Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.     

Isolation and properties of immobilized alkaline phosphatase from E. coli

Preparations of alkaline phosphatase from E. coli, immobilized on Sepharose, with a specific activity of 40-60 U/g wet weight were obtained. The immobilized enzyme was stable up to 50 degrees C; at higher temperatures it was inactivated. At 70 degrees most of the activity was lost for 1 h. The substrate (AMP) stabilized the enzyme. In the temperature range from 30 to 40 degrees C activation of the enzyme was observed, especially pronounced in the presence of the substrate. The pH optimum of the immobilized enzyme activity (7.8-8.2) is shifted towards the acid region, as compared to the soluble enzyme (8.0-8.6). The kinetic parameters for inhibition by the reaction product were determined using the integral Michaelis-Menten equation. KmAMP was found to be higher in case of the immobilized enzyme as compared to the soluble one (5.02 X 10(-4) M and 1.85 X 10(-5) M, respectively), which seems to be associated with diffusion limitations.     

Immobilization of Escherichia coli cells containing aspartase activity with kappa-carrageenan. Enzymic properties and application for L-aspartic acid production.

Whole cells of Escherichia coli having high aspartase (L-asparate ammonialyase, EC 4.3.1.1) activity were immobilized by entrapping into a kappa-carrageenan gel. The obtained immobilized cells were treated with glutaraldehyde or with glutaraldehyde and hexamethylenediamine. The enzymic properties of three immobilized cell preparations were investigated, and compared with those of the soluble aspartate. The optimum pH of the aspartase reaction was 9.0 for the three immobilized cell preparations and 9.5 for the soluble enzyme. The optimum temperature for three immobilized cell preparations was 5--10 degrees C higher than that for the soluble enzyme. The apparent Km values of immobilized cell preparations were about five times higher than that of the soluble enzyme. The heat stability of intact cells was increased by immobilization. The operational stability of the immobilized cell columns was higher at pH 8.5 than at optimum pH of the aspartase reaction. From the column effluents, L-aspartic acid was obtained in a good yield.     

温和气单孢菌YH311硫酸软骨素裂解酶的分离纯化与固定化

通过硫酸铵沉淀、QAESephadex-A50柱层析及Sephadex-G150凝胶过滤等纯化步骤,对源自温和气单孢菌YH311的ChSase进行了分离纯化。结果表明,ChSase经上述纯化步骤后被纯化了55倍,其最终纯度可达95%以上,比活为31.86u/mg。经SDSPAGE及IFE测定可知该酶的分子量约为80kD,等电点为4.3～4.8。将纯化后的ChSase用海藻酸钠或纤维素固定化后,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高：固定化酶用80℃水浴处理120min或于4℃冰箱放置30d后仍可保留50%以上的相对活力;但固定化酶的收率较低,仅为18.56%和18.86%。     

Immobilization of chitosan-invertase neoglycoconjugate on carboxymethylcellulose-modified chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on a carboxymethylcellulose-coated chitin support via polyelectrolyte complex formation. The yield of immobilized protein was determined to be 72% and the enzyme retained 68% of the initial invertase activity. The optimum temperature for invertase was increased by 5 degrees C and its thermostability was enhanced by about 9 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 12.6-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The prepared biocatalyst retained 98% and 100% of the original catalytic activity after 10 cycles of reuse and 70 h of continuous operational regime in a packed bed reactor, respectively. The immobilized enzyme retained 95% of its activity after 50 days of storage at 37 degrees C.     

Production and properties of beta-mannanase by free and immobilized cells of Aspergillus oryzae NRRL 3488

Seven fungi were tested for production of mannanases. The highest mannanase activities were produced by Aspergillus oryzae NRRL 3488 after 7 days in static cultures. Mannanases were induced by gum locust bean (1.0%). The highest mannanase activity was produced when a mixture of peptone, urea and ammonium sulphate was used as nitrogen source. Zn2+ or Co2+ favoured enzyme production. The immobilized cells on Ca-alginate and agar were able to produce beta-mannanase for four runs with a slight decrease in the activity. The optimum temperature for enzyme reaction was 50-55 degrees C at pH 6.0. In the absence of substrate the enzyme was thermostable retaining 75% activity for 1 h at 50 degrees C, and 68% activity for 1 h at 60 degrees C.     

Immobilization of cycloisomaltooligosaccharide glucanotransferase for the production of cycloisomaltooligosaccharides from dextran

Immobilization of cycloisomaltooligosaccharide glucanotransferase (CITase) and its application in the production of cycloisomaltooligosaccharides (CIs) from dextran were studied. Among various carrier materials examined, the enzyme adsorbed physically on Chitopearl BCW-3505 showed the highest activity (1.75 U/ml carrier). The activity remaining was 35%. The maximum CI yield in batch reactions at 0.2, 2 and 10% dextran was 28, 24 and 12%, respectively. The maximum CI yield at 2% dextran (24%) was slightly less than that with the free enzyme under the same conditions (26%). The concentration of linear oligosaccharides, the byproducts in the reaction mixture, was greater with the immobilized CITase than the free enzyme. The immobilized CITase was less thermostable than the free enzyme by about 10 degrees C. The pattern of influence of Ca(2+) concentration on the thermostability differed between the free and immobilized CITase. A Ca(2+) concentration of 50-100 mM was optimum for the thermostability of the immobilized CITase, 10-50 mM for the free enzyme. CIs were produced continuously by a column system packed with the immobilized enzyme at 40 degrees C with a space velocity (SV) of 6 h(-1). The three quarters life time was 4 weeks. We think that relatively long life time at fast SV was accomplished and CI production cost by this method should be lower than the batch reaction. This is the first report on immobilization of CITase.     

The preparation and characterization of an immobilized l-glutamic decarboxylase and its application for determination of l-glutamic acid

This paper is to study the preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) and develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO(2) electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The dynamic response of Na(2)HPO(4)-citric acid buffer system selected is much better than that of the others, 0.10 M HAc-0.10 M NaAc and 0.10 M sodium citrate-0.10 M citric acid. The initial rate of the enzyme reaction v(0) in this buffer system is 1.76 mol. l(-1) min(-1), moreover, the rate of the enzyme reaction appears linear in the first 4 min. The optimum adsorption equilibrium time is around 6 h. The amount of enzyme adsorbed on CM-CADB resin affects the response to substrate L-glutamic acid, the widest range of linearity is obtained with over 30 mg (GDC)/g(resin). The GDC activity immobilized on CM-CADB reaches a maximum when the immobilization temperature was kept around 40 degrees C. pH was kept at 4.4 when measuring the activity of the immobilized GDC. No variation of the activity of immobilized GDC is observed when the capacity is over 2.5 meq/g.(CM-CADB resin). The properties of the immobilized enzyme on CM-CADB were characterized. No significant improvement can be achieved when the substrate concentration exceeds 12.00 mmol/l, where the activity of immobilized GDC is equal to 1.58 mmol/l.min.g. The optimum pH is found to be 5.2, which changes 0.2 unit, comparing with that of the free GDC (5.0). The optimum temperature is found to be around 48 degrees C, which is lower than that of free GDC (55 degrees C). The critical temperature of the free GDC and the immobilized GDC is approximately 50 degrees C and 45 degrees C, respectively. The half-life of the activity is 127 days when the immobilized enzyme was stored in the cold (4 degrees C). An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO(2) electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamic acid. The determination conditions are that the buffer solution is 0.10 M Na(2)HPO(4)-0.05 M citric acid at pH 4.4 and t = 37 degrees C. The limit of detection is 1.0 x 10(-)(5) M. The linearity response is in the range of 5 x 10 (-2) - 5 x 10 (-5) M. The equation of linear regression of the calibration curve is y = 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamic acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

Characterization and evaluation of Aspergillus oryzae lactase coupled to a regenerable support

A derivative of crosslinked Sepharose, p-(N-acetyl-L-tyrosine azo) benzamidoethyl-CL-Sepharose 4B, was synthesized and used for the selective immobilization of thermostable lactase from Aspergillus oryzae.Preparations of soluble and immobilized lactase were evaluated under initial velocity conditions in a batch process. Immobilization had no significant effect on the pH optimum at 50 degrees C or kinetic parameters at pH 4.5 or pH 6.5 and 50 degrees C. At pH 4.5, the soluble enzyme possessed maximum activity at 60 degrees C and the immobilized at 55 degrees C; at pH 6.5 both showed maximum activity at 55 degrees C. The activation energy, entropy, and enthalpy decreased significantly with immobilization at pH 4.5 but not at pH 6.5. When the immobilized enzyme was placed in a packed-bed reactor, the effect of temperature on activity was altered as reflected by a marked decrease in the thermodynamic parameters of activation at both pH levels. Upon immobilization there was also a dramatic increase in the apparent thermal stability of the lactase, and the mean half-life at 50 degrees C was increased from 7.2 to 13 days at pH 4.5 and from 3.8 to 16 days at pH 6.5.     

Immobilization of the serine protease from Thermomonospora fusca YX on porous glass

As much as 84% of the thermostable serine protease from Thermomonospora fusca strain YX was covalently attached to silanized glass using glutaraldehyde. The immobilized protease exhibited a higher temperature optimum (86 degrees C) and pH optimum (9.4) for activity compared to soluble YX-protease (80 degrees C and pH 9.0, respectively). Immobilization improved enzyme thermo-stability above 90 degrees C and reduced inactivation during prolonged storage (9% loss of activity after 90 days at 12 degrees C). A continuous-flow column reactor packed with immobilized protease readily hydrolyzed casein over broad ranges of temperature and pH.     

Immobilized cyclomaltodextrin glucanotransferase of an alkalophilic Bacillus sp. No. 38-2

Cyclomaltodextrin glucanotransferase [1,4-alpha-D-glucan-4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), E.C.-2.4.1.19] of an alkalophilic Bacillus sp. No. 38-2 (ATCC 21783), which contains three types of enzymes (acid, neutral, and alkaline enzymes), was immobilized on synthetic adsorption resin. No distinguishing changes in pH or thermal stabilities of enzyme were observed due to the immobilization. Since acid-enzyme activity had disappeared, the optimum pH of immobilized enzyme was 9.0. Optimum temperature for the enzyme activity changed from 50 to 55 degrees C. The enzyme converted starch to cyclodextrins without significant loss of activity under the conditions of continuous reaction for about two weeks by using the column system (60 degrees C at pH 8.0). About 63% of soluble starch solution [4% (w/v)] was changed to cyclodextrins, as tested so far.     

Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

The rate of celluose degradation, limited due to the inhibition by cellobiose, can be increased by the hydrolysis of cellobiose to glucose using immobilized beta-glucosidase. Production of beta-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. A study of the immobilization of beta-glucosidase containing cells of Pichia etchellsii on various solid supports was conducted and immobilization by entrapment in calcium alginate gel beads was found to be the most simple and efficient method. A retention of 96.5% of initial activity after ten sequential batch uses of the immobilized preparation was observed. The pH and temperature optima for free and immobilized cells were the same, i.e., 6.5 (0.05M Maleate buffer) and 50 degrees C, respectively. Even though the temperature optimum was found to be 50 degrees C, the enzyme exhibits a better thermal stability at 45 degrees C. Beads stored at 4 degrees C for six months retain 80% of their activity. Kinetic studies performed on free and immobilized cells shown that glucose is a noncompetitive product inhibitor.The immobilized preparation was found to be limited by pore diffusion but exhibited no film-diffusion resistance during packed bed column indicated by a low dispersion number of 0.1348. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to the cellobiose hydrolysis system. The rate of reaction with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. An effectiveness factor of 0.49 was obtained for a particle diameter of 2.5 mm. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column was found to fall rapidly with increase in conversion rate indicating that the operating conditions of the column would have to be a compromise between high conversion rates and reasonable productivity. A half-life of over seven days was obtained at the operating temperature of 45 degrees C in continuous operation of the packed bed reactor. However, the half-life in the column was found to be greatly affected by temperature, increasing to over seventeen days at a temperature of 40 degrees C and decreasing to less than two days at 50 degrees C.     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24 h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.     

Laccase stabilization by covalent binding immobilization on activated polyvinyl alcohol carrier

AIMS: Attempts were made to immobilize laccase from Panus conchatus. METHODS AND RESULTS: The laccase was immobilized on carboxylated polyvinyl alcohol (PVA) activated by N-hydroxysuccinimide (N-HSI) in aqueous solution at different pHs, temperatures, and with different reaction times. An optimum condition for laccase immobilization is at pH 3.2, 40 degrees C and 12 h, respectively. Immobilization of laccase increased optimal pH for reaction with 2, 2'-azinobis (3-ethylbenzthiazoline-6-solfonate) (ABTS) and pH stability. Immobilized laccase proved to be reacted consecutively 17 times with only a 50% decrease on activity and be used in removal of 2,4,6-trichlorophenol (TCP). CONCLUSIONS: It was possible to immobilize the laccase on carboxylated polyvinyl alcohol by activation with N-hydroxysuccinimide in HAc-NaAc buffer. The immobilized laccase is both stable and reusable. SIGNIFICANCE IMPACT OF THE STUDY: The results indicate that this immobilized laccase can be used in the removal of poisonous effluent from pulp bleaching mills.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.