Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst

Bioprocess and Biosystems Engineering - N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) were immobilized for synthesis of N-acetylneuraminic acid (Neu5Ac) on three...     

Use of porous glass fiber as a support for biocatalyst immobilization

Summary Porous glass fiber has a very high surface area and good mechanical properties that make it an excellent support for biocatalyst immobilization. By packing aligned glass fibers in a tubular reactor such that the fibers are all parallel to the axis of the tube, the resulting pressure drop is considerably smaller than for a similar bed of packed beads. The utility of this support was demonstrated by immobilizing -glucoamylase by silane-glutaraldehyde coupling, and measuring its activity toward converting maltose to glucose. Using optimized immobilization conditions, an enzyme loading of 1.5 mg protein perm ² surface area was obtained, with an activity of 370 units/g glass at 50°C. The half-life of the immobilized glucoamylase was more than twice as long as that of the free enzyme.     

Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance

Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.     

Ionic liquid-reconstituted cellulose composites as solid support matrices for biocatalyst immobilization

Preparation of cellulose-polyamine composite films and beads, which provide high loading of primary amines on the surface allowing direct one-step bioconjugation of active species, is reported using an ionic liquid (IL) dissolution and regeneration process. Films and bead architectures were prepared and used as immobilization supports for laccase as a model system demonstrating the applicability of this approach. Performance of these materials, compared to commercially available products, has been assessed using millimeter-sized beads of the composites and the lipase-catalyzed transesterification of ethyl butyrate.     

Reversible, covalent immobilization of enzymes by thiol-disulphide interchange.

1. alpha-Amylase and alpha-chymotrypsin have been immobilized by covalent attachment to mercaptohydroxypropyl ether agarose gel. The technique involves two steps: (a) thiolation of the enzymes by methyl 3-mercaptopropioimidate, (b) coupling of the thiolated enzymes to a mixed disulphide derivative of agarose obtained by reacting mercaptohydroxypropyl ether agarose with 2,2'-dipyridyl disulphide. 2. The immobilization technique can be performed so that most of the inherent activity of the enzymes is conserved. However, diffusion limitations and steric factors prevent full manifestation of the immobilized activities. 3. Immobilized alpha-amylase was used in a packed-bed reactor for the continuous hydrolysis of starch. When the enzymically active gel had lost its activity it could be regenerated in situ by reductive uncoupling of the inactive protein and attachment of a new portion of thiolated alpha-amylase.     

Principles,techniques, and applications of biocatalyst immobilization for industrial application

Immobilization is one of the most effective and powerful tools used in industry, which has been studied and improved since the last century. Various immobilization techniques and support materials have been used on both laboratory and industrial scale. Each immobilization technique is applicable for a specific production mostly depending on the cost and sensibility of process. Compared to free biocatalyst systems, immobilization techniques often offer better stability, increased activity and selectivity, higher resistance, improved separation and purification, reuse of enzymes, and consequently more efficient process. Recently, many reviews have been published about immobilization systems; however, most of them have focused on a specific application or not emphasized in details. This review focuses on most commonly used techniques in industry with many recent applications including using bioreactor systems for industrial production. It is also aimed to emphasize the advantages and disadvantages of the immobilization techniques and how these systems improve process productivity compared to non-immobilized systems.     

High-yield method for immobilization of enzymes

Two types of polyethylenimine-coated glass microbeads (13–44 μm) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were performed by adsorption followed by treatment with glutaraldehyde. The immobilized-enzyme activities per unit support of all of the enzymes tested were compared with and found to be superior to the immobilized activities attainable on aminopropyl-activated glass microbeads. When enzyme was present in less than saturating amounts, the coated beads were able to remove 100% of the glucose oxidase activity initially present in the immobilization solution, with 78–87% of that activity expressed on the support surface. Bound glucose oxidase was more stable to thermal inactivation than native enzyme.     

Improved yield of antibiotics by immobilization of cells of myxobacteria

Strains of myxobacteria were investigated for antibiotic production in submerged culture using free-cells and cells immobilized by entrapment in kappa-carrageenan and by adsorption onto a plastic surface. Yield increases of 25–49% were obtained from immobilized cells and the highest yields were from entrapped cells.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Nanodevices for the immobilization of therapeutic enzymes

Therapeutic enzymes are one of the most promising applications of this century in the field of pharmaceutics. Biocatalyst properties can be improved by enzyme immobilization on nano-objects, thereby increasing stability and reusability and also enhancing the targeting to specific tissues and cells. Therapeutic biocatalyst–nanodevice complexes will provide new tools for the diagnosis and treatment of old and newly emerging pathologies. Among the advantages of this approach are the wide span and diverse range of possible materials and biocatalysts that promise to make the matrix–enzyme combination a unique modality for therapeutic delivery. This review focuses on the most significant techniques and nanomaterials used for enzyme immobilization such as metallic superparamagnetic, silica, and polymeric and single-enzyme nanoparticles. Finally, a review of the application of these nanodevices to different pathologies and modes of administration is presented. In short, since therapeutic enzymes constitute a highly promising alternative for treating a variety of pathologies more effectively, this review is aimed at providing the comprehensive summary needed to understand and improve this burgeoning area.     

Kinetically controlled synthesis of dipeptides using ficin as biocatalyst.

The application of the sulfhydryl protease ficin as biocatalyst is proposed as a novel method for enzyme-catalyzed synthesis of dipeptides. The negligible peptidase but considerable esterase activity at alkaline pH facilitated the kinetically controlled formation of peptide bonds by coupling the ester substrates Z-Ala-OMe and Z-Gly-OMe with L-alanine, D-alanine, L-glutamine, D-glutamine and L-Cys(acetamidomethyl) respectively. The reaction is accomplished without the occurrence of secondary peptide hydrolysis. Under optimum reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 4.8% ethanol, 40 degrees C), the peptide yields ranged from 5 to 91%, depending on the structure of the amino and/or carboxyl component. No racemization was observed in the enzymatic reaction. Application of short-chain peptides has been advocated recently in clinical nutrition. Ficin-catalyzed peptide synthesis might be an attractive biotechnological approach for the synthesis of suitable dipeptides in this respect.     

Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH? group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.     

Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.     

石油降解酶的筛选、固定化及添加表面活性剂强化其对石油的降解

在石油开采和加工过程中产生的含油污泥是石油化工行业中主要的污染源,会对周边生态环境造成持续性的危害。生物法降解被认为是一种有效且可持续的技术而备受关注。目前的研究多聚焦在微生物法降解石油,而酶法降解鲜有报道。【目的】本研究旨在优选石油高效降解酶,并深入分析其降解特性,以期构建一套酶法降解石油的体系。【方法】基于分子对接模拟技术,分析酶与石油类常见底物的结合可能性及结合模式,通过石油降解实验优选降解酶;通过添加表面活性剂及酶固定化进一步提高酶对石油的降解效果。【结果】利用分子对接模拟及实验验证获得5种具有石油降解能力的生物酶,其中枯草芽孢杆菌(Bacillus subtilis)来源的漆酶BsLac对石油的降解率最高,72 h可降解34.1%的石油;进一步研究发现,表面活性剂的添加均可促进BsLac对石油的降解,其中槐糖脂的促进作用尤为显著。当槐糖脂终浓度为1 000 mg/L时,石油的降解率最高,为46.3%;然而,2,2''-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐的添加对BsLac降解石油的促进作用并不明显;固定化结果表明,以花生壳为吸附载体固定化的BsLac对石油的降解率最高,为56.3%。【结论】本研究基于分子对接模拟和实验筛选获得了石油降解酶BsLac,固定化的BsLac可实现石油的高效降解,为生物酶法降解石油的进一步探索奠定了实验和理论基础。     

Effect of hydrophobicity degree of polymer particles on lipase immobilization and on biocatalyst performance

Abstract

Many studies describe the advantages of using hydrophobic particles on lipase immobilisation. However, many of these works neglect the effect of other variables of the supports, such as specific area and porosity, on the biocatalyst performance, and do not evaluate the influence of the hydrophobicity level of the particles on the biocatalysts’ activity as a single variable. Thus, the focus of the present work was to evaluate the effect of the hydrophobicity degree of polymeric particles on the biocatalysts’ activities, mitigating the influence of other variables. The study was divided into two steps. Firstly, distinct particles, exhibiting different composition and hydrophobicity levels, were used for the immobilization of a commercial lipase B from Candida antarctica (CAL-B). Then, distinct core-shell polymeric particles presenting different functional compounds on the surface were produced, using as comonomers styrene, divinylbenzene, 1-octene, vinylbenzoate and cardanol. Such particles were subsequently used for CAL-B immobilisation and the performance of the biocatalysts was evaluated on hydrolysis (using p-nitrophenyl laurate, as substrate) and esterification (using ethanol and oleic acid, as substrate) reactions. Based on the screening step, it was observed that for non-porous particles the correlation coefficients between the hydrophobicity level of the supports and the biocatalysts performance, for both hydrolysis and esterification reactions, were very low (0.32 and 0.45, respectively). It highlights that there was no significant correlation between these variables and that, probably, the chemical composition of the polymeric chains affects more significantly the biocatalyst performance. Then, analysing the subsequent stage, it was observed that small changes in the surface composition of the core-shell particles result in significant changes on the textural properties of the supports (specific area ranging from 1.2?m².g^?1 to 18.3?m².g^?1; and contact angles ranging from 71° (hydrophilic particles) to 92° (hydrophobic supports) when polymer films were put in contact with water). Such particles were also employed on CAL-B immobilization and it was noticed that higher correlation coefficients were achieved for hydrolysis (ρ?=?0.53) and esterification (ρ?=?0.74) reactions. Therefore, it is shown that the hydrophobicity degree of such supports starts to affect more effectively the biocatalysts performance when other textural features of the supports become more significant, such as specific area and porosity.     

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process.Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications.     

Overview on immobilization of enzymes on synthetic polymeric nanofibers fabricated by electrospinning

The arrangement and type of support has a significant impact on the efficiency of immobilized enzymes. 1-dimensional fibrous materials can be one of the most desirable supports for enzyme immobilization. This is due to their high surface area to volume ratio, internal porosity, ease of handling, and high mechanical stability, all of which allow a higher enzyme loading, release and finally lead to better catalytic efficiency. Fortunately, the enzymes can reside inside individual nanofibers to remain encapsulated and retain their three-dimensional structure. These properties can protect the enzyme's tolerance against harsh conditions such as pH variations and high temperature, and this can probably enhance the enzyme's stability. This review article will discuss the immobilization of enzymes on synthetic polymers, which are fabricated into nanofibers by electrospinning. This technique is rapidly gaining popularity as one of the most practical ways to fibricate polymer, metal oxide, and composite micro or nanofibers. As a result, there is interest in using nanofibers to immobilize enzymes. Furthermore, present research on electrospun nanofibers for enzyme immobilization is primarily limited to the lab scale and industrial scale is still challanging. The primary future research objectives of this paper is to investigate the use of electrospun nanofibers for enzyme immobilization, which includes increasing yield to transfer biological products into commercial applications.     

Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization.

The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios. This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion. A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process. The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.     

A new method for immobilization of pectic enzymes

Summary Polygalacturonase and pectinesterase have been successfully immobilized on gamma alumina by activation of the support with glutaraldehyde at pH 3.0. The half life of the enzymes increased by four and two fold compared to the immobilization on gamma alumina without activation.