Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

Production of palatinose using<Emphasis Type="Italic"> Serratia plymuthica</Emphasis> cells immobilized in chitosan

In recent decades, the production of palatinose has aroused great interest since this structural isomer of sucrose has interesting potential. We describe a simple and effective method of immobilizing Serratia plymuthica cells in chitosan. The sucrose isomerase activity of immobilized preparations was enhanced many times by activation with fresh nutrient medium and subsequent drying. The preparations obtained were physically very stable with high enzyme activity and excellent operational stability. The effect of temperature, pH and substrate concentration on enzyme activity of the immobilized cells was investigated. Using immobilized cells, a complete conversion of sucrose (40% solution) into palatinose was achieved in 4 h in a "batch"-type enzyme reactor. The use of free or immobilized cells had no effect on the composition of the solution, in particular the sugar content. The palatinose content was 80% and that of trehalulose 7%.     

Performance of Aspergillus niger B 03 β-xylosidase immobilized on polyamide membrane support

The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters K_m, V_max and K_i were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.     

Ribonuclease production by free and immobilizedAspergillus clavatus cells

Several fungal strains ofAspergillus andPenicillium were immobilized by cryopolymerization in polyvinyl alcohol cryogel beads.Aspergillus clavatus was the best producer of extracellular ribonuclease. Enzyme productivity and growth of free and immobilized cells in shake flasks and agitated bioreactor were studied. Ribonuclease production and growth behaviour depended on concentrations of glucose, peptone and soybean in the culture medium. Enzyme production was influenced by agitation and aeration intensity. In repeated batch, shake-flask cultures, the immobilized cells showed 2 to 3.5 times higher enzyme activity than free cells. The optimal conditions in a bioreactor were at 150 rev/min agitation speed and 0.5 vol/vol.min aeration. Enzyme productivity of immobilized cells (237 units/g dry mycelium.h) was 2.1 times higher than the productivity of free cells in a bioreactor, and 2.3 times higher than that of a shake-flask culture.R.J. Manolov is with the Institute of Microbiology, Department of Enzymes, Bulgarian Academy of Sciences, Georgy Bonchev Street 26, 1113 Sofia, Bulgaria.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

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.     

Characterization of microbial endo-β-glucanase immobilized in alginate beads

Endo-β-glucanase (endo-β-1,4-glucano-glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH-activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilizatíon. The enzyme immobilized in alginate beads showed a K_m value (1.02% w/v) lower than that of the enzyme (1.31%). The apparent V_max of immobilized cellulase preparations (238.3 μmol glucose/ml × h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60°C) of the free and entrapped enzymes remained unaltered. In contrast, the half-life of the endoglucanase immobilized in calciumalginate beads was 4.6 h at 55°C and 5.4 h at 60°C, while that of the free enzyme was 3.0 h at 55°C and 1.2 h at 60°C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and β-glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250–720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized β-glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co-immobilization of both types of enzymes reduced by hydrolytic efficiency to half.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Enantioselective behavior of lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> immobilized in different supports

Considering the extraordinary microbial diversity and importance of fungi as enzyme producers, the search for new biocatalysts with special characteristics and possible applications in biocatalysis is of great interest. Here, we report the performance in the resolution of racemic ibuprofen of a native enantioselective lipase from Aspergillus niger, free and immobilized in five types of support (Accurel EP-100, Amberlite MB-1, Celite, Montmorillonite K10 and Silica gel). Amberlite MB-1 was found to be the best support, with a conversion of 38.2%, enantiomeric excess of 50.7% and enantiomeric ratio (E value) of 19 in 72 h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (E = 23) in a shorter reaction period (48 h) at 35°C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8 months of storage at 4°C and could be reused at least six times.     

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.     

Isozymes of <Emphasis Type="Italic">α</Emphasis>-amylases from newly isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> CKB19: Production from immobilized cells

The aim of this study was to produce two isozymes of α-amylase by immobilization of a newly isolated soil bacterium. The bacterium was identified as Bacillus thuringiensis CKB19 on the basis of its 16S rRNA profile. Enzyme production by free cells increased linearly with cell growth up to 34 h in starch containing enriched liquid media. The active bacterial cells were immobilized in Caalginate beads, and operational stability of the entrapped cell was optimized for amylase production. Enzyme production was optimal at an alginate concentration of 2 g% (w/v), calcium chloride concentration of 1 M, and with 300 beads (each bead contained 2 × 10⁷ cells)/250 mL flask. Amylase production by the immobilized cells was about 3 times higher than free cell fermentation after 34 h of incubation. It was observed that the immobilized bacterium secreted two different amylases (Am-I and Am-II) into the culture fluid. The molecular masses of Am-I and Am-II were 59.6 and 44.7 kd, respectively, and showed optimum activity at pH 5.0 and 9.0. Both amylases showed optimum activity at 40°C and were stable at the same temperature, with losses of only 10 and 20% (for Am I and Am II, respectively) of their original activities after 24 h of incubation. Further, both amylases were salt tolerant (up to 4 M NaCl) and hydrolyzed raw starchy foods into glucose. All these characteristics make this enzyme mixture suitable for use as a digestive aid and for the improvement of digestibility of animal feed ingredients.     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

Continuous Production of Dextran from Immobilized Cells of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> KIBGE HA1 Using Acrylamide as a Support

The cells of L. mesenteroides KIBGE HA1 were immobilized for the production of dextran on acrylamide gel and gel concentration was optimized for maximum entrapment. Sucrose at substrate concentration of 10% produced high yield of dextran at 25°C with a percent conversion of 5.82 while at 35°C it was 3.5. However, increasing levels of sucrose diminished dextran yields. The free cells stopped producing dextran after 144 h, while immobilized cells continued to produce dextran even after 480 h. Molecular mass distribution of dextran from free cells indicate that it is identical to that of blue dextran while the molecular mass of dextran from immobilized cells is lower than that of free cells.     

Catalytic properties of the immobilized Talaromyces thermophilus β-xylosidase and its use for xylose and xylooligosaccharides production

The present study explores the efficiency of Talaromyces thermophilus β-xylosidase, in the production of xylose and xylooligosaccharides. The β-xylosidase was immobilized by different methods namely ionic binding, entrapment and covalent coupling and using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the best support material for β-xylosidase immobilization; it gave the highest immobilization and activity yields (94%, 87%, respectively) of initial activity, and also provided the highest stability, retaining 94% of its initial activity even after being recycled 25 times. Shifts in the optimal temperature and pH were observed for the immobilized β-xylosidase when compared to the free enzyme. The maximal activity obtained for the immobilized enzyme was achieved at pH 8.0 and 53 °C, whereas that for the free enzyme was obtained at pH 7.0 and 50 °C. The immobilized enzyme was more thermostable than the free β-xylosidase. We observed an increase of the K_m values of the free enzyme from 2.37 to 3.42 mM at the immobilized state. Native and immobilized β-xylosidase were found to be stimulated by Ca²⁺, Mn²⁺ and Co²⁺ and to be inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, EDTA and SDS. Immobilized enzyme was found to catalyze the reverse hydrolysis reaction, forming xylooligosaccharides in the presence of a high concentration of xylose. In order to examine the synergistic action of xylanase and β-xylosidase of T. thermophilus, these two enzymes were co-immobilized on chitosan. A continuous hydrolysis of 3% Oat spelt xylan at 50 °C was performed and better hydrolysis yields and higher amount of xylose was obtained.     

Immobilization of Xylan-Degrading Enzymes from Scytalidium thermophilum on Eudragit L-100

Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared to 6.5 in case of free enzyme. Immobilization increased the t_1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The K_m and V_max values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol) as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification of xylan was also improved by xylanase immobilization.     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Immobilization of the recombinant invertase INVB from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on Nylon-6

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for of immobilized and free re-INVB, respectively. values for immobilized and free enzymes were 6.1 × 10² and 1.2 × 10⁴ s⁻¹, respectively, and immobilized re-INVB showed of 158.73 μmol h min⁻¹ mg⁻¹. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.