Yoshiki Sano

Immobilization of microorganisms with PVA hardened by iterative freezing and thawing

Abstract In order to utilize polyvinyl alcohol (PVA) as a gel matrix for the immobilization of microorganisms, PVA was subjected to iterative freezing-thawing. The effects of the procedure on the mechanical characteristics of the PVA hydrogel and the stability of the immobilized microorganisms were investigated. PVA showed rubber-like elasticity after iterative freezing-thawing. Gel strength increased with the iteration number of freezing-thawing until seven iterations. Although the activities of both the free and immobilized cells decreased during the iteration of freezing-thawing, addition of cryoprotectants such as glycerol and skim milk was effective for preventing the decrease in activity.

Release of thermophilic α-amylase from transformed Escherichia coli by addition of glycine

Abstract For the purpose of the efficient extraction of thermophilic α-amylase, we investigated the effect of glycine on the extracellular release of a foreign gene product from a recombinant Escherichia coli . Regardless of the medium, carbon source, growth phase and host tested, about 70–80% of α-amylase was released from E. coli HB101/pHI301 by the addition of glycine. Distribution of α-amylase activity in E. coli HB101/pHI301 revealed that the extracellular release of the enzyme significantly depended on a decrease of the activity in the periplasm. Based on the effect of the glycine concentration and the time-course of the release, the optimal condition of glycine treatment may be 3 h-incubation in 1% glycine. For simplification of the purification process, we tested the release of α-amylase from E. coli HB101/pHI301 in a solution containing only glycine instead of growth media containing glycine. The former was sufficiently effective for the release of the enzyme. This result suggests a simple and easy method for the purification of the enzyme.

Mechanical and kinetic properties of PVA hydrogel immobilizing β-galactosidase

Abstract The mechanical and kinetic properties of PVA (polyvinyl alcohol) hydrogel prepared by the iterative freezing and thawing (IFT) method were studied in order to assess its applicability as an immobilizing support. The PVA hydrogel showed rubber-like elasticity and the Youngs modulus of the gel increased with increasing polymer concentration. The gel strength greatly improved with the number of freezing and thawing iterations ( n ). Thermal treatment of 10% gel with n = 2 for 10 min at 45°C caused a significant loss of strength, but at n = 7 no change in gel strength was observed. A β-galactosidase-producing recombinant E. coli was permeabilized by toluene and the kinetic characteristics of immobilized whole cells in the PVA hydrogel were investigated using ONPG (2-nitrophenyl-β- d -galactopyranoside) as a substrate under negligible intraparticle diffusion resistance. Compared with free cells, the Michaelis constant of the β-galactosidase was increased by the entrapment in PVA hydrogel, although that of the free cells decreased with the addition of 3% PVA into the reaction mixture. The kinetic parameters of both free and immobilized cells were not influenced by n until it reached 7 times, and the PVA concentration did not affect the kinetic parameters of the immobilized cells. The optimal pH of the enzyme activity was not changed by immobilization, although the activity profile was broader than that of the free cells.

Encapsulation of Biocatalyst with PVA Capsules

Abstract PVA capsules were prepared by dropping PVA solution into cold methanol contained in a glass cylinder and subsequent incubation in methanol. The diameter of the capsules decreased slightly with incubation time due to dehydration. The membrane thickness of the capsules was measured by the immobilization of living cells and was found to increase almost proportionally to increasing incubation time in the methanol. Encapsulation of toluene-permeabilized cells having β-galactosidase activity as an enzyme was attempted under various conditions. The activity and stability of the enzyme in the capsules in phosphate buffer at 28°C were examined. In a preliminary experiment, methanol inactivated the activity of the free enzyme. However, PVA repressed the inactivation. The activity of the capsules decreased with increasing incubation time, probably due to inactivation of the enzyme activity by methanol and the increase of intraparticle diffusion resistance as a result of growth in membrane thickness. Incubation of the capsules at 0°C and a methanol temperature gradient in the cylinder used for the preparation of the capsules were effective in improving the enzyme activity of the capsules. The long-term stability of the enzyme activity of the capsules was better than that of free cells, and was improved by incubation at room temperature. Although a temperature gradient in the methanol used in capsule preparation enhanced the activity, it reduced the stability of the capsules.

Effective diffusivity of glucose in PVA hydrogel

Abstract Effective diffusivity of glucose in PVA hydrogel beads prepared by an iterative freezing and thawing method was determined by a curve fitting the theoretical diffusion model in a well-mixed solution, and the effects of iteration number of freezing and thawing ( n ) on the diffusion and partition coefficients were examined. The diffusivity (4.10 ± 0.20 × 10 −4 cm 2 /min) in PVA gel at n = 2 was almost the same as that in water (4.08 × 10 −4 cm 2 /min), and decreased with n . The partition coefficient also gradually decreased with n . These results may be attributable to an increase in crystallinity due to syneresis during iterative freezing and thawing.

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Abstract Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.

Simultaneous Release and Purification of Gene Product in an Aqueous Two-Phase System

Abstract For simplification of cell disruption and the subsequent aqueous two-phase separation process, simultaneous release and purification of β-galactosidase from a recombinant Escherichia coli were investigated using a PEG-phosphate aqueous two-phase system containing glycine. The enzyme was released in the two-phase system by the addition of glycine, the percentage of the release increasing with incubation time. The effects of PEG and phosphate concentrations on the release were investigated in a buffer solution. Although the release was enhanced by the addition of PEG, phosphate repressed it. In order to ascertain the optimum operational conditions, the effects of various parameters on the partition coefficient (top/bottom) of the enzyme were investigated. The enzyme was preferentially partitioned to the top phase in a 13% PEG 6000-10% phosphate system, but the partition coefficient was significantly decreased by the addition of NaCl. It was slightly decreased by adding the supernatant of a sonicate of the host cells, but significantly increased with decreasing PEG molecular weight. The addition of glycine to the two-phase system favorably enhanced the partition of the enzyme to the top phase.

Immobilization of β-Galactosidase with Polyvinyl Alcohol

Abstract Immobilization of β-galactosidase with PVA (polyvinyl alcohol) hardened by freezing-thawing was attempted and the stability of the immobilized enzyme was investigated. Afterwards the freezing-thawing procedure, leakage of the enzyme from PVA hydrogel beads, and faster activity drop of the gel beads were observed during prolonged incubation. The stability of the immobilized enzyme treated with boric acid at an adequate concentration restored to the extent of the free enzyme, although higher concentrations of boric acid inhibited the free enzyme.

Immobilization of a Thermostable Enzyme Using a Sol-Gel Preparation Method

A thermostable β-galactosidase was immobilized with tetramethoxysilane (TMOS) using a sol-gel preparation method. The thermal stability of free and immobilized enzymes changed with enzyme concentration, suggesting an effect of contaminants in the enzyme solution. The thermal stability of the immobilized enzyme was affected by preparation conditions and was much lower than that of free enzyme. The use of high concentrations of magnesium in the preparation enhanced the thermal stability of the immobilized enzyme, although high concentrations of magnesium in the incubation buffer reduced the thermal stability. A cell-free extract of E. coli B cells exhibiting no β-galactosidase activity was found to be most effective in improving the thermal stability of both the free and immobilized enzyme. The pH of incubation buffer during thermal treatment and preparation of immobilized enzyme significantly affected enzyme activity and thermal stability. The highest activity and stability were obtained at pH 6. Immobilization and thermal treatment had no effect on the Michaelis constants independent of the enzyme concentration and this suggests that immobilizing support and the cell-free extracts have insignificant effect on the kinetic properties of the immobilized enzyme.

Effect of polyvinyl alcohol on kinetic and thermal properties of β-galactosidase

The effect of PVA (polyvinyl alcohol) with various degrees of polymerization and saponification on the kinetic and thermal characteristics of β-galactosidase was investigated. With ONPG (2-nitrophenyl-β- d -galactoside) as a substrate, the enzyme activity increased with increasing PVA concentration. While the influences of the properties of PVA on Michaelis constants were insignificant, the maximum reaction rate of the enzyme increased with increasing degrees of polymerization and saponification. The addition of PVA enhanced the thermal stability of the enzyme, and it increased with increasing PVA concentration. Dependency of the thermal stability on the characteristic properties of PVA was not remarkable.