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Removal of Pharmaceutical Pollutants from Municipal Sewage Mediated by Laccases
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2020
Thomas Hahn, Fabian Haitz, Jan Gajewski, Marius Mohr, Marc Beckett, Susanne Zibek
Adsorptive binding to a resin is a fast-and-easy way to achieve an immobilization. However, the bond between the laccase and the carrier is weaker than one obtained with covalent linkage and leads to leakage of the laccase. A covalent bond between carrier material and laccase avoids leaching. Furthermore, in order to achieve higher surgical stability, a covalent immobilization is beneficial. With regard to the carrier material, amine functionalization is an option since the enzyme can be bound by simple dialdehyde linkage performed with glutardialdehyde (GA) which acts as a linker and a cross-linker at the same time. Alternatively, carboxy-functionalized resins can be applied requiring activation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimid (EDC) and n-hydroxysuccinimid (NHS). The application of epoxy-preactivated carriers can also be used for immobilization and does not need any further treatment. A self-immobilization strategy, that does not require any kind of carrier, is provided by the formation of cross-linked enzyme aggregates (CLEAs). CLEAs meet the overall requirements and guarantee high activity with simultaneous improvement of the operative stability and are thus ideal for the given purpose. Independent from the kind of linkage used, retention of the carrier material with immobilized laccase or CLEAs is mandatory.
Role of Chitosan Nanotechnology in Biofuel Production
Published in Madan L. Verma, Nanobiotechnology for Sustainable Bioenergy and Biofuel Production, 2020
Meenu Thakur, Rekha Kushwaha, Madan L. Verma
Most of the cross-linking methods use the multifunctional agents and utilize their stable interactions for cross-linking (Ahmad and Sardar 2015). This method is devoid of support matrix which ensures 100% enzyme activity. The major problems associated are conformational changes and less retainment of enzyme activity (Ahmad and Sardar 2015). Sheldon (2007) has derived the cross-linked enzyme aggregates (CLEAs) method for effective enzyme immobilization. Non-ionic polymers and organic solvents can be used for preparing cross-linked enzyme aggregates which is a simple and effective method. Enzymes such as lipases from Rhizomucor miehei and Thermomyces lanuginosus can be precipitated using ammonium sulfate, SDS and glutaraldehyde (Lopez-Serrano et al. 2002). By these immobilization methods, the hydrolytic activities of aggregates can increase by two to three-folds.
Advances in Nanobiocatalysis Strategies for Lipase Immobilization and Stabilization
Published in Mahmood Aliofkhazraei, Advances in Nanostructured Composites, 2019
Patel Vrutika, Ashok Pandey, Christian Larroche, Datta Madamwar
Carrier-free biocatalysts are novel type enzyme catalysts bearing the advantages of the high concentration of active enzyme within the biocatalyst particle and reduced cost (Roessl et al. 2010). In this case, the enzyme protein constitutes its own support so that concentrations close to the theoretical packing limit are obtained (Cao 2005). Therefore, carrier-free immobilized enzymes are advantageous as catalysts in processes where high productivity and yield is required or in the case of labile enzymes that cannot be properly stabilized by conventional immobilization to solid supports (Illanes et al. 2009). Carrier-free immobilized enzymes are prepared by direct chemical crosslinking of the protein containing the enzyme, using mainly glutaraldehye as crosslinking agent. This strategy has been applied to cross linked enzymes in solution (CLEs), to cross linked enzyme crystals (CLECs) and more recently, to cross linked enzyme aggregates (CLEAs). CLEAs have advantages over CLEs of better mechanical properties and higher yields of activity and are simpler and much cheaper to produce than CLECs, which require a purified crystal protein as starting material.
An effective immobilization of β-glucosidases by partly cross-linking enzyme aggregates
Published in Preparative Biochemistry & Biotechnology, 2022
Yuefeng Deng, Jie Ouyang, Hu Liu, Jianjun Wang, Yihui Zhu, Ziqian Chen, Chengli Yang, Dali Li, Kefeng Ma
In addition to the immobilization carrier, the choice of immobilization methods should be taken into account. Traditional methods for enzymes immobilization are varied, which mostly are divided into: adsorption,[23] encapsulation,[24,25] crosslinking[26,27] and covalent immobilization.[28] In the last years, as a newly developing branch of immobilized enzyme technology, cross-linked enzyme aggregates (CLEAs) have gathered widespread attention thanks to its simple preparation, high specific activity, and better stability.[29] CLEAs were aggregates formed by intermolecular multipoint cross-linking between enzyme molecules,[30] with glutaraldehyde or other reagent as crosslinking agent.[31] Although the carrier-free CLEAs have many characteristics, they are mechanically fragile and difficult to recycle,[32,33] so their industrial application are seriously limited. Therefore, the immobilization of CLEAs on the carrier can not only has good mechanical resistance, but has high enzyme loading and enhanced stability.[34–36] As the carrier of immobilized CLEAs, MTL has excellent mechanical properties and easy recovery, which seems to make up for the the shortcomings of carrier-free CLEAs wonderfully.
Cross-linked esterase aggregates (CLEAs) using nanoparticles as immobilization matrix
Published in Preparative Biochemistry and Biotechnology, 2019
Nithyakalyani Doraiswamy, Mahalakshmi Sarathi, Gautam Pennathur
Cross-linked Enzyme Aggregates (CLEAs) is one of the recent carrier-free immobilization techniques[5] and has attracted attention for the ease and sturdiness of the method employed.[3] CLEAs are produced through extensive cross-linking of the enzyme aggregates held together through covalent bonds with bifunctional agents, usually glutaraldehyde.[6,7] CLEAs have proved to have enhanced stability compared to the corresponding soluble enzyme.[2] This enhanced stability is through the multipoint attachment of enzyme molecules and rigidity enforced on its tertiary structure.[2] It is a highly advantageous technique with greater volumetric activity and increased specific activity,[8] but there are certain shortcomings like difficulty in handling and recoverability,[2,9] hence improvement to this technique is carried out and is known as supported CLEAs strategies.[3,10] The properties of the nano-sized particles such as their excellent dispersibility with lower mass transfer resistance make them suitable matrices in the supported CLEAs strategy.[1,3] Their flexible and rigid nature allows them to withstand the high-pressure conditions in the reactor configuration improving the stability of the immobilized enzymes and catalytic efficiency.[3]
Improving operational stability of thermostable Pythium myriotylum secretory serine protease by preparation of cross-linked enzyme aggregates (CLEAs)
Published in Preparative Biochemistry & Biotechnology, 2020
Aswati R. Nair, Geethu Chellapan
For many commercial applications, crude enzymes are preferred over purified preparations due to their availability, low cost, and operational stability.[19] However, the presence of other competing enzymes, low stereo-selectivity, and cellular contaminants adds to irreproducibility amongst batches.[43] These problems are circumvented by immobilization of enzymes on carrier, entrapment or cross-linking of molecules.[15,17,19,24] Carrier-based immobilization based on magnetic nanoparticles have yielded stable biocatalysts,[19,20] but these pose problems with respect to enzyme loading capacity, enzyme leakage, fragility to abrasion and applicability in reactors with mechanical stirring.[19] Contrarily, carrier-free immobilization strategies especially development of cross-linked enzyme aggregates (CLEAs) except for investing significant efforts in optimizing preparation conditions, offer many advantages like application to a wide range of enzymes, efficient, simple and low-cost approach.[15,16,19,24,25,44] Nevertheless, the development of an optimum immobilization protocol to enhance the stability of enzymes characterized by diverse sources with different specificities is largely empirical and requires detailed characterization. In this context, present experiments were undertaken to enhance stability of spPm1, characterized in earlier studies to possess appreciable natural stability at high temperature and pH,[14] by optimizing the CLEA development protocol.