Phospholipase D production using immobilized cells of Streptoverticillium cinnamoneum

Summary Production of phospholipase D (PLD) by Streptoverticillium cinnamoneum immobilized within porous particles was investigated in repeated batch fermentation. The enzyme productivity in repeated batch fermentation was 2.2-fold that obtained in batch fermentation without immobilization, since many of the immobilized cells could be utilized as seed cells for each subsequent batch cycle.     

Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale three-phase fluidized bed reactor

The main objective of this work was to investigate the removal of aqueous phenol using immobilized enzymes in both bench scale and pilot scale three-phase fluidized bed reactors. The enzyme used in this application was a fungal tyrosinase [E.C. 1.14.18.1] immobilized in a system of chitosan and alginate. The immobilization matrix consisted of a chitosan matrix cross-linked with glutaraldehyde with an aliginate-filled pore space. This support matrix showed superior mechanical properties along with retaining the unique adsorptive characteristics of the chitosan. Adsorption of the o-quinone product by the chitosan reduced tyrosinase inactivation that is normally observed for this enzyme under these conditions. This approach allowed reuse of the enzyme in repeated batch applications. For the bench scale reactor (1.2-l capacity) more than 92% of the phenol could be removed from the feed water using an immobilized enzyme volume of 18.5% and a residence time of the liquid phase of 150 min. Removal rates decreased with subsequent batch runs. For the pilot scale fluidized bed (60 l), 60% phenol removal was observed with an immobilized enzyme volume of 5% and a residence time of the liquid phase of 7 h. Removal decreased to 45% with a repeat batch run with the same immobilized enzyme.     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

Production of alkaline protease with immobilized cells of bacillus subtilis PE-11 in various matrices by entrapment technique

The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric productivity with calcium alginate was 15.11 U/mL/hour, which was 79.03% higher production over the conventional free-cell fermentation. Similarly, the specific volumetric productivity by repeated batch fermentation was 13.68 U/mL/hour with k-Carrageenan, 12.44 U/mL/hour with agar-agar, 11.71 U/mL/hour with polyacrylamide, and 10.32 U/mL/hour with gelatin. In the repeated batch fermentations of the shake flasks, an optimum level of enzyme was maintained for 9 days using calcium alginate immobilized cells. From the results, it is concluded that the immobilized cells ofB subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation. The alginate immobilized cells ofB subtilis PE-11 can be proposed as an effective biocatalyst for repeated usage for maximum production of alkaline protease. Published: October 21, 2005     

Immobilization of goat brain aminopeptidase B in calcium alginate beads

Aminopeptidase B, an arginyl aminopeptidase, was purified from goat brain with a purification factor of ～280 and a yield of 2.7%. It was entrapped in calcium alginate together with bovine serum albumin. The optimal conditions for immobilization for maximum activity yield were 1% CaCl₂ and 2.5% alginate. The immobilized enzyme retained ～62% of its initial activity and could be used for five successive batch reactions with retention of 30% of the initial activity. The pH and temperature optima of the free and immobilized enzyme were pH 7.4, 45°C and pH 7.8, 50°C respectively, while the pH and thermal stability as well as the stability of the enzyme in organic solvents were improved significantly after entrapment. The K_m value for the immobilized enzyme was about twofold higher than that of the soluble enzyme. Because of this increased stability, the immobilized enzyme may be useful in the meat processing industry.     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

Entrapment of purified novel dextransucrase obtained from newly isolated Acetobacter tropicalis and its comparative study of kinetic parameters with free enzyme

Abstract

Purified Acetobacter tropicalis dextransucrase was immobilized in different matrices viz. calcium-alginate, κ-carrageenan, agar, agarose and polyacrylamide. Calcium-alginate was proved to be superior to the other matrices for immobilization of dextransucrase enzyme. Standardization of immobilization conditions in calcium-alginate resulted in 99.5% relative activity of dextransucrase. This is the first report with such a large amount of relative activity as compared to the previous reports. The immobilized enzyme retained activity for 11 batch reactions without a decrease in activity which suggested that enzyme can be used repetitively for 11 cycles. The dextransucrase was also characterized, which revealed that enzyme worked best at pH 5.5 and 37?°C for 30?min in both the free as well as immobilized state. Calcium-alginate immobilized dextransucrase of A. tropicalis showed the K_m and V_max values of 29?mM and 5000?U/mg, respectively. Free and immobilized enzyme produced 5.7?mg/mL and 2.6?mg/mL of dextran in 2?L bench scale fermenter under optimum reaction conditions. This immobilization method is very unconventional for purified large molecular weight dextran-free dextransucrase of A. tropicalis as this method is used usually for cells. Such reports on entrapment of purified enzyme are rarely documented.     

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.     

Stabilization and activity-enhancement of mandelate racemase from Pseudomonas putida ATCC 12336 by immobilization

Mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12336 was efficiently immobilized through ionic binding onto DEAE- and TEAE 23-cellulose. The activity of the immobilized enzyme was significantly enhanced as compared to the native protein, i.e., 2.7- and 2.5-fold, respectively. DEAE-cellulose-immobilized mandelate racemase could be efficiently used in repeated batch reactions for the racemization of (R)-mandelic acid under mild conditions.     

Thermo-sensitive amphiphilic block copolymer poly (styrene-<Emphasis Type="Italic">b</Emphasis>-N-isopropylacrylamide) with switchable catalytic activity immobilizing pectinase

Pectinase was immobilized onto thermo-sensitive amphiphilic block copolymers poly(styrene-b-Nisopropylacrylamide) PS-b-poly(N-isopropylacrylamide) (PNIPAM) by covalent attachment. Biochemical studies have found that the stability of the PS-b-PNIPAM support is not impeded by the bound proteins despite that up to 242.5 mg of enzyme is immobilized per gram of carrier particles. The immobilized enzyme retained nearly 65% of its initial activity over 30 days, and the optimum temperature and pH also increased to the range of 60 ∼ 70°C and 4.0 ∼ 6.0, respectively. The immobilized enzyme also exhibited great operational stability, and more than 60% residual activity was observed in the immobilized enzyme after 10 batch reactions. Moreover, the lower critical solution temperature of the PS-b-PNIPAM support could be switched on or off by a small change in solution temperature. Thus, the immobilized pectinase could be recovered and showed durable activity during the recycle process.     

Immobilization of acetylcholinesterase in nanofibrous PVA/BSA membranes by electrospinning

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted continuous attention in the field of enzyme immobilization. In this study, acetylcholinesterase (AChE) has been successfully immobilized in PVA nanofibers via electrospinning of a mixture of AChE, BSA as an enzyme stabilizing additive and PVA. The maximum activity recovery of immobilized AChE was about 40%. In comparison with free enzyme, the immobilized AChE showed improved stability while retaining a considerable amount of activity at lower pH values. Moreover, the immobilized AChE retained >34% of its initial activity when stored at 30°C for 100 days and retained 70% of its initial activity after ten consecutive reactor batch cycles.     

Enhanced production of a thermostable mannanase by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> on various membranes

Cells of the thermophilic Bacillus subtilis WY34 were immobilized on various formaldehyde-activated polymer membranes and the immobilized cells were used for the production of thermostable mannanase in flasks. The results showed that polyethersulfone membranes (PES) and nylon-6 membranes were the most suitable supports for cell immobilization to produce the mannanase. Moreover, PES and nylon-6 membranes immobilized cells provided 1.78- and 1.74-fold higher mannanase activity compared to the control after 4 days of cultivation, respectively. The immobilized cells on PES and nylon-6 membranes had good stability and retained 131.5 and 114.3% of ability of enzyme production even after six cycles of repeated batch fermentation, respectively. Active cell growth was observed by scanning electron microscopy (SEM) after 16 days (four cycles) repeated batch cultivation. Therefore, the membrane-immobilized cells of B. subtilis WY34 can be proposed as an effective biocatalyst for repeated usage for production of the thermostable mannanase.     

Improvement of acid phosphatase production by immobilization of Humicola lutea mycelium in polyurethane sponge

Acid phosphatase production by the fungus Humicola lutea 120-5, immobilized in polyurethane sponge, was studied under semicontinuous shake flask fermentation and compared to the enzyme secretion by free cells. The effect of parameters such as the carrier content and the duration of the batch in repeated batch experiments on the phosphatase production half-life was investigated. The best results were obtained with 1.0 g of sponge cubes (about 1.0 cm per side) per culture flask using 72 h runs. In these conditions the half-life of enzyme production by immobilized biocatalyst was 15 sequential cycles (45 days) compared to three cycles (9 days) for the free mycelium. The maximal phosphatase titre registered in free cell fermentation was 2500 U/l (i.e. 100%), while the relative enzyme activity of the optimal immobilized system was over 100% during the whole half-life time of 45 days. Significant improvement (200–215%) in the yield was observed in one-third of this period or 15 days. The supernatant medium obtained at any stage of the repeated batch cultures did not contain free cells and, due to the low pH (3.0–3.5), the whole process was carried out without any bacterial contamination. In comparison with free cell fermentation, the significant improvement of the acid phosphatase production by polyurethane sponge-immobilized H. lutea mycelium as well as its operation stability was confirmed by scanning electron microscopy.     

Properties of fibre-entrapped aspartase

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) was extracted and purified from Escherichia intermedia cells. The enzyme was entrapped in cellulose triacetate porous fibres and the properties of the immobilized enzyme compared with those of the free enzyme. Similar behaviour was observed with regard to optimum pH, temperature, heat stability and kinetic constants. The stability of the entrapped enzyme was tested under operating conditions in a series of batch reactions. Good results were obtained for both the stability and the efficiency of the immobilized enzyme. The potential use of aspartase fibres for the production of l-aspartic acid is discussed.     

Prediction of continuous reactors performance based on batch reactor deactivation kinetics data of immobilized lipase

Experiments on deactivation kinetics of immobilized lipase enzyme fromCandida cylindracea were performed in stirred batch reactor using rice bran oil as the substrate and temperature as the deactivation parameter. The data were fitted in first order deactivation model. The effect of temperature on deactivation rate was represented by Arrhenius equation. Theoretical equations were developed based on pseudo-steady state approximation and Michaelis-Menten rate expression to predict the time course of conversion due to enzyme deactivation and apparent half-life of the immobilized enzyme activity in PFR and CSTR under constant feed rate policy for no diffusion limitation and diffusion limitation of first order. Stability of enzyme in these continuous reactors was predicted and factors affecting the stability were analyzed.     

Immobilization and stabilization of papain on poly(hydroxyethyl methacrylate-ethylenglycol dimethacrylate) beads grafted with epoxy functional polymer chains via surface-initiated-atom transfer radical polymerization (SI-ATRP)

Poly(hydroxyethyl methacrylate–ethylen glycol dimethacrylate), p(HEMA–EGDMA), beads were prepared by suspension polymerization, and were decorated with fibrous poly(glycidyl methacrylate), p(GMA), via surface initiated-atom transfer radical polymerization (SI-ATRP). The functional epoxy groups of the beads were used for covalent immobilization of papain. The average amount of immobilized enzyme was 18.7 mg/g beads. The immobilized enzyme was characterized by temperature, pH, operational and storage stability experiments. The maximum velocity of the free and immobilized enzymes (V_max) and Michaelis–Menten constant (K_m) values were determined as 10.7 and 8.3 U/mg proteins and 274 and 465 μM, respectively. The immobilized papain was operated in a batch reactor, and it was very effective for hydrolysis of different proteins (i.e., casein and cytochrom c).     

A new method of whole microbial cell immobilization

Summary A simple method for producing gelatin-immobilized microbial cells is described. The microorganism used as an example was baker's yeast (Saccharomyces cerevisiae). The gel particles containing these cells were utilized as an immobilized enzyme (invertase) both in stirred batch and packed bed systems.     

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.     

Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis

Summary Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%–100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity.     

活性炭吸附固定化粪产碱杆菌青霉素G酰化酶

目的：以活性炭为载体固定化粪产碱杆菌来源的青霉素G酰化酶,考察固定化酶的性质。方法：对影响酶固定化的因素优化筛选,确定有显著影响的因素：pH、离子强度、酶量、固定化时间进行L934的正交实验,获得最佳固定化条件,并对固定化酶的最适反应温度、pH及批次稳定性进行研究。结果：最佳固定化条件为：载体0.3g,酶量5mL,总反应体系为12mL,离子强度1mol/L,温度4℃,pH 7.0,固定化40h;最高固定化酶活性为135.9U/g湿载体。固定化酶性最适反应温度为55℃,最适pH为10,重复使用12次后没有活性损失。结论：活性炭吸附固定化青霉素G酰化酶的活性高,批次反应稳定,具有工业应用潜力。