Enzyme immobilisation in biocatalysis: why, what and how

Xylanase are responsible enzyme for hydrolysis of xylan into many beneficial products such as xylose, xylitol and xylooligosaccharides. Due to industrial potential of xylanase, a large number of studies have become interested in their immobilisation to reduce the cost of enzyme. Immobilised enzymes are currently the object of interest due to its benefits over soluble or free enzyme applied in enzymatic hydrolysis. The aims of this study were to determine the suitable method for xylanase immobilisation and to identify the significant parameter which affecting the immobilisation yield by fractional factorial design (FFD). Immobilised xylanase was prepared using a single immobilisation techniques of entrapment and covalent binding and also a combination of entrapment and covalent binding techniques. The immobilisation conditions for xylanase which includes of sodium alginate concentration, calcium chloride concentration, agitation rate and enzyme loading were screened using FFD experimental design to determine the most significant parameters in affecting the efficiency of immobilised xylanase. The analysis of xylanase activity was determined using dinitrosalicyclic acid (DNS) method. The xylanase enzyme was successfully immobilised by entrapment in sodium alginate beads and covalent binding on the surface of beads by glutaraldehyde. The combination of entrapment and covalent binding showed the highest immobilisation yield of 65.81 % compared to a single technique which contributes only 31.99 % and 48.53 %. Glutaraldeyhde concentration showed the most significant parameters compared to the others parameter which gives about 69.01 % of contribution on the xylanase immobilisation yield. The study shows the efficiency of enzyme immobilisation could be improved by a combination of immobilisation techniques and determination of the most significant factors for xylanase immobilisation.

Enzyme immobilization on different supports has emerged as an efficient and cost-effective tool to improve their stability and reuse capacity. This work aimed to produce a stable immobilized multienzymatic system of xylanase and filter paper-ase (FPase) onto magnetic chitosan using genipin as a cross-linking agent and to evaluate its biochemical properties and reuse capacity. A mixture of chitosan magnetic nanoparticles, xylanase, and FPase was covalently bonded using genipin. Immobilization yield and efficiency were quantified. The activity of free and immobilized enzymes was quantified at different values of pH, temperature, substrate concentration (Km and Vmax), and reuse cycles. The immobilization yield, immobilization efficiency, and activity recovery were 145.3% ± 3.06%, 14.8% ± 0.81%, and 21.5% ± 0.72%, respectively, measured as the total hydrolytic activity. Immobilization confers resistance to acidic/basic conditions and thermal stability compared to the free form. Immobilization improved 3.5-fold and 78-fold the catalytic efficiency (Kcat/Km) of the xylanase and filter paper-ase activities, while immobilized xylanase and FPase could be reused for 34 min and 43 min, respectively. Cross-linking significantly improved the biochemical properties of immobilized enzymes, combined with their simplicity of reuse due to the paramagnetic property of the support. Multienzyme immobilization technology is an important issue for industrial applications.

Alpha glucosidases are multifunctional glycoside hydrolases with hydrolysis and transglycosylation ability. It can be utilized for the glycosidic bond synthesis or glycosylation of Ascorbic acid/Vitamin C to its stable analogue, Ascorbic acid 2 glucoside (AA2G), a compound with wide applications in cosmetics and pharma. The application of α-glucosidases for industrial scale transglycosylation is limited due to the low transglycosylation yield of free enzymes. Enzyme immobilization techniques could enable the development of efficient, reusable catalysts. Only a few glycoside hydrolases have been studied in immobilized form for transglycosylation reactions, and α-glucosidases are probably the least explored in this form. Transglycosylation activity of immobilized α-glucosidase from Aspergillus carbonarius BTCF 5 was studied for AA2G synthesis, where different immobilization techniques like calcium alginate encapsulation, adsorption on chitosan beads, covalent cross-linking on magnetic nanoparticles, and cross-linked enzyme aggregates (CLEA) were employed for the immobilization. The immobilization yield of calcium alginate encapsulated enzyme, enzyme immobilized on Fe-MNP support, enzyme immobilized on chitosan beads and as CLEA were 107%, 99%, 46% and 486%, respectively. CLEA was identified as the best immobilization technique for this bi-substrate reaction due to the high immobilization yield and activity retention (30% activity retained after 5 consecutive cycles). Enzyme immobilization increased the transglycosylation activity by 38%, yielding 118 mM AA2G against 72 mM by the free enzyme. This indicates the potential of immobilized α-glucosidase as a catalyst for synthesizing AA2G at an industrial scale.

Enzymatic degradation of biomass is preferred over chemical methods for environmental protection. However, in most cases, a chemical pretreatment is still required to help improve enzyme accessibility of the biomass. Therefore, it is necessary to develop highly efficient enzymes for those reasons and to save costs accordingly. Costs can be further reduced if the enzymes are recycled. XynR8(N58D) is an engineered and highly active xylanase originating from the rumen fungus Neocallimastix patriciarum. In this study, XynR8(N58D) was extracellularly over-expressed by Pichia pastoris. The resulting crude enzyme solution, with a purity of XynR8(N58D) of over 80%, was used to prepare the cross-linked enzyme aggregates (CLEA) of XynR8(N58D). Specific activity of the crude enzyme solution was 11,599.39 IU/mg, and that of CLEA was 6129.42 IU/mg. The immobilization yield and the immobilization efficiency were 62.68 ± 16.20% and 84.31 ± 3.11%, respectively. The thermostability, stress tolerance, and shelf life of CLEA were significantly better than those of the free enzyme. The free enzyme and CLEA both effectively decomposed insoluble xylan and autoclaved corncob powders. Therefore, XynR8(N58D) made into CLEA not only maintained high activity, but also improved its stability and permitted its reuse or recyclability. Both forms of the enzyme can effectively degrade lignocellulosic biomass without relying on chemical pretreatment to reduce recalcitrance of the biomass structure. The application of XynR8(N58D) and its CLEA to biomass degradation can help establish an eco-friendly and more cost-effective process.

Route to the synthesis of enantiomerically pure ethyl (S)-3-cyano-5-methylhexanoate, (S)-5, a key chiral intermediate for Pregabalin has been improved. The racemic β-cyano diester, 3 was prepared in 98% purity via gelatine catalysed Knoevenagel condensation of diethylmalonate with isovaleraldehyde followed by hydrocyantion of α,β-unsaturated diester 14 using acetone cyanohydrin and K2CO3. Racemic diethyl 2-(1-cyano-3-methylbutyl)malonate, rac-3, has been resolved using lipase from Thermomyces lanuginosus immobilised in form of crosslinked enzyme aggregates, CLEAs. The CLEAs were made by employing commercial soymilk as an additional protein source and a reaction was carried out in a moving basket reactor. The immobilised enzyme was found to be stable in many organic solvents and temperature up to 50 °C. The resolution reaction was studied in a basket reactor at 50% substrate loading in calcium acetate buffer, pH 7.5 at 30 °C by using 20% w/w enzyme loading. The apparent kinetic parameters were V max,app = (8.74 ± 0.43) mM/h/g and K m,app = (1.5 ± 0.07) M (correlation coeff. r = 0.98). The desired ethyl (S)-3-cyano-5-methylhexanoate, (S)-5 is obtained in 90-92% theoretical yield and e.e > 99%. The advantages of this improved process are mild reaction conditions; an alternate method for hydrocyanation step avoiding the use of highly toxic potassium cyanide at large scale operation and an immobilised enzyme that can be reused for at least 11 recycles.

Cross-linked enzyme aggregates (CLEAs) of α-galactosidase, partially purified from maize (Zea mays) flour, were prepared. The impact of various parameters on enzyme activity was examined to optimize the immobilization procedure. Biochemical characterization of the free and immobilized enzyme was carried out. Stability (thermal, pH, storage and operational stability) and reusability tests were performed. The potential use of the free enzyme and the CLEAs in hydrolysis processes of raffinose-type oligosaccharides present in soymilk was investigated. α-galactosidase CLEAs were prepared with 47% activity recovery under optimum conditions [1:5 (v/v) enzyme solution:saturated ammonium sulfate solution ratio; 7.5mg protein and 0.1% (v/v) glutaraldehyde, 6h, 4°C, 150 rpm]. α-galactosidase CLEAs exhibited increased stability in comparison to the free enzyme. The CLEAs and the free enzyme showed a maximum activity at 40°C and their optimal pH values were5.5 and 6.0, respectively. Kinetic constants (KM , Vmax and kcat ) were calculated for the free enzyme and the CLEAs in the presence of p-nitrophenyl-α-d-galactopyranoside, stachyose, melibiose and raffinose. The effect of various chemicals and sugars on enzyme activity showed that both enzyme forms were significantly inhibited by HgCl2 and galactose. The CLEAs hydrolyzed 85% of raffinose and 96% of stachyose. The α-galactosidase CLEAs, with their satisfactory enzymatic characteristics, have much potential for use in the food and feed industry. © 2019 Society of Chemical Industry.

Enzymes are commonly used as biocatalysts for various biological and chemical processes in industrial applications. However, their limited operational stability, catalytic efficiency, poor reusability, and high-cost hamper further industrial usage. Thus, crosslinked enzyme aggregates (CLEAs) are developed as a better enzyme immobilization tool to extend the enzymes' operational stability. This immobilization method is appealing because it is simpler due to the absence of ballast and permits the collective use of crude enzyme cocktails. CLEAs, so far, have been successfully developed using a variety of enzymes, viz., hydrolases, proteases, amidases, lipases, esterases, and oxidoreductase. Recent years have seen the emergence of novel strategies for preparing better CLEAs, which include the combi- and multi-CLEAs, magnetics CLEAs, and porous CLEAs for various industrial applications, viz., laundry detergents, organic synthesis, food industries, pharmaceutical applications, oils, and biodiesel production. To better understand the different strategies for CLEAs' development, this review explores these strategies and highlights the relevant concerns in designing innovative CLEAs. This article also details the challenges faced during CLEAs preparation and solutions for overcoming them. Finally, the trending strategies to improve the preparation of CLEAs alongside their industrial application trends are also discussed.

Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non‐aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross‐linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross‐linked enzyme aggregates (CLEAs).

In the past couple of decades, cross linked enzyme aggregates (CLEAs) have emerged as a novel and versatile carrier free immobilization technique. The immobilization of enzymes as cross linked enzyme aggregates (CLEAs) involves precipitation of an enzyme from aqueous solution followed by cross linking with a bi-functional reagent. It is worth noting that many parameters alter the enzyme precipitation and the aggregate cross linking and hence affect the activity and stability of CLEAs. Therefore to endorse CLEAs for industrial application, each newly synthesised CLEA is characterized. This review intends to investigate the effects of various parameters, such as the nature and purity of the enzyme, the nature and amount of precipitant, the nature and amount of cross linker, the cross linking time, the pH and temperature during CLEA preparation and washing and separation techniques on the activity and stability of CLEAs. The major parameters such as catalytic properties, particle size and morphology, stability and reusability required for approval of industrial applicability of newly synthesized CLEAs are critically reviewed. Furthermore the scope of CLEAs in non-aqueous solvent, the development of one pot cascade processes and the design of different types of enzyme reactors is also discussed.

Effect of the degree of cross-linking on the properties of different CLEAs of penicillin acylase

Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase

Different Methods of Acid Phosphatase Immobilization for Its Application in FIA Systems with Potentiometric Detection

Silanized maghemite for cross-linked enzyme aggregates of recombinant xylanase from Trichoderma reesei

Immobilized enzymes are used in analytical chemistry and as catalysts for the production of chemicals, pharmaceuticals and food. Because of their particular structure, immobilized enzymes require optimal conditions, different from those of soluble enzymes. Particle size, particle-size distribution, mechanical and chemical structure, stability and the catalytic activity, used for immobilization, must be considered. Generally, cellulases are used in various industries, including food, brewery and wine, agriculture, textile, detergent, animal feed, pulp and paper, and in research development. For the industrial application of cellulase, its immobilization, which allows the conditions of repeated use of the enzyme alongside retaining its activity, has been recently investigated. Celullase was immobilized with the use of glutaraldehyde, a covalent cross-linking agent in to cross-linked enzyme aggregates (CLEAs). The stability and activity of cross-linked cellulase, exposed to carbon dioxide under high pressure, were studied. Efficiency of enzyme immobilization was determined using Bradford method (Bradford, 1976). The activity of cross-linked cellulase was determined by spectrophotometric method.

Enzyme immobilization technology as a tool to innovate in the production of biofuels: A special review of the Cross-Linked Enzyme Aggregates (CLEAs) strategy