Micro‐ and Nanocarriers for Immobilization of Enzymes

Two types of micro‐ and nanocarriers for immobilization of enzymes for biotechnological and biomedical applications are described: magnetic nanoparticles and cross‐linked enzyme aggregates (CLEAs). Nanosized structures with their large surface and smaller size volume ratio, which is dependent on their strong magnetic dipole, give key features that make magnetic nanoparticles useful in many biotechnological and biomedical applications. They are therefore used as carriers to which different active substances can bind. The preparation of the magnetic nanoparticles, possible surface coating methods, and functionalization with different materials are described. Enzyme immobilization methods, such as adsorption, affinity binding, chelation, or metal binding or covalent binding, enable the preparation of efficient and stable enzyme bound to magnetic nanoparticles. Such a product may be used among bioreactor applications for targeted drug delivery in biosensors or bioimaging and magnetic resonance imaging. Preparation of CLEAs, the microsized enzyme structures without a carrier, is described as well. Their main advantage is very simple preparation, where two steps, precipitation of the enzyme and cross‐linking, are joined. A broad spectrum of enzymes for CLEA preparation has been used and many biotechnological reactions are catalyzed. The improvement in CLEA preparation to enhance their stability and operability is also shown

Enzyme immobilization on surface modified magnetic carriers

Doktorska disertacija zajema dva dela, v prvem delu smo se osredotočili na sintezo magnetnega nosilca, modificiranega z organskim polimerom karboksimetil dekstranom (CMD). V sintezni postopek smo vpeljali tri različne koncentracije CMD (0,25 g/mL, 0,40 g/mL in 0,50 g/mL CMD) ter sintetizirali tri različne modificirane magnetne nanodelce (CMD1-MNPs, CMD2-MNPs in CMD3-MNPs). Sintetizirane CMD-MNPs smo okarakterizirali z različnimi analiznimi metodami: Fourier-transformirano infrardečo spektroskopijo (FT-IR), termogravimetrično analizo (TGA), vrstično elektronsko mikroskopijo (SEM), energijsko disperzijsko spektroskopijo (EDS), transmisijsko elektronsko mikroskopijo (TEM), z meritvami dinamičnega sipanja svetlobe (DLS). Magnetne lastnosti smo določili z elektronsko paramagnetno resonanco (EPR) in vibracijskim magnetometrom (VSM). Uspešno smo sintetizirali magnetne nosilce CMD-MNPs z ozkimi porazdelitvami nanovelikosti od 27-30 nm. S FT-IR analizo smo določili prisotnost karboksilnih in hidroksilnih skupin na površini CMD-MNPs, kar potrjuje prisotnost polimerne prevleke CMD na sintetiziranih MNPs. Z meritvami EPR in VSM smo dokazali, da imajo sintetizirani CMD-MNPs magnetne lastnosti ter feromagnetni sistem. Določili smo inhibitorne lastnosti CMD-MNPs na rast dveh bakterijskih kultur. Inhibitorne učinke na rast testnih mikroorganizmov smo zaznali pri CMD3-MNPs, medtem ko ostali CMD-MNPs in neprevlečeni MNPs ne izkazujejo antimikrobne učinkovitosti. Proučevali smo tudi inhibitorne lastnosti MNPs modificiranih s hitozanom (HIT-MNPs) in aminosilanom (AMS-MNPs) na petih različnih bakterijskih kulturah, pri katerih nismo zaznali inhibiornega učinka na rast izbranih mikororganizmov. Za nadaljnje raziskave smo izbrali CMD3-MNPs, na katerega smo vezali encim alkohol dehidrogenazo (ADH). V drugem delu doktorske disertacije smo nosilec CMD3-MNPs površinsko funkcionalizirali z epiklorohidrinom (EClH). Optimalna koncentracija EClH je znašala 4 % (v/v). Proučevali smo vpliv različnih procesnih parametrov na preostalo aktivnost in učinkovitost imobilizacije ADH na CMD3-MNPs. Pri optimalnih pogojih imobilizacije ADH na CMD3-MNPs smo dosegli 89,6 % preostalo aktivnost imobilizirane ADH in 99,5 % učinkovitost imobilizacije. Nadaljevali smo s postopkom koimobilizacije, pri čemer smo na funkcionaliziran nosilec CMD3-MNPs koimobilizirali encim ADH in kofaktor β-nikotinamid adenin dinukleotid (β-NAD). S spreminjanjem procesnih parametrov smo proučevali njihov vpliv na preostalo aktivnost imobilizirane ADH s kofaktorjem β-NAD na CMD3-MNPs in učinkovitost koimobilizacije. Pri optimalnih pogojih koimobilizacije ADH in β-NAD na CMD3-MNPs smo dosegli 73,3 % preostalo aktivnost ADH ter 93,8 % učinkovitost imobilizacije. Izvedli smo še študijo termične stabilnosti proste ADH, ADH imobilizirane na CMD3-MNPs in ADH koimobilizirane z β-NAD na CMD3-MNPs pri različnih temperaturah. ADH imobilizirana na CMD3-MNPs je ohranila skoraj 60 % svoje začetne aktivnosti po 24. urah inkubacije pri temperaturah 20 °C in 40 °C. ADH koimobilizirana z β-NAD na CMD3-MNPs je pri temperaturi 30 °C ohranila 75,4 % začetne aktivnosti, pri 50 °C pa 66,5 % začetne aktivnosti po 5. urah inkubacije. Proučili smo še stabilnost ADH imobilizirane na CMD3-MNPs in ADH koimobilizirane z β-NAD na CMD3-MNPs, ki smo ju skladiščili pri 4 °C. Po treh tednih sta obe obliki imobilizrane ADH ohranili 60 % svoje začetne aktivnosti. Magnetne nosilce HIT-MNPs in AMS-MNPs smo funkcionalizirali z mrežnim povezovalcem glutaraldehidom (GA) in amino-donorjem pentaetilenheksaminom (PEHA), na katere smo imobilizirali encim β-galaktozidazo (β-GAL) ter optimirali koncentraciji GA in PEHA. Pri kombinaciji obeh GA in PEHA smo dosegli hiperaktivacijo encima (128,9 %), do katere pride zaradi konformacijskih sprememb encima. Hiperaktivacijo smo dosegli tudi pri imobilizaciji β-GAL na AMS-MNPs. Najvišjo preostalo aktivnost β-GAL smo dosegli, kadar smo kot mrežni povezovalec uporabili 20 % (v/v) PEHA (154,4 %).Doctoral thesis consists of two parts, in the first part synthesis of magnetic carrier and modification with organic polymer carboxymethyl dextran (CMD) was performed. Three different CMD concentrations were applied into the synthesis process (0,25 g/mL, 0,40 g/mL in 0,50 g/mL CMD), which resulted in three different modified magnetic carriers (CMD1-MNPs, CMD2-MNPs and CMD3-MNPs). All CMD-MNPs were characterized with various analytical methods: Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDX), transmission electron microscopy (TEM), dynamic light scattering (DLS). Magnetic properties were analyzed with electron paramagnetic resonance (EPR) and magnetization measurements with vibrating sample magnetization (VSM). CMD-MNPs were successfully synthesized with average diameters from 27-30 nm and size distribution revealed most consistent sizes of CMD3-MNPs. Hydroxyl and carboxyl groups were confirmed on the CMD-MNPs surface, which confirms the presence of CMD layer on the surface of CMD-MNPs. EPR and VSM measurements confirmed magnetic properties of all CMD-MNPs and a ferromagnetic system. Inhibition properties were determined of all CMD-MNPs on two different bacterial cultures, where CMD3-MNPs displayed inhibition zone, confirming antimicrobial properties, whereas with other CMD-MNPs no inhibition was detected. Toxicity was also determined on MNPs coated with chitosan (HIT-MNPs) and aminosilane (AMS-MNPs) on five different bacterial cultures, where no inhibition was detected. CMD3-MNPs was chosen for further investigation of enzyme immobilization in the second part of doctoral thesis, because of its most favourable properties. In second part of doctoral thesis nano-carrier CMD3-MNPs was surface functionalized with epoxy cross-linking, using epoxy cross-linker epichlorohydrin (EClH) to covalently bind enzyme alcohol dehydrogenase (ADH). With optimization of process parameters EClH with 4 % (v/v) was used, which resulted in the highest residual activity of immobilized ADH. Epoxy-functionalized CMD3-MNPs were used in immobilization protocol, where effect of process parameters was investigated on the residual activity and immobilization efficiency of ADH. After successful optimization ADH immobilized onto CMD3-MNPs managed to obtain 89,6 % of residual activity and 99,5 % of immobilization efficiency. Further on, co-immobilization of enzyme ADH and cofactor β-nicotinamide adenine dinucleotide (β-NAD) was performed. Again, effect of process parameters on residual activity and immobilization efficiency of co-immobilization were investigated. After successful optimization, ADH co-immobilized with β-NAD onto CMD3-MNPs managed to obtain 73,3 % of residual activity and 93,8 % of immobilization efficiency. Thermal stability of immobilized ADH at different temperatures was investigated. ADH immobilized onto CMD3-MNPs obtained almost 60 % of its initial activity after 24 hours at 20 °C and 40 °C, ADH co-immobilized with β-NAD onto CMD3-MNPs managed to obtain 75,4 % of its initial activity at 30 °C and 66,5 % of its initial activity at 50 °C after 5 hours. Storage stability of ADH immobilized onto CMD3-MNPs and ADH co-immobilized with β-NAD onto CMD3-MNPs were investigated at 4 °C, where both obtained almost 60 % of its initial activity after three weeks. Magnetic carriers HIT-MNPs and AMS-MNPs were functionalized with cross-linker glutaraldehyde (GA) and amino-donor pentaethylenehexamine (PEHA). Functionalized HIT-MNPs and AMS-MNPs were immobilized with enzyme β-galactosidase (β-GAL). When using combination of 0,5 % (v/v) GA and 30 % (v/v) PEHA 128,9 % of residual activity was achieved, which resulted in hyper-activation of enzyme due to its conformational changes. Hyper-activation was achieved also with immobilizing β-GAL onto AMS-MNPs and the highest residual activity was obtained with with 20 % (v/v) of PEHA (154,4 %)

Multifunctional iron oxide nanoparticles as promising magnetic biomaterials in drug elivery

A wide range of applications using functionalized magnetic nanoparticles (MNPs) in biomedical applications, such as in biomedicine as well as in biotechnology, have been extensively expanding over the last years. Their potential is tremendous in delivery and targeting systems due to their advantages in biosubstance binding. By applying magnetic materials-based biomaterials to different organic polymers, highly advanced multifunctional bio-composites with high specificity, efficiency, and optimal bioavailability are designed and implemented in various bio-applications. In modern drug delivery, the importance of a successful therapy depends on the proper targeting of loaded bioactive components to specific sites in the body. MNPs are nanocarrier-based systems that are magnetically guided to specific regions using an external magnetic field. Therefore, MNPs are an excellent tool for different biomedical applications, in the form of imaging agents, sensors, drug delivery targets/vehicles, and diagnostic tools in managing disease therapy. A great contribution was made to improve engineering skills in surgical diagnosis, therapy, and treatment, while the advantages and applicability of MNPs have opened up a large scope of studies. This review highlights MNPs and their synthesis strategies, followed by surface functionalization techniques, which makes them promising magnetic biomaterials in biomedicine, with special emphasis on drug delivery. Mechanism of the delivery system with key factors affecting the drug delivery efficiency using MNPs are discussed, considering their toxicity and limitations as well

Bioethanol Production by Enzymatic Hydrolysis from Different Lignocellulosic Sources

As the need for non-renewable sources such as fossil fuels has increased during the last few decades, the search for sustainable and renewable alternative sources has gained growing interest. Enzymatic hydrolysis in bioethanol production presents an important step, where sugars that are fermented are obtained in the final fermentation process. In the process of enzymatic hydrolysis, more and more new effective enzymes are being researched to ensure a more cost-effective process. There are many different enzyme strategies implemented in hydrolysis protocols, where different lignocellulosic biomass, such as wood feedstocks, different agricultural wastes, and marine algae are being used as substrates for an efficient bioethanol production. This review investigates the very recent enzymatic hydrolysis pathways in bioethanol production from lignocellulosic biomass

Toxicity of magnetic chitosan micro and nanoparticles as carriers for biologically active substances

Nanoparticles of inorganic magnetic core surrounded by layers of functional coatings are potential representatives of nanostructures for immobilization of bio-substances. Magnetic nanoparticles (MNPs) are often bound in aggregates due to a strong magnetic dipole, which has a lot of advantages, such as large surface area for binding biologically active substances. Chitosan is a polysaccharide polymer that is non-toxic, hydrophilic, biocompatible and has hydroxy and amino groups in its structure. Because of these chemical and biological properties it is a desirable bio-product for immobilization of enzymes and for binding of other biologically active substances. Magnetic micro and nanoparticles were synthesized with chitosan by three different methodsmicroemulsion process, suspension cross-linking technique and covalent binding of chitosan. Toxic effect of the prepared magnetic particles was determined as well and was examined on five different bacterial culturesEscherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae. At concentrations of 10-30 mg of magnetic particles per 0.5 McFarland Standard solution of E. coli and per 400 CFU of S. aureus, P. aeruginosa, E. faecalis in K. pneumonia, no inhibition on the chosen bacterial cultures was detected.Nanodelci iz magnetnega anorganskega jedra, oblečeni z večslojno funkcionalno prevleko, predstavljajo pomemben razred nanostrukturiranih delcev za vezavo biosubstanc. Zaradi možnega magnetnega dipola se magnetni nanodelci pogosto združujejo v skupke, kar pa ima številne prednosti, kot na primer velika površina, na katero lahko pritrdimo različne biološke komponente. Hitozan je polisaharidni polimer, ki je nestrupen, hidrofilen, biokompatibilen in vključuje prisotnost hidroksilne in amino skupine v svoji strukturi. Zaradi naštetih kemijskih in bioloških lastnosti spada hitozan med zaželjene biomateriale za imobilizacijo encimov in vezavo drugih biološko aktivnih substanc. Magnetne nanodelce, prevlečene s hitozanom, smo pripravili po treh različnih postopkihs postopkom mikroemulzije, s postopkom suspenzijske zamreževalne tehnike ter s postopkom kovalentne vezaven hitozana. Toksikološke vplive pripravljenih magnetnih delcev smo preverili na petih različnih bakterijskih kulturahEscherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis in Klebsiella pneumoniae. Pri koncentracijah 10-30 mg magnetnih delcev na 0.5 McFarland standardne raztopine bakterijske kulture E.coli in na 400 CFU bakterijskih kultur S. aureus, P. aeruginosa, E. faecalis in K. pneumonia do inhibicije rasti mikroorganizmov ni prišlo

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from <i>T. reesei</i>: A Stable and Efficient Biocatalyst

Cross-linked enzyme aggregates (CLEAs) represent an effective tool for carrier-free immobilization of enzymes. The present study promotes a successful application of functionalized magnetic nanoparticles (MNPs) for stabilization of cellulase CLEAs. Catalytically active CLEAs and magnetic cross-linked enzyme aggregates (mCLEAs) of cellulase from Trichoderma reesei were prepared using glutaraldehyde (GA) as a cross-linking agent and the catalytic activity and stability of the CLEAs/mCLEAs were investigated. The influence of precipitation agents, cross-linker concentration, concentration of enzyme, addition of bovine serum albumin (BSA), and addition of sodium cyanoborohydride (NaBH3CN) on expressed activity and immobilization yield of CLEAs/mCLEAs was studied. Particularly, reducing the unsaturated Schiff’s base to form irreversible linkages is important and improved the activity of CLEAs (86%) and mCLEAs (91%). For increased applicability of CLEAs/mCLEAs, we enhanced the activity and stability at mild biochemical process conditions. The reusability after 10 cycles of both CLEAs and mCLEAs was investigated, which retained 72% and 65% of the initial activity, respectively. The thermal stability of CLEAs and mCLEAs in comparison with the non-immobilized enzyme was obtained at 30 °C (145.65% and 188.7%, respectively) and 50 °C (185.1% and 141.4%, respectively). Kinetic parameters were determined for CLEAs and mCLEAs, and the KM constant was found at 0.055 ± 0.0102 mM and 0.037 ± 0.0012 mM, respectively. The maximum velocity rate (Vmax) was calculated as 1.12 ± 0.0012 µmol/min for CLEA and 1.17 ± 0.0023 µmol/min for mCLEA. Structural characterization was studied using XRD, SEM, and FT-IR. Catalytical properties of immobilized enzyme were improved with the addition of reducent NaBH3CN by enhancing the activity of CLEAs and with addition of functionalized aminosilane MNPs by enhancing the activity of mCLEAs