Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

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.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Urease immobilization on macroporous silicas

Urease was immobilized on macroporous silicas using gamma-aminopropyl triethoxysilane and glutaraldehyde. The amount of protein on the surface, the structure of pores of the support and the purity of the initial enzyme were varied, the enzymic activity of the immobilized preparations being controlled. After the immobilization of sufficiently large quantities of the enzyme (about 3 mg protein per m2 support) about 35% of the specific activity was retained. The maximum activity per unit weight of the support was observed for silicagels and silochromes with the mean diameter of pores 70-90 nm and the specific surface area about 70 m2/g. The use of purified urease produced highly active preparations of the immobilized enzyme (17,000 U per g dry support). Freeze-drying of the immobilized enzyme in the presence of sorbitol yielded dry preparations retaining their activity.     

Enhanced yeast immobilization by nutrient starvation

Saccharomyces uvarum NRRL Y1347 cells were immobilized in a porous support. Cell loadings of up to 600 mg dry cell/g support or 70 mg dry cell/cm³ support were obtained. Starvation in a marine environment increased the adhesion strength of immobilized cells.     

Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Summary Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell -galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.M. Decleire et al.: Hydrolysis of whey by immobilized whole cells of Kluyveromyces bulgaricus     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Compositions and compositional-behavioural relationships of enzymes immobilized on porous inorganic supports via titanium(IV) species

The compositions and compositional-behavioural relationships of glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) immobilized on titanium(IV)-activated porous inorganic supports have been investigated for several transition metal activation techniques based on the metal-link/chelation method developed by our group. The highest activity (239 Ug^?1 matrix) of immobilized glucoamylase was obtained with the hydrous titanium(IV) oxide derivative of the support when this and a 15% w/v TiCl₄ solution were dried at 45°C in vacuum for 30 h. However, the immobilized enzyme preparation displayed a very unstable behaviour, as did also the preparation which was obtained by drying the mixture of support and transition metal solution at atmospheric pressure. This was mainly due to an enzyme deactivation by titanium inhibition instead of enzyme loss in substrate solution. When amination and carbonylation steps were included in the immobilization technique much more stable preparations were obtained, mainly when the support was activated by drying at 45°C with a 15% w/v TiCl₄ solution ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1495}\text{h}$) although with a lower initial activity (35.6 Ug^?1 matrix). The pure TiCl₄ support activation rather than TiCl₄/HCl solution support activation led to less stable immobilized enzyme preparations (washing and amination solvent chloroform, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 365}\text{h}$; washing and amination solvent water, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 276}\text{h}$) than the preparation obtained with the dried titanium(IV)-activated support. This was due to loss of enzyme-titanium(IV) complex in solution, as the interactions between the titanium(IV) and the silanol groups of the porous silica are weak. However, the amination (with 1,6-diaminohexane) and carbonylation (with glutaraldehyde) steps always led to immobilized enzyme preparations with constant specific activities and protein/titanium(IV) ratio. This suggests that the spacing effect introduced by these reactions removes the titanium(IV) inhibition of glucoamylase.     

Biospecific immobilization of mannan-penicillin G acylase neoglycoenzyme on Concanavalin A-bead cellulose

The matter of this work was to evaluate possibilities of biospecific immobilization of synthetic mannan-penicillin G acylase neoglycoconjugate on Concanavalin A support. The conjugate containing 37% (w/w) of yeast mannan was prepared. Significant biospecific interaction of this neoglycoenzyme with Con A was confirmed by precipitation method. The biospecific sorption of conjugate was investigated using Concanavalin A-triazine bead celluloses MT-100 with different content of Con A (from 1.4 to 9.8 mgCon A/gwet support). The results obtained under optimal conditions were compared with those from covalent immobilization of PGA. The sorbent capacity was observed higher for covalent binding of enzyme. On the other hand, the biospecifically immobilized neoglycoenzyme retained a greater amount of initial activity. The maximum amount of 6.6mgimmobilizedneoglycoenzyme/gwet Con A-sorbent (18.1 U/g) was achieved. The amount as well as activity of immobilized mannan-penicillin G acylase was increased by its two multiple layering on surface of sorbent (10.1mg, respectively, 23.5 U/gwet sorbent). Determined storage and operational (using flow calorimetric method) stabilities of biospecifically immobilized enzyme, were similar, possibly somewhat higher that those of covalent bound penicillin G acylase.     

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.     

Tris–sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris–sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.     

Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus

Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.     

Alcohol production by magnetic immobilized yeast

Summary Saccharomyces cerevisiae was immobilized in calcium alginate gel together with varying concentrations of iron oxide, in the form of magnetite or a colloidal ferrite suspension, Ferrofluid. Inclusion of magnetic material apparently had no adverse effect on the yeast cells as judged from their fermentation capacity, their operational stability as well as their ability to propagatein situ in the presence of nutrients. The usefulness of magnetic preparations in viscous or particle containing media is discussed.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Non-porous magnetic supports for cell immobilization

A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/l · h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.     

Could the improvements in the alcoholic fermentation of high glucose concentrations by yeast immobilization be explained by media supplementation?

Summary The maximal concentration of ethanol produced during the fermentation of 320 g/l glucose bySaccharomyces bayanus was higher when the yeast cells were immobilized either by adsorption on celite or by entrapment in k-carrageenan beads (from 10.5% with free cells up to 14.5% and 13.1% (v/v) respectively). This increase was due to medium supplementation with the compounds present in the immobilization supports.     

Characterization of a Saccharomyces cerevisiae mutant with enhanced production of beta-D-fructofuranosidase

The present study focused on the improvement of Saccharomyces cerevisiae through random mutagenesis for enhanced production of beta-D-fructofuranosidase (FFase) using sucrose salt media. Sixty strains of S. cerevisiae were isolated from different fruits and soil samples and screened for FFase production. Enzyme productivity of different yeast isolates ranged from 0.03 to 1.10 U/ml. The isolate with the highest activity was subjected to ultraviolet (UV) radiation and mutagenesis using N-methyl N-nitro N-nitroso guanidine (MNNG). One mutant produced FFase at a level of 17.8+/-0.9 U/ml. The MNNG-treated isolate was exposed to ethyl methane sulphonate (EMS), and a mutant with an enzyme activity of 25.56+/-1.4 U/ml was obtained. Further exposure to UV radiation and chemicals yielded a mutant exhibiting an activity of 34.12+/-1.8 U/ml. After optimization of incubation time (48 h), sucrose concentration (5.0 g/L), initial pH (6.0) and inoculum size (2.0% v/v), enzyme production reached 45.65+/-4.6 U/ml with a noticeable greater than 40-fold increase compared to the wild-type culture. On the basis of kinetic variables, notably Q(p) (0.723+/-0.2U/g/h), Y(p/s) (2.036+/-0.05 U/g) and q(p) (0.091+/-0.02 U/g yeast cells/h), the mutant S. cerevisiae UME-2 was found to be a hyperproducer of FFase (LSD 0.054, p0.05).