Reasons for the high stability of fumarase activity ofBrevibacterium flavum cells immobilized with k-carrageenan gel

Whole cells ofBrevibacterium flavum having high fumarase activity were immobilized using K-carrageenan. The reason for the high stability of fumarase activity of immobilized cells was investigated. One of main reasons for stabilizing fumarase activity by immobilization using K-carrageenan against organic solvents such as ethanol and acetone was the lower concentration of these solvents in the carrageenan gel compared with that in outer bulk solution. The stabilization of fumarase activity in the immobilized cells against protein-denaturing reagents was found to be related to rheological properties of K-carrageenan gel. Another reason for stabilizing fumarase activity by immobilization with K-carrageenan was to protect the cells from lysis. When immobilized cells were freeze-thawed, their fumarase activity increased and operation stability decreased. Therefore, one reason for the high decay of fumarase activity caused by the freeze-thawing may be a change in the pore size of the K-carrageenan gel. Fumarase activity and the operational stability of immobilized cells was found to depend on gelling conditions. Therefore, the steric structure of the K-carrageenan gel may be related to the decay of fumarase activity.     

Cephalexin synthesis using immobilizedXanthomonas citri cells

For continuous production of cephalexin, whole cells ofXanthomonas citri were immobilized by entrapment in polyacrylamide gel and kappa-carrageenan gel. It wasfound that cells immobilized with kappa-carrageenan showed better thermal stability compared to those immobilized by polyacrylamide gel. The cells immobilized with kappa-carrageenan were treated with glutaraldehyde and hexamethylenediamine to prevent gel destruction during prolonged operation. By immobilizing intact cells, the optimal temperature for the synthetic enzyme reaction shifted higher by 8°C and the optimal pH became broader around 6.2 In continuous operation, the immobilized cells retained better operational stability at 25 than at 37°C, and also showed maximal conversion up to 83% at 25°C.     

Immobilized cells in microbial nitrate reduction

The microorganismPseudomonas denitrificans was immobilized in alginate. These immobilized cells were capable of reducing 0.8 mg NO ₃ ^- /min/g wet weight of cells.     

Stabilization of fumarase activity ofBrevibacterium flavum cells by immobilization with k-carrageenan

Whole cells ofBrevibacterium flavum having fumarase activity were immobilized using K-carrageenan. The stabilities of fumarase activity in the immobilized cells against external factors, including heat, pH, organic solvents, and protein denaturing reagents, were compared with those of free cells and native enzyme. The stabilities of fumarase activity in immobillized cells against external factors were highest, and those of native enzyme were lowest. In the “gel-state,” K-carrageenan-immobilized cells showed a much higher stabilization effect for external factors than “sol-state” immobilized cells.     

Continuous production of L-aspartic acid

For the continuous production ofl-aspartic acid from fumaric acid and ammonia by the action of aspartase, the enzyme extracted fromEscherichia coli orE. coli cells having high aspartase activity were immobilized by various methods. In 1973 we succeeded in the industrial production ofl-aspartic acid usingE. coli cells immobilized with polyacrylamide gel. For the improvement of this process, we developed a novel technique using κ-carrageenan as the immobilizing matrix forE. coli cells. Further, EAPc-7 strain, having higher aspartase activity, was contracted from the parentE. coli by continuous cultivation with a definite medium. The aspartase activity was about seven times higher than that of the parent cells. In 1982 we changed from the conventional method to the improved method, using EAPc-7 strain immobilized with κ-carrageenan.     

Use of hydrogen-sensitive Pd-MOS materials in biochemical analysis

A metal oxide semiconductor sensitive to hydrogen was used to monitor H₂ production from immobilizedClostridium acetobutylicum cells and from immobilized hydrogenase on a continuous basis.     

Affinity chromatographic purification of lentil lectin using immobilized yeast cells

A model system for evaluating macroporous supports containing immobilized whole cells in affinity Chromatographic applications is described. Whole cells were immobilized in a polyacrylamide network in a two-step polymerization process. The affinity system discussed consists of immobilized cells ofSaccharomyces cervisiae in the purification of lentil lectin fromLens culinaris.     

Determination of phenol and catechol concentrations with oxygen probes coated with immobilized enzymes or immobilized cells

The enzyme phenol 2-hydroxylase was immobilized on Sepharose and used in conjunction with an O₂ electrode for quantitating phenol. Similarly, catechol 1,2-oxygenase was used for quantitating catechol. A third probe was prepared by immobilization ofTrichosporon cutaneum cells rather than purified phenol 2-hydroxylase for phenol quantitation. The whole cell system gave results comparable to the immobilized enzyme system.     

Characterization of bioconversion of fumarate to succinate by alginate immobilized Enterococcus faecalis RKY1

In this study, the immobilization characteristics of Enterococcus faecalis RKY1 for succinate production were examined. At first, three natural polymers—agar, κ-carrageenan, and sodium alginate—were tried as immobilizing matrices. Among these, sodium alginate was selected as the best gel for immobilization of E. faecalis RKY1. Efficient conditions for immobilization were established to be with a 2% (w/v) sodium alginate solution and 2-mm-diameter bead. The bioconversion characteristics of the immobilized cellsat various pH values and temperatures were examined and compared with those of free cells. The optimum pH and temperature of the immobilized cells were the same as for free cells, 7.0 and 38°C respectively, but the conversion ratio was higher by immobilization for all the other pH and temperature conditions tested. When the seed volume of the immobilized cells was adjusted to 10% (v/v), 30 g/L of fumarate was completely converted tosuccinate (0.973 g/g conversion ratio) after 12 h. In addition, the immobilized cells maintained a conversion ratio of >0.95 g/g during 4wk of storageat 4°C in a 2% (w/v) CaCl₂ solution. In repetitive bioconversion experiments, the activity of the immobilized cells decreased linearly according to the number of times of reuse.     

Immobilized plant cells

Plant cells have been immobilized in alginate, where they have been shown to retain their biological activity. Such systems can be utilized for bioconversions.     

Denitrification and removal of heavy metals from waste water by immobilized microorganisms

Pseudomonas fluorescens, immobilized on soft polyvinyl chloride granules containing up to 35% softeners as carbon source, was used for simultaneous removal of nitrate and heavy metals. In typical continuous column operation, a 100 mg/L nitrate input solution was reduced to a 20 mg/L output at a feeding rate of 1500 mL/h, with a capacity of 14 kg/day/m³, and with an efficiency of 79%. In the same column, Pb(NO₃)₂ concentration was reduced from 1.0 to 0.05−0.1 mg/L and ZnSO₄ concentration was reduced from 10 to 5 mg/L.Pseudomonas aeruginosa immobilized on an O₂ plasma-treated melt blown polypropylene web was used for removing 95% of a 1.7 nCi PuCl₄ activity from a nuclear plant waste water in a batch operation.     

Immobilization of isolated and cellular hydrogenase ofD. desulfuricans in radiation polymerized polyacrylamides

Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells.     

Gibberellic acid production by free and immobilized cells in different culture systems

Gibberellic acid production was studied in different fermentation systems. Free and immobilized cells of Gibberella fujikuroi cultures in shakeflask, stirred and fixed-bed reactors were evaluated for the production of gibberellic acid (GA₃). Gibberellic acid production with free cells cultured in a stirred reactor reached 0.206 g/L and a yield of 0.078 g of GA₃/g biomass.     

Production of α-keto acids with alginate-entrapped whole cells of the yeastTrigonopsis variabilis

The yeast,Trigonopsis variabilis, was immobilized by entrapment in alginate. The immobilized cells containing high amounts of D-amino acid oxidase were used to convert D-amino acids to their corresponding α-keto acids.     

ImmobilizedE. coli Alkaline Phosphatase

We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg²⁺. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.     

Entrapment of living microbial cells in covalent polymeric networks

The kinetics of oxidative phenol degradation with microbial cellsCandida tropicalis, immobilized in a polyacrylamide and polymethacrylamide matrix, were mathematically simulated assuming zero-order and Michaelis-Menten rate equations. For zero-order kinetics an expanded equation for catalytic effectiveness as a function of the Thiele modulus, Biot number, and partition coefficients was derived and compared with numerical solutions for Michaelis-Menten kinetics. Errors with regard to the zero-order approximation become negligible ifc _o/K _M >2. Experimentally determined catalyst activities as a function of particle size and cell concentration were compared to calculated ones. Additional experiments to determine the diffusion and oxygen consumption ratios have been carried out in an effort to resolve the physical parameters to be used in the above mentioned calculations. Furthermore, experiments on cell growth during reincubation with nutrients and oxygen are reported; an increase in activity up to a factor of ten was observed. These experiments demonstrate that the microbial cells are entrapped in the polymer matrix in the living state.     

Preparation,characterization, and potential application of an immobilized glucose oxidase

A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50°C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50°C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.     

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.     

Immobilization,characterization, and laboratory-scale application of bovine liver arginase

Arginase isolated from beef liver was covalently attached to a polyacrylamide bead support bearing carboxylic groups activated by a water-soluble carbodiimide. The most favorable carbodiimide wasN-cyclohexyl-Nt’-(methyl-2-p-nitrophenyl-2-oxoethyl) aminopropyl carbodiimide methyl bromide, but for practical purposes,N-cyclohexyl-Nt’-morpholinoethyl carbodiimide methyl tosylate was used. The optimal conditions for the coupling procedure were determined. The catalytic activity of the immobilized arginase was 290–340 U/g solid or 2.9–3.4 U/mL wet gel. The pH optimum for the catalytic activity was pH 9.5, the apparent temperature maximum was at 60°C and K_mapp was calculated to be 0.37M L-arginine. Immobilization markedly improved the conformational stability of arginase. At 60°C, the pH for maximal stability was found to be 8.0. The immobilized arginase was used for the production of L-ornithine and D-arginine.     

Cell biosensor for detection of phenol in aqueous solutions

A microbial sensor for concentration measurement of phenol in aqueous solutions has been developed. Phenol-utilizing cellsPseudomonas putida GFS-8 immobilized in poly(vinyl)alcohol cryogel were used as a biological transducer. Relationships between phenol concentration in the activating medium and endogenic cell respiration have been established. Cell respiration and phenol concentration in the assay solution positively correlated at a phenol concentration range of 0.1–2.0 mg/L and were linearly dependent in the range of 0.1–1.0 mg/L. A Clark membrane electrode was the physiochemical transducer. The assay may be completed within 5 min. The cells oxidize phenol, pyrocatechol, mesityl oxide, aniline, and do not react with a number of xenobiotics, sugars, and alcohol. With the exception of aniline, most components found in waste waters from phenol production affect neither the assay process nor the ability of these cells to use phenol as exogenic respiratory substrate. The immobilized cells retained their ability to utilize phenol as an exogenic respiratory substrate for up to 1 mo.