Immobilization of microbial protease on vermiculite

Vermiculite, an inert and cheap solid support material, was used in the immobilization of protease by adsorption. Adsorption of protease on vermiculite saturated with potassium, calcium and aluminium was studied. Aluminium saturated vermiculite adsorbed maximum amount of enzyme at pH 6.5. The maximum adsorption of enzyme on cationic vermiculite occurred within one hour at 30°C. When the temperature was increased there was a two fold increase in the adsorption of the enzyme. From the Freundlich isotherm data, the values of k and n were computed.     

Immobilization of urease on vermiculite

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses.     

Immobilization of alkaline protease on nylon

The parameters involved in immobilization of alkaline protease on nylon using glutaraldehyde as coupling agent and the characteristics of the immobilized enzyme were investigated. Optimum temperature and pH of both free and immobilized enzyme for the degradation of protein was found. Immobilized enzyme showed better thermal stability than the free enzyme. The reusability and storage stability of the immobilized enzyme was also studied.     

Development of novel robust nanobiocatalyst for detergents formulations and the other applications of alkaline protease

Alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized rattle-type magnetic core@mesoporous shell silica (RT-MCMSS) nanoparticles by physical adsorption and covalent attachment. However, the covalent attachment approach was superior for NPST-AK15 protease immobilization onto the activated RT-MCMSS-NH₂ nanoparticles and was used for further studies. In comparison to free protease, the immobilized enzyme exhibited a shift in the optimal temperature and pH from 60 to 65 °C and pH 10.5–11.0, respectively. While free protease was completely inactivated after treatment for 1 h at 60 °C, the immobilized enzyme maintained 66.5 % of its initial activity at similar conditions. The immobilized protease showed higher k _cat and K _m , than the soluble enzyme by about 1.3-, and 1.2-fold, respectively. In addition, the results revealed significant improvement of NPST-AK15 protease stability in variety of organic solvents, surfactants, and commercial laundry detergents, upon immobilization onto activated RT-MCMSS-NH₂ nanoparticles. Importantly, the immobilized protease maintained significant catalytic efficiency for ten consecutive reaction cycles, and was separated easily from the reaction mixture using an external magnetic field. To the best of our knowledge this is the first report about protease immobilization onto rattle-type magnetic core@mesoporous shell silica nanoparticles that also defied activity-stability tradeoff. The results clearly suggest that the developed immobilized enzyme system is a promising nanobiocatalyst for various bioprocess applications requiring a protease.     

米曲霉F-81产中性蛋白酶的固定化条件及其特性

以海藻酸钠为载体,戊二醛为交联剂固定化米曲霉F-81产中性蛋白酶,研究了固定化条件及固定化酶的性质。结果表明,固定化的最佳条件为:固定化时间1 h、海澡酸钠浓度4%、戊二醛浓度9%、CaCl2浓度0.7 mol/L。在此条件下固定化的中性蛋白酶活力为游离酶活力的68%。固定化酶的最适作用温度为65℃,最适作用pH值为7.0。60℃下酶稳定性较好,80℃下处理60 min,粗酶中几乎检测不到酶活力;中性蛋白酶pH稳定范围为6.5-9.5。Km值为24.83 mg/mL,最大反应速率Vmax为0.043 12 mg/min。     

Protease production by free and immobilized cells of the cold-adapted yeast Cryptococcus victoriae CA-8

The present study was performed to produce the protease using free and immobilized cells of locally isolated cold-adapted psychrotolerant yeast Cryptococcus victoriae CA-8. Cell immobilization was performed using sodium alginate as entrapping agent. The best conditions for enzyme production by both free and immobilized cells of the yeast were temperature of 15°C and initial pH of 8.0. The optimal incubation times were 72 and 96 h for immobilized and free cells, respectively. Immobilized cells were reused in 3 successive reaction cycles without any loss in the maximum protease activity. Little decreases in the protease activity were observed in 4 and 5 cycles. Under the optimized conditions, the maximum enzyme activities were determined as 12.1 and 13.5 U/mL for free and immobilized cells, respectively. This is a first attempt on cold-active alkaline protease production by free and/or immobilized cells of yeasts. Besides, the protease activity of the yeast C. victoriae CA-8 was investigated for the first time in the present study.     

Digestion of protein substrates by subtilisin: immobilization changes the pattern of products

Subtilisin BPN' (Bacillus protease strain N') was immobilized on glass-bead carriers of controlled pore size by the glutaraldehyde method. The Vmax and Km values of the synthetic substrate were similar for immobilized and free enzymes. However, the hydrolytic patterns of immobilized and free enzymes toward casein and carboxymethylated lysozyme were different. The free enzyme rapidly hydrolyzed the substrate in the early stage of the reaction to produce peptides of various sizes. The immobilized enzyme, however, slowly digested the casein and lysozyme during digestion; even in the late stage of digestion the original substrates were present in the reaction mixture. The peptide size produced by immobilized enzyme depended on the pore size of the carrier; enzyme immobilized on glass of smaller pore size produced smaller peptide products. These phenomena found with our system of immobilized protease and a protein substrate can be explained by a multiple attack mechanism, in which the substrate that has been forced to enter the matrix is attacked many times by the protease to be completely hydrolyzed, because the substrate and the intermediate-sized product are trapped inside the matrix under reduced diffusion movement. To explain the effective digestion that forms amino acids, we have proposed that a multiple type of attack is responsible for the intracellular protein degradation that takes place in cellular organelles in which hydrolytic enzymes are entrapped.     

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology

The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.     

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K_m, V_m, and k_cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16^th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG^≠ (activation free energy) and ΔG^≠_E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG^≠_ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS^≠ value implied that the immobilized form of the enzyme was more ordered than its free form.     

A novel support for the immobilization of lipase and the effects of the details of its preparation on the hydrolysis of triacylglycerides

Summary The lipase from Candida cylindracea was immobilized by its adsorption on the internal surface of hydrophobic microporous poly(styrene-divinylbenzene) supports prepared by the concentrated emulsion polymerization method. The prepared supports have a surface area of the order of 200 m²/g. The immobilized enzyme catalyst is used for the hydrolysis of triacylglycerides. The effects of the amounts of surfactant and divinylbenzene used in the preparation of the hydrophobic support on the adsorption capacity for lipase and on the activity of the immobilized lipase have been investigated. The activity of the immobilized enzyme per enzyme molecule can be higher than that of the free lipase.     

Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes

In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produced a protease useful for the deproteinization of shrimp and crab shell wastes. The optimal culture conditions for P. aeruginosa K-187 to attain the highest protease activity were investigated and discussed. The highest protease activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prior to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinization. The percent of protein removal for shrimp and crab shell powder (SCSP) after 7-day incubation was 72%, while that of natural shrimp shell (NSS) and acid-treated SCSP was 78% and 45%, respectively. In contrast, with the protease produced under pre-optimization conditions, the percent of protein removal for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectively. For comparison, three other protease-producing microbes were tested for crustacean waste deproteinization. However, they were shown to be less efficient in deproteinization than P. aeruginosa K-187. The crude protease produced by P. aeruginosa K-187 can be covalently immobilized on a reversibly soluble polymeric support (hydroxypropyl methycellulose acetate succinate). The immobilized enzyme was soluble above pH 5.5 but insoluble below pH 4.5. Immobilization efficiency was 82%. The immobilized enzyme was stable between pH 6 and 9 and at temperatures below 60 degrees C. The optimum pH and temperature for the immobilized enzyme was pH 8 and 50 degrees C. The half-life of the immobilized enzyme was 12 days, longer than that of free protease (8 days). The utilization of the immobilized enzyme for the deproteinization of SCSP has resulted in a 67% protein removal. By contrast, SCSP protein removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, and Sephacryl S-200 gel-permeation chromatography. The enzyme had a molecular weight estimated to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was active from pH 7 to 9 and its optimal pH was 8.     

Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles

Mucor javanicus lipase was effectively immobilized on silica nanoparticles which were prepared by Stöber method. Glycidyl methacrylate (GMA), which bears a reactive epoxide group, was incorporated onto the surface of the nanoparticles and the epoxide groups were directly used for multipoint coupling of the enzyme. We also introduced amine residues by coupling ethylene diamine (EDA) to the epoxide group of GMA. M. javanicus lipase was covalently immobilized onto the amine-activated silica nanoparticles by using glutaraldehyde (GA) or 1,4 phenylene diisothiocyanate (NCS) as a coupling agent. The lipase loading capacities of the EDA-GA and EDA-NCS nanoparticles (81.3 and 60.9 mg g⁻¹, respectively) were much higher than that of the unmodified GMA nanoparticles (18.9 mg g⁻¹). The relative hydrolytic activities in an aqueous medium of the lipases immobilized on EDA-GA and EDA-NCS attached silica nanoparticles (115% and 107%, respectively) were significantly high and almost in the same range with the free enzyme. This may be due to the improvement of the enzyme–substrate interaction by avoiding the potential aggregation of free lipase molecules. The immobilized lipases were also more resistant to temperature inactivation than the free form. This work demonstrates that the size-controlled silica nanoparticles can be efficiently employed as host materials for enzyme immobilization leading to high activity and stability of the immobilized enzymes.     

Stabilization of alpha-chymotrypsin by covalent immobilization on amine-functionalized superparamagnetic nanogel

Stabilization of alpha-chymotrypsin (CT) by covalent immobilization on the amine-functionalized magnetic nanogel was studied. The amino groups containing superparamagnetic nanogel was obtained by Hoffman degradation of the polyacrylamide (PAM)-coated Fe(3)O(4) nanoparticles prepared by facile photochemical in situ polymerization. CT was then covalently bound to the magnetic nanogel with reactive amino groups by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide as coupling reagent. The binding capacity was determined to be 61mg enzyme/g nanogel by BCA protein assay. Specific activity of the immobilized CT was measured to be 0.93U/(mgmin), 59.3% as that of free CT. The obtained immobilized enzyme had better resistance to temperature and pH inactivation in comparison to free enzyme and thus widened the ranges of reaction pH and temperature. The immobilized enzyme exhibited good thermostability, storage stability and reusability. Kinetic parameters were determined for both the immobilized and free enzyme. The value of K(m) of the immobilized enzyme was larger than did the free form, whereas the V(max) was smaller for the immobilized enzyme.     

Some properties of free and immobilized alpha-amylase from Penicillium griseofulvum by solid state fermentation

Alpha-amylase was produced from Penicillium griseofulvum by an SSF technique. Alpha-amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5 degrees C and 40 degrees C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver-Burk plots for both enzymes. Km values were 9.1 mg mL(-1) for free enzyme, and 7.1 mg mL(-1) for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the a-amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.     

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.     

Improved biochemical characteristics of crosslinked β-glucosidase on nanoporous silica foams

Large mesoporous cellular foam (LMCF) materials were synthesized using the microemulsion templating route. For the enzyme stabilization, β-glucosidase was immobilized onto mesocellular silica foams (MCFs) in a simple and effective way, a process achieved using enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of crosslinked enzyme aggregates (CLEAs) of nanometer scale. The structural and chemical properties of these prepared materials were characterized by TG, CPMAS NMR and nitrogen adsorption measurements. The crosslinked immobilizates retained activity over wider ranges of temperature and pH than those of the free enzyme. Kinetic parameter (K_m) of the immobilized β-glucosidase is lower than that of its free counterpart. The resulting CLEA was proved to be active and recyclable up to 10 cycles without much loss in activity. This demonstrates its prospects for commercial applications. The immobilizate exhibited enhanced storage stability characteristics than the native enzyme. In contrast to adsorbed GL and covalently bound glucosidase, the resulting crosslinked enzyme aggregates (CLEAs) showed an impressive stability with high enzyme loadings.     

The use of crude lipase in deprotection of C-terminal protecting groups

A crude lipase, Newlase F, was used to remove C-terminal protecting groups from dipeptide esters. Hydrolysis of dipeptide n-heptyl esters with Newlase F was conducted in aqueous media containing acetonitrile. The optimum pH and temperature of lipase in Newlase F were 7.0 and 30 °C, respectively. Low level acetonitrile promoted the hydrolysis of dipeptide n-heptyl esters, while high level acetonitrile inhibited the hydrolysis. However, the protease activity in Newlase F was significantly inhibited by acetonitrile. Lipase in Newlase F worked better in a medium containing water-miscible organic solvents than in water-immiscible ones. N-terminal protecting groups were not affected by the protease in the crude enzyme. It was found that the protease in Newlase F did not hydrolyze amide bond with hydrophilic amino acids on either side under these conditions (pH 7.0, room temperature). Newlase F may consequently be used widely in the synthesis of peptide conjugates. The crude enzyme was immobilized on SBA-15 mesoporous molecular sieve. The lipase activity of immobilized preparation was more active on hydrolysis of C-terminal protecting groups and stable than the free enzyme. The immobilization also reduced the protease activity.     

Raw Starch-digestive Chitin-immobilized Amylase from a Protease—Glycosidase-less Mutant of Aspergillus awamori var. kawachi

The α-amylase and glucoamylase produced by a protease-, glycosidase-less mutant HF-15 of Aspergillus awamori var. kawachi were found to be adsorbable onto chitin. This adsorption was pH-independent, different from the adsorption onto raw corn starch. The binding between amylases and chitin was so tight that a chitin-immobilized amylase was obtained without the aid of a cross linking agent, glutaraldehyde, and it retained more than 90% of the original activity of the free enzyme. The immobilized amylase digested gelatinized potato starch, glycogen and even raw corn starch to the same high extent as glucose similar to the free enzyme, but it was different from the unbound crude enzyme in the lack of transglucosidase activity, and slightly different in pH- and thermo-stabilities. An experiment using the immobilized amylase for alcohol fermentation demonstrated the possibility of recycling the enzyme for raw starch saccharification.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.