej časti bol študovaný efekt tweenov na rozpustnej a imobilizovanej forme RA lipázy. Imobilizácia spočívala v priamej adsorpcii enzýmu na neupravený nosič. Ako nosič bol použitý oxidovaný grafén ošetrený tweenom (tween 20, 60, 80). Aktivita enzýmu bola stanovená spektrofotometricky za pomoci substrátu p-nitrofenyl laurátu. Zvýšenie aktivity voľnej lipázy (104 % oproti maximálnej hodnote) bolo zaznamenané pri použití tweenu 20 o koncentrácii vysoko nad hodnotou kritickej micelárnej koncentrácie (10 mmol•dm-3). Na základe štúdie imobilizačných podmienok, boli nastavené ideálne parametre pre dosiahnutie účinnej imobilizácie v spojení s čo najvyššou lipolytickou aktivitou (koncentrácia enzýmu 0,1 mg•ml-1, fosfátový tlmivý roztok pH 7,2, koncentrácia tweenu 10,8 mmol•dm-3, čas imobilizácie 1 hodina). Obe formy lipázy vykazovali maximálnu aktivitu pri 35 °C. Optimálne pH sa u imobilizovanej lipázy posunulo na hodnotu 8, v porovnaní s voľnou formou, ktorej pH optimum bolo stanovené na 9. Tepelná stabilita vykazovala približne rovnaký priebeh u oboch foriem skúmanej hydrolázy. Avšak v prípade štúdia stability enzýmu pri dlhodobej úschove bolo po imobilizácii zistené výrazné zlepšenie tohto parametru.The diploma thesis deals with the issue of the effect of tweens on enzymatic activity of model hydrolase both free and immobilized on carbon-based carrier. In theoretical part, structural features, mechanism of action, and specialty applications of microbial lipases are reviewed along with detergent chemistry, with emphasize on tween family of detergents belonging into non-ionic surfactant group. In experimental part, effect of tweens on soluble as well as immobilized hydrolase was examined. Immobilization of commercial preparation of lipase was performed by non-covalent adsorption on graphene oxide as a carrier treated with different tweens (tween 20, 60, 80). The activity was determined spectrophotometrically by p-nitrophenyl laurate assay. Enhancement of soluble Rhizopus arrhizus lipase activity (activity coupling of 104 %) was observed at tween 20 concentration of 10 mmol•dm-3, which is highly above critical micelle concentration of this detergent. On the base of screening study, immobilization protocol comprised the incubation of soluble enzyme at concentration of 0.1 mg•ml-1 in phosphate buffer (pH 7.2) with tween 20 (10.8 mmol•dm-3) and the carrier for one hour. Both soluble and immobilized lipase exhibited maximum activity at approx. 35 °C. Optimal pH of immobilized lipase shifted to 8 compared to soluble form for which pH optimum at 9 was determined. Thermal stability profile follows almost same trend for both soluble and immobilized enzyme samples. The interactions between carrier and enzyme are suggested to be mainly non–covalent (adsorption, electrostatic interactions). No protein leaching was observed under studied conditions, and significant improvement of storage stability of immobilized lipase was achieved (activity retention of 41 % after 110 days) in comparison with soluble lipase (activity retention of 16 % after 42 days).

Liquid chromatographic strategies for separation of bioactive compounds in food matrices

Nowadays, there is an increasing attention for nutraceuticals and, in general, bioactive compounds naturally present in food. Indeed, the possibility of preserving human health and preventing disease (e.g., cardiovascular diseases, cancer etc.) by the intake of healthy food is attractive for both consumers and food industries. In turn, research in this field was also prompted significantly, with the aim of characterizing these bioactive compounds and ascribe to them a specific activity. The bioactive compounds can belong to several chemical classes. However, their chemical diversity and presence in complex matrices, such as food, make it challenging both their isolation and characterization. To tackle this issue, efficient separation systems are needed, which are mainly based on chromatography. In this context, this mini-review aims to provide the reader with an overview of the most relevant and recent approaches for the separation of the most common bioactive compounds in food, in particular polyphenols, phenols, carotenoids, and peptides, by liquid chromatography approaches. © 2018 by the authors

Biodiesel via in situ wet microalgae biotransformation: Zwitter-type ionic liquid supported extraction and transesterification

The production of biodiesel derived from microalgae is among the most forthcoming technologies that provide an ecologic alternative to fossil fuels. Herein, a method was developed that enables the direct extraction and conversion of algal oil to biodiesel without prior isolation. The reaction occurs in aqueous media catalyzed by immobilized Candida antarctica lipase B (Novozyme 435). Zwitter-type ionic liquids were used as cocatalyst to improve the selectivity and reactivity of the enzyme. In a model reaction with sunflower oil, 64% biodiesel was obtained. Applying this method to a slurry of whole-cell Chlorella zof ingiensis in water resulted in 74.8% of lipid extraction, with 27.7% biotransformation products and up to 16% biodiesel. Factors that reduced the lipase activity with whole-cell algae were subsequently probed and discussed. This "in situ" method shows an improvement to existing methods, since it integrates the oil extraction and conversion into an one-pot procedure in aqueous conditions. The extraction is nondisruptive, and is a model for a greener algae to biodiesel process

Synthesis of a novel polyester building block from pentoses by tin-containing silicates

C5-Sugars form a new bio-monomer (trans-2,5-dihydroxy-3-pentenoic acid methyl ester), which can undergo enzymatic copolymerization with E6-HH to form multifunctional polymers.</p

Development of new antibiotics against Bacillus anthracis

Tese de doutoramento, Química (Química Orgânica), Universidade de Lisboa, Faculdade de Ciências, 2018The rise of bacterial resistance has driven the search for new antimicrobials employing not only innovative mechanisms of action, but also new characteristics such as high selectivity or multitargeting capabilities, whilst the search for new biological targets continues. Past work demonstrated the potential of carbohydrate chemistry, with a new family of surface-active deoxy sugars showing promising antimicrobial activity and surprising selectivity towards the Bacillus genus. These characteristics, and the absence of correlation between MIC and CMC values for the most active compound, motivated the creation of a small library of compounds with the optimal structural features for these alkyl deoxy glycosides. Simultaneously, the referred antimicrobial and surfactant characteristics showed remarkable differences when compared to the literature of similar alkyl glycosides, hence the study of a possible mechanism of action, independent of the expected surfactant effect was undertaken. The synthesis of antimicrobial derivatives was target oriented, and the methodology applied successfully provided the desired analogs in good yields. The evaluation of antimicrobial activity allowed to establish the essential structural features of this alkyl deoxy glycoside family, leading to dodecyl 2,6-dideoxy-α-L-arabino-hexopyranoside (1) as the ideal compound for the biological studies. This compound showed to be ineffective in the disinfection of surfaces and lack sporicidal activity, however, bacterial mutants resistant to the main families of antibiotics revealed unaltered susceptibility (MIC=16 μg/mL), and no resistance development was observed after continuous exposure. The study of the mode of action was initiated by kinetic characterization of the bactericidal action, followed by potential target assessment by measuring the differential metabolic response when exposed to compound 1, defining the main metabolic pathways affected. By establishing a hierarchical relationship model between them, the most probable targets were identified: ABC and PTS transport systems and the amino acyl t-RNA biosynthesis. The creation of mutant libraries helped to understand that the existence of a specific molecular target is very unlikely, since no significant alteration of the susceptibility of all mutant libraries to compound 1 was observed. Hence, the importance of the bacterial ultrastructures to the antibacterial activity was assessed and the susceptibility of protoplasts and spheroplasts revealed that, for Gram-negative bacteria, the ultrastructures enveloping the cytoplasmic membrane are responsible for the resistance to this compound, while in Gram-positive bacteria they have no effect. This also means that the specificity observed to Bacillus spp., here exemplified S. aureus, results from the characteristics of the bacterial membrane. The impact of compound 1 in the cellular envelope was achieved using AFM techniques, unequivocally demonstrating that these compounds cause alterations of cell surface morphology and cellular damage, even at sub inhibitory concentrations. Additionally, an opportunity emerged to apply this expertise in carbohydrate synthesis to develop new tools for early Alzheimer’s disease diagnosis and possible therapeutics, aiming to improve solubility and bioavailability of known bioactive molecules while providing additional selectivity. A simple, low cost synthetic strategy was paramount to the challenge proposed: the glucoconjugation of fluorescein, quinolone, curcumin and cyclohexylpiperazine derivatives. The methodologies chosen resulted in the successful glucosilation, particularly for the latter. In summary, the multidisciplinary work here reported denotes the capabilities and importance of carbohydrate chemistry for all life sciences. Allowed to successfully establish a correlation between the structure and the bioactivity of alkyl deoxy glycosides, fully characterized the exhibited bactericidal action and provided essential information regarding the peculiarities of the mechanism of action, to be used as guidelines for future work. Moreover, it demonstrated potential in assisting the development of new tools against AD.Fundação para a Ciência e a Tecnologia (FCT); Companhia Industrial Produtora de Antibióticos (CIPAN

Carbohydrate-Based Amphiphiles: Resource for Bio-based Surfactants

International audienc