Biocatalysts Enzymes Research Articles

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed.

Magnetic Nanocomposite Thin Film Photocatalyst and Cell Extract Enzyme Biocatalyst in Application of Nanobiotechnology for Development of a Photo-Bio Desulfurization System

A photo-bio hybrid system was developed using iron oxide nanocomposite thin film as photocatalyst and cell debris and crude cell extract enzyme as biocatalyst. The iron oxide nanocomposite was prepared by a sol-gel approach and coated on glass by spin-coating technique. The nanocomposite has photocatalytic property, which is used under very mild reaction conditions. The iron oxide nanocomposites were characterized by X-ray diffraction (XRD), Fourier transmission infrared (FT-IR), UV–Vis spectroscopy (DRS), and field emission scanning electron microscopy (FESEM). Advance oxidative desulfurization using iron oxide nanocomposite thin film as photocatalyst and cell debris and crude cell extract enzyme as biocatalyst showed that 83% efficiency for desulfurization of dibenzothiophene as a model sulfur compound. These results suggest that use of the constructed nanobiotechnology can be transformed into green technology.

Enzymatically catalyzed poly(butylene succinate)(PBS) copolymers as building blocks for degradable scaffolds

Event Abstract Back to Event Enzymatically catalyzed poly(butylene succinate)(PBS) copolymers as building blocks for degradable scaffolds Miroslawa El Fray1, Monika Malesa1 and Aleksandra Kaczmarek1 1 West Pomeranian University of Technology Szczecin, Biomaterials and Microbiological Technologies, Poland Introduction: An increasing exploitation of regenerative potential of living tissues is observed from last two decades since temporary, biodegradable polymeric templates (scaffolds) are being developed for tissue engineering[1]. Biodegradable polymers are synthesized from non-toxic monomers, but toxic catalysts are frequently used like in case of biodegradable aliphatic polyesters where highly toxic tin compounds are used[2]. Therefore, stiff but biodegradable polymers are needed for tissue engineering and other biomedical applications. In our work we focus on much safer enzymatic biocatalysts which are proposed for the synthesis of aliphatic polyester, poly(butylene succinate)(PBS) and its copolymers. Materials and Methods: Poly(butylene succinate)(PBS) and its copolymers containing ethylene glycol sequences (PBS-EG) and dilinoleic acid sequences (PBS-DLA) were prepared by enzymatic polymerization of succinic acid ester (diethyl succinate)(DES), 1,4-butanediol (1,4-BD) and poly(ethylene glycol) (PEG_3000) or dilinoleic acid (DLA) in organic solvent (diphenyl ether). Lipase B derived from Candida antarctica strain, trade name Novozym 435th (N435), was used as a catalyst. The hard segments (PBS) to soft segments (EG or DLA) ratio based on stoichiometric calculations was 50:50 wt%. The reaction mixture was dissolved in chloroform to remove the unreacted monomers and impurities and then the microspheres in which the enzyme catalyst was immobilized were separated. Samples chemical structure were assessed by using ATR-FTIR and NMR analysis, degradation and contact angle measurement. Sample mechanical characterization was performed by tensile test. Preliminary cell viability tests have been performed by using fibroblasts. Results and Discussion: The PBS-EG and PBS-DLA copolymers were synthesized using Candida Antarctica from lipase B. Analysis of chemical structure with 1H NMR and ATR FT-IR spectroscopy confirmed the presence of specific bonds and groups, characteristic for ester and ether groups. The contact angle measurements on both materials revealed differences in hydrophilicity thus indicating that depending from the nature of copolymer building block, degradation profile can be adjusted. This can be achieved along with mechanical properties changes since PBS-DLA copolymer showed to be more flexible as compared to PBS-EG. Positive results from the preliminary cell viability tests have been also obtained. Conclusions: Enzymatically catalyzed copolymers of poly(butylene succinate), namely PBS-EG and PBS-DLA has been obtained. Results showed that these materials have different surface and mechanical properties depending from the chemical structure of soft segment building block. Their degradation profile and mechanical properties can also be adjusted thus making them an interesting candidate for degradable scaffolds, specifically for heart tissue engineering. This work was supported by the National Science Centre under the HARMONIA scheme (UMO-2014/14/M/ST8/00610).

Cytochrome P450 enzymes: understanding the biochemical hieroglyphs

Cytochrome P450 (CYP) enzymes are the primary proteins of drug metabolism and steroid biosynthesis. These crucial proteins have long been known to harbor a cysteine thiolate bound to the heme iron. Recent advances in the field have illuminated the nature of reactive intermediates in the reaction cycle. Similar intermediates have been observed and characterized in novel heme-thiolate proteins of fungal origin. Insights from these discoveries have begun to solve the riddle of how enzyme biocatalyst design can afford a protein that can transform substrates that are more difficult to oxidize than the surrounding protein architecture.

The Complete Oxidation of Glycerol to CO2: A Hybrid Enzymatic and Organic Electrocatalytic Cascade

There has been a significant interest lately in the complete oxidation of biofuels to CO2. The use of multiple biocatalysts allows for deeper biofuel oxidation and thus maximizes the energy density that can be extracted per molecule of biofuel. However, there are still many biofuels for which a suitable biocatalyst is not known. We have recently taken an alternative approach to combine the high catalytic activity of enzymatic biocatalysts with the promiscuous nature of small molecule organic catalysts. We demonstrate the complete electrochemical oxidation of the biofuel glycerol to CO2 using a hybrid enzymatic and small-molecule catalytic system. Combining an enzyme, oxalate oxidase, and an organic oxidation catalyst, 4-amino-TEMPO, we are able to electrochemically oxidize glycerol at a carbon electrode, while collecting as many as 16 e-s per molecule of fuel. Additionally, we investigate the anomalous electrocatalytic properties that allow 4-amino-TEMPO to be active under the acidic conditions required for oxalate oxidase to function.

Simultaneous saccharification and fermentation of cellulose in ionic liquid for efficient production of α-ketoglutaric acid by Yarrowia lipolytica.

Ionic liquids (ILs) are benign solvents that are highly effective for biomass pretreatment. However, their applications for scale-up biorefinery are limited due to multiple expensive IL recovery and separation steps that are required. To overcome this limitation, it is very critical to develop a compatible enzymatic and microbial biocatalyst system to carry the simultaneous saccharification and fermentation in IL environments (SSF-IL). While enzymatic biocatalysts have been demonstrated to be compatible with various IL environments, it is challenging to develop microbial biocatalysts that can thrive and perform efficient biotransformation under the same conditions (pH and temperature). In this study, we harnessed the robust metabolism of Yarrowia lipolytica as a microbial platform highly compatible with the IL environments such as 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]). We optimized the enzymatic and microbial biocatalyst system using commercial cellulases and demonstrated the capability of Y. lipolytica to convert cellulose into high-value organics such as α-ketoglutaric acid (KGA) in the SSF-IL process at relatively low temperature 28 °C and high pH 6.3. We showed that SSF-IL not only enhanced the enzymatic saccharification but also produced KGA up to 92% of the maximum theoretical yield.

Identification, characterization, and immobilization of an organic solvent-stable alkaline hydrolase (PA27) from Pseudomonas aeruginosa MH38.

An organic solvent-stable alkaline hydrolase (PA27) from Pseudomonas aeruginosa MH38 was expressed, characterized, and immobilized for biotechnological applications. Recombinant PA27 was expressed in Escherichia coli as a 27 kDa soluble protein and was purified by standard procedures. PA27 was found to be stable at pH 8–11 and below 50 °C. It maintained more than 80% of its activity under alkaline conditions (pH 8.0–11.0). Furthermore, PA27 exhibited remarkable stability in benzene and n-hexane at concentrations of 30% and 50%. Based on these properties, immobilization of PA27 for biotechnological applications was explored. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. Interestingly, immobilized PA27 displayed improved thermal/chemical stabilities and high reusability. Specifically, immobilized PA27 has improved thermal stability, maintaining over 90% of initial activity after 1 h of incubation at 80 °C, whereas free PA27 had only 35% residual activity. Furthermore, immobilized PA27 showed higher residual activity than the free enzyme biocatalysts against detergents, urea, and phenol. Immobilized PA27 could be recycled 20 times with retention of ~60% of its initial activity. Furthermore, macroscopic hydrogel formation of PA27 was also investigated. These characteristics make PA27 a great candidate for an industrial biocatalyst with potential applications.

Improved Interfacial Electron Transfer in Modified Bilirubin Oxidase Bio-Cathodes

Very promising area of enzymatic fuel cell (EFC) research is in regards to a family of enzymes known as multi-copper oxidases (MCOs), which are capable of performing oxygen reduction reaction (ORR). Recently, MCOs have been shown to compare favorably to other state of the art ORR catalysts due to their activity at moderate temperatures and neutral pH at which they operate1. However, despite continuing research and on-going advancements in biocatalysis, relatively low catalyst stability (e.g. decreasing enzyme activity) and low current density, compared with traditional fuel cells, are impeding widespread application of EFCs. Specifically, enzyme biocatalyst activity and subsequent cell potential and current density decrease over time due to the inherently short lifetime of the enzyme and/or low interfacial electron transfer rate between the electrode and the enzyme. Improving enzyme stability and optimizing the interfacial electron transfer at the electrode is a key area of EFC research.The interfacial electron-transfer of MCOs is dependent on the spacing between the enzyme (the T1-Cu center) and the electrode surface2. To decrease the gap between the T1-Cu and the electrode, the enzyme should be “oriented” with the T1-Cu facing the underlying electrode surface. In this study a hypothesis was developed for orienting the T1-Cu site of Bilirubin oxidase (BOx) towards the electrode surface. Substrates of BOx, such as bilirubin, can be artificially attached to the electrode and subsequently used as “orienting agents” in the development of enzymatic oxygen reducing cathodes (Fig. 1)3. Two approaches were investigated in order to further improve current generation through favorable orientation of BOx on the electrode: i) “scale up” the substrate molecule through utilization of polymers or ii) “scale down” the substrate structure by the utilization of functional analogues.Multi-wall carbon nanotubes (MWNTs) were modified subsequently by physical adsorption of tethering 1-pyrenebutanoic acid succinimidyl ester (PBSE) and orienting agent (bilirubin). The interactions of bilirubin and PBSE with the MWNT scaffold are provided by π – π stacking. At the same time the tethering agent is attached to the enzyme by enzyme`s amino groups. The interaction of bilirubin and the enzyme is ensured by the key-lock principle. Thus the nanotube matrix modified by the application of tethering and orienting agents provides enhanced electrode stability and improved electron transfer efficiency showing increased bio-cathode performance (Fig. 2). The BiRu-PBSE modified cathode showed increase in current density, compared to the PBSE, bilirubin, and unmodified BOx cathodes of 0.4, 3.2, and a 9.5 times, respectively.As a macromolecular analogue of bilirubin a short-chained chemically polymerized polypyrrole was also tested. Unfortunately, the polypyrrole-modified cathode did not induce favorable enzyme orientation, most likely due to: i) the hydrophobicity of polypyrrole; ii) the lack of any carboxyl/negatively charged groups in the polypyrrole molecule; iii) the inhibition of the attached polypyrrole to the electronic communication between BOx and the electrode.The “scale down” approach involved the use of two bilirubin functional analogues, pyrrole-2-carboxaldehyde and 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde (DPy-Carb), for enzyme orientation along with PBSE as the tethering agent. The DPy-Carb-PBSE-BOx modified cathode had a significantly higher generated current density compared to both the PBSE-BOx and BiRu-PBSE-BOx scaffolds (Fig. 3).The orientation efficiency of the enzyme after the modification procedure with DPy-Carb and PBSE was determined to be 90%. Therefore, int can be concluded that the enhanced performance of the modified cathodes is most likely due to improved interfacial electron transport as a result of favorable T1-Cu site orientation towards the electrode surface.Favorable orientation of bilirubin oxidase on a MWNT matrix and subsequent increase in electrocatalytic activity was achieved through cathode modification with a cross-linker and the natural substrate bilirubin or its artificial analogues as orienting agents. The described herein approach can be successfully applied for other reducing enzymes from the family of MCOs used at the cathode in the development of enzymatic biofuel cells.

Immobilization of phospholipase a1 using a polyvinyl alcohol-alginate matrix and evaluation of the effects of immobilization

The paper presents the synthesis and performance of an immobilized phospholipase A1 with practical application for oil degumming. The polyvinyl alcohol (PVA) had a number of properties indicating this polymer as a good enzyme carrier. The combination with alginate made a macro-porous structure, evidenced by SEM analyses. When the process time in boric acid solution was 30 minutes, the results revealed that beads prepared with 10% (w/v) PVA and 2% (w/v) sodium alginate in 4% (w/v) boric acid and 2% (w/v) calcium chloride solution exhibited high immobilized enzyme activity, immobilization yield and stability. The pH and temperature optimum for the PVA-alginate immobilized phospholipase A1were 5.6 and 58 °C, respectively. The enzyme immobilized in the beads retained 50.37% of the initial activity in the eighth cycle. The enzyme biocatalyst immobilized in the beads retained 78.58% of the initial activity after storing 6 weeks at 4 °C.

Hybrid graphite film–carbon nanotube platform for enzyme immobilization and protection

A novel platform consisting of a multilayered substrate, activated graphite-like carbon film, and dense forest of long, vertically-aligned multiwall carbon nanotubes grown by the chemical vapor deposition is designed, fabricated, and tested for covalent immobilization of enzymatic biocatalysts with the aim of protecting them from shear forces and microbial attacks present in bioreactors. The covalent bonding ensures enzyme retention in a flow, while the dense nanotube forest may serve as a protection of the enzymes from microbial attack without impeding the flow of reactants and products. This platform was demonstrated for the two reference enzymes, horseradish peroxidase and catalase, which were immobilized without degrading their biological activity. This combination of an activated carbon layer for an efficient immobilization of biocatalysts with a protective layer of inert carbon nanotubes could dramatically improve the efficiency and longevity of enzymatic bio-catalysis employed in a large variety of advanced biotechnological processes.

Structural and functional importance of local and global conformational fluctuations in the RNase A superfamily

Understanding the relationship between protein structure and flexibility is of utmost importance for deciphering the tremendous rates of reactions catalyzed by enzyme biocatalysts. It has been postulated that protein homologs have evolved similar dynamic fluctuations to promote catalytic function, a property that would presumably be encoded in their structural fold. Using one of the best-characterized enzyme systems of the past century, we explore this hypothesis by comparing the numerous and diverse flexibility reports available for a number of structural and functional homologs of the pancreatic-like RNase A superfamily. Using examples from the literature and from our own work, we cover recent and historical evidence pertaining to the highly dynamic nature of this important structural fold, as well as the presumed importance of local and global concerted motions on the ribonucleolytic function. This minireview does not pretend to cover the overwhelming RNase A literature in a comprehensive manner; rather, efforts have been made to focus on the characterization of multiple timescale motions observed in the free and/or ligand-bound structural homologs as they proceed along the reaction coordinates. Although each characterized enzyme of this architectural fold shows unique motional features on a local scale, accumulating evidence from X-ray crystallography, NMR spectroscopy and molecular dynamics simulations suggests that global dynamic fluctuations, such as the functionally relevant hinge-bending motion observed in the prototypical RNase A, are shared between homologs of the pancreatic-like RNase superfamily. These observations support the hypothesis that analogous dynamic residue clusters are evolutionarily conserved among structural and functional homologs catalyzing similar enzymatic reactions.

Oxyfunctionalization of aliphatic compounds by a recombinant peroxygenase from Coprinopsis cinerea

The goal of this study is the selective oxyfunctionalization of aliphatic compounds under mild and environmentally friendly conditions using a low-cost enzymatic biocatalyst. This could be possible taking advantage from a new peroxidase type that catalyzes monooxygenase reactions with H2 O2 as the only cosubstrate (peroxygenase). With this purpose, recombinant peroxygenase, from gene mining in the sequenced genome of Coprinopsis cinerea and heterologous expression using an industrial fungal host, is tested for the first time on aliphatic substrates. The reaction on free and esterified fatty acids and alcohols, and long-chain alkanes was followed by gas chromatography, and the different reaction products were identified by mass spectrometry. Regioselective hydroxylation of saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (only at the ω-2 position in myristoleic acid). Alkyl esters of fatty acids and monoglycerides were also ω-1 or ω-2 hydroxylated, but di- and tri-glycerides were not modified. Fatty alcohols yielded hydroxy derivatives at the ω-1 or ω-2 positions (diols) but also fatty acids and their hydroxy derivatives. Interestingly, the peroxygenase was able to oxyfunctionalize alkanes giving, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule. The predominance of mono- or di-hydroxylated derivatives seems related to the higher or lower proportion of acetone, respectively, in the reaction medium. The recombinant C. cinerea peroxygenase appears as a promising biocatalyst for alkane activation and production of aliphatic oxygenated derivatives, with better properties than the previously reported peroxygenase from Agrocybe aegerita, and advantages related to its recombinant nature for enzyme engineering and industrial production.

CYP63A2, a Catalytically Versatile Fungal P450 Monooxygenase Capable of Oxidizing Higher-Molecular-Weight Polycyclic Aromatic Hydrocarbons, Alkylphenols, and Alkanes

Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).

Biocatalytic alternative for bio-glycerol conversion with alkyl carbonates via a lipase-linked magnetic nano-particles assisted process

Bio-glycerol (B-Gly) was converted to glycerol carbonate (GlyC) as an important added-value product using an advanced biocatalytic process involving the glycerol (Gly) carbonylation with dimethyl carbonate (DMC) assisted by a heterogeneous enzyme biocatalyst (lipase covalently attached on magnetic nano-particles surface). The biocatalytic process was set up optimizing the experimental parameters (e.g. molar ratio of the reagents, temperature, incubation time and catalyst content). In addition, the efficiency of DMC as carbonylation agent was evaluated against diethyl carbonate (DEC) and dibenzyl carbonate (DBC). B-Gly was tested as a raw material for GlyC synthesis in the set up biocatalytic process using commercial pure Gly (S-Gly) as reference. B-Gly was produced based on a conventional transesterification process applied to both crude and residual sun-flower oil. Crude sun-flower oil was the typical commercial one and residual sun-flower oil was that recovered from the cooking process. Comparable performances were obtained using B-Gly from residual oil and S-Gly (e.g. conversion of Gly of 36% and 45% for selectivity in GlyC of 90% and 92%, respectively), while the use of B-Gly from crude residual oil led to a Gly conversion of only 27% with selectivity in GlyC of 95%.

Purification, Characterization and Application of a Cold- Adapted Phospholipase A1 from Bacillus Cereus Sp. AF-1

ABSTRACTThis paper reports the purification, characterization and application of an extracellular phospholipase A1 that functions at low temperature, from Bacillus cereus sp. AF-1 isolated from soil near the oil mill in Tianjin, China. The enzyme was purified 56-fold from fermentation supernatant, with 30.19 % recovery (specific activity 1195.24 U/mg). The purified enzyme has a molecular weight of 20 kDa. Using phospholipid as substrate, the enzyme showed maximum specific activity (Vmax) of 1.302 mmol·L−1mg−1 proteins with its corresponding Km value of 11.11 mg·mL−1. The enzyme was identified as phospholipase A1 by analyzing the free fat acid of enzymic hydrolysates which was extracted with a two-phase heptane-isopropyl alcohol—water system. The optimal temperature and pH for phospholipase A1 were 35°C and 7.5, respectively. The enzyme biocatalyst retained 85% of the initial activity after 10 weeks of storage at 4°C. The phosphorus content of the crude oil could be reduced to 7 mg·kg−1 when the optimal set of variables was: enzyme dosage of 0.4 mg kg−1 and reaction time of 3 h.

Carbon nanotube-modified biocatalytic microelectrodes with multiscale porosity

Macropores were introduced into nanotube matrices via polystyrene bead templates, and the resulting matrix was applied to carbon fiber microelectrodes as a porous medium for immobilization of enzymatic biocatalysts. The macropores were found to increase the electrochemically active surface area by twofold at a nominal polystyrene mass fraction of 73%. The modified electrodes were further coated with biocatalyst hydrogel comprising glucose oxidase, redox polymer, and crosslinker to create a glucose oxidizing bioanode. Glucose oxidation current density also increased two fold after introduction of the macropores. Focused ion beam cut cross-sections reveal complete adsorption of the enzyme-hydrogel matrix into the CNT layer. This templating technique is a promising approach to the maximization of surface area and transport in bioelectrodes.

Cross-Linked Enzyme Aggregates of Naringinase: Novel Biocatalysts for Naringin Hydrolysis

Cross-linked enzyme aggregates (CLEAs) have emerged as interesting biocatalyst design for immobilization. These new generation enzyme biocatalysts, CLEAs, in addition to exhibiting good mechanical stability, can be highly active, since they do not include large amounts of foreign particulate nonenzymatic material and may have increased stability. Naringinase (NGase) is an enzyme complex with high potential in pharmaceutical and food industries. In fact, NGase can be used in the biotransformation of steroids, of antibiotics and mainly on glycosides hydrolysis. In this paper, the formation of CLEAs was tried using ammonium sulphate, polyethylene glycol 6000 and tert-butyl alcohol as precipitant agents and glutaraldehyde as cross-linking agent, at different pH, time, and temperature conditions. However, among the precipitant agents tested, only tert-butyl alcohol cross-linked with glutaraldehyde allowed the formation of CLEAs, at pH 4.0 and at temperature between 7 and 10°C. Different enzyme loadings were tested. The NGase-CLEAs were highly effective in naringin hydrolysis. The operational stability of the NGase-CLEAs aggregates was studied through six successive reutilizations.

Biological production of vanillin using enzyme and whole-cell biocatalysts

Biological production of vanillin using enzyme and whole-cell biocatalysts

Immobilization of enzyme biocatalyst on natural halloysite nanotubes

Abstract In this study, we use natural halloysite nanotubes as novel support materials to immobilize enzymes. Two typical industrial enzymes (α-amylase and urease) with different sizes were immobilized in channels of the nanotubes through simple physical adsorption. After 60 min heating, both immobilized enzymes retained more than 80% activity. Stored for 15 days, the immobilized enzymes still showed more than 90% activity. More than 55% initial activity of the enzyme was retained after 7 cycles. The immobilized enzymes exhibited thermal stability, good storage stability and reusability, which indicate that halloysite is a promising support material for enzyme immobilization.

Recent advance in the support and technology used in enzyme immobilization

In the industrial biotechnology, the skill to prepare the immobilized enzyme with stability has proven to be one of the key steps in rendering an enzymatic process that is economically viable. Availability of the immobilized enzyme biocatalyst with improved activity and stability is also expected to reduce the expense of products. If properly designed, enzyme immobilization is also a very powerful tool to enhancing almost all enzyme properties, e.g., activity, stability, specificity and selectivity, reduction of inhibition. Due to the above advantages, enzyme immobilization has been a focus for biologists, chemists and enterprisers. This review is focused on advances over the last several years in enzyme immobilization. Several new type of carriers and technology have been implemented to improve traditional enzyme immobilization, which aimed to enhance enzyme loading, activity and stability to decrease the enzyme biocatalyst cost in industrial biotechnology. These include cross-linked enzyme aggregates (CLEAs), microwave-assistant immobilization, click chemistry technology, mesoporous support and single enzyme nanoparticle. In addition, with the growing attention paid to cascade enzymatic reaction and in vitro synthetic biology, it is possible that multi-enzyme co-immobilization would be one of the next goals in the future. Key words: Enzyme immobilization, cross-linked enzyme aggregates (CLEAs), microwave irradiation, mesocellular siliceous foams (MCFs), click chemistry, single enzyme nanoparticle.