Enzymatic in situ saccharification of cellulose in aqueous-ionic liquid media

The enzymatic saccharification of a model cellulosic substrate, Avicel PH-101, using an ionic liquid (IL), 1-ethyl-3-methylimidazolium diethylphosphate, was explored. After mixing the IL solution of cellulose with different volumes of 10 mM citrate buffer (pH 5.0), cellulase was directly added to the aqueous-IL mixture at 40°C. When the volume of IL to water was greater than 3:2, little cellulase activity was observed. However, decreasing the volume ratio markedly enhanced enzymatic activity: an IL to water ratio of 1:4 (v/v) resulted in over 70% of the starting amount of cellulose (10 mg/ml) being converted to glucose and cellobiose.     

Galacto-oligosaccharide production byβ-galactosidase in hydrophobic organic media

Summary Production of galacto-oligosaccharide (GO), including trisaccharide and tetrasaccharide, was performed using a -galactosidase in water-hydrophobic solvent mixtures. A maximum GO concentration of 45% (w/w) was attained in a 95% cyclohexane/5% water mixture from a 55% (w/w) of lactose at 60°C and pH 6.0, while a maximum of 38% GO in aqueous media. GO production decreased with an increase in surfactant concentration. The optimum water content for GO production showed a broad range from 2.5 to 10% (v/v). Solvent properties, such as log P and the dipole moment, had no relation to GO production.     

An improved FIA-system for measuring α-amylase in cultivation media

Summary This paper describes a flow-injection analysis (FIA) system for measuring -amylase in cultivation media. It is based on measuring the decolorization of an iodine-starch complex. The FIA-system is optimized with respect to stability and reproducibility. Monitoring of -amylase during batch cultivations with Aspergillus oryzae indicates that -amylase is constitutively expressed at high glucose concentration.     

Enzymes in lyotropic liquid crystal — A new method of bioconversion in non-aqueous media

Summary A new method has been developed for the use of enzymes as catalysts in organic solvents. The enzyme and cofactor are entrapped in a lyotropic liquid crystal which is insoluble in an organic solvent. The biocatalyst is significantly stabilized in this biphasic reaction system and product recovery is facilitated.     

What can we learn by studying enzymes in non-aqueous media?

What is the role of water in enzyme structure and function? One approach to answers should come from studies in which the amount of water present is a variable. In the absence of bulk liquid water, effective monitoring of enzyme action requires an alternative fluid medium through which substrates and products may be transported. The past 20 years have seen quite extensive study of enzyme behaviour when reactants are transferred via a bulk phase that is an organic liquid, a supercritical fluid or a gas. Some lipases, at least, remain highly active with only a few, if any, residual water molecules. Many enzymes seem to require larger amounts of water, but still not a liquid water phase. There are hysteresis effects on both the amount of bound water and the observed catalytic activity. Increasing hydration promotes mobility of the enzyme molecule, as revealed by various techniques, and there are correlations with catalytic activity. There are other plausible roles for hydration, such as opening up proton conduction pathways.     

How do additives affect enzyme activity and stability in nonaqueous media?

The catalytic activities of lyophilized powders of alpha-chymotrypsin and Candida antarctica lipase were found to increase 4- to 8-fold with increasing amounts of either buffer salts or potassium chloride in the enzyme preparation. Increasing amounts of sorbitol in the chymotrypsin preparation produced a modest increase in activity. The additives are basically thought to serve as immobilization matrices, the sorbitol being inferior because of its poor mechanical properties.Besides their role as supports, the buffer species were indispensable for the transesterification activity of chymotrypsin because they prevented perturbations of the pH during the course of the reaction. Hence, increasing amounts of buffer species yielded a 100-fold increase in transesterification activity. Effects of pH changes were not as predominant in the peptide synthesis and the lipase-catalyzed reactions.Immobilization of the protease on celite resulted in a remarkable improvement of transesterification activity as compared to the suspended protease, even in the absence of buffer species. Immobilization of the lipase caused a small improvement of activity. The activity of the immobilized enzymes was further enhanced 3-4 times by including increasing amounts of buffer salts in the preparation.The inclusion of increasing amounts of sodium phosphate or sorbitol to chymotrypsin rendered the catalyst more labile against thermal inactivation. The denaturation temperature decreased with 7 degrees C at the highest content of sodium phosphate, as compared to the temperature obtained for the denaturation of the pure protein. The apparent enthalpy of denaturation increased with increasing contents of the additives. The enhancement of hydration level and flexibility of the macromolecule upon addition of the compounds partly provides the explanation for the observed results. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 67-76, 1997.     

A whole-cell process for the production of ε-caprolactone in aqueous media

ε-Caprolactone is an industrially important intermediate produced in multi-10,000 ton scale annually with broad applications. We report on a whole-cell biocatalytic conversion of cyclohexanol to ε-caprolactone using the combination of alcohol dehydrogenase (ADH) with two stability-improved variants (QM and M15) of the Baeyer-Villiger monooxygenase CHMO with a special focus on process development at the 200 mM scale. Influence of parameters such as volumetric mass transfer co-efficient, stirrer speed and catalytic loading (amount of E. coli whole-cells expressing ADH and CHMO) on the process efficiency were studied and optimised. This resulted in over 98% conversion, a product titer of 20 g L^–1 and an isolated product amount of 9.1 g (80%). This corresponds to a space-time yield of 1.1 g L^–1 h⁻¹ and a reaction yield (mole of product per mole substrate) of 0.9. Comparing the two CHMO variants a significant difference in catalytic yield (weight of product to weight of catalyst; 0.6 vs 0.3) was observed without any inherent changes in the process. Hence, the reported process can accommodate in the future improved variants of the CHMO.     

Glycosylated α-chymotrypsin as a catalyst for kyotorphin synthesis in water-organic media

-Chymotrypsin was covalently modified with cellobiose by chemical means. After adsorption on to a porous polyamide support, both the native and the glycosylated immobilized derivatives were used to synthesize a kyotorphin derivative (N-benzoyl-l-tyrosyl-l-argininamide) in acetonitrile/water. Glycosylated chymotrypsin gave a 125% increase in product formation (750 nmol mg^–1 catalyst in 3 h) at 60% (v/v) acetonitrile/water. Maximal protective effect of this glycosylation process was at 70% (v/v) acetonitrile/water, at which concentration the half-life of the glycosylated enzyme was 20-times longer than that of the native form (52 min and 2.8 min, respectively).     

Enzymatic reactions in aqueous-organic media. VI. Peptide synthesis by α-chymotrypsin in hydrophilic organic solvents

The peptide synthesis from N-acetyl-L-tyrosine ethyl ester and amino acid amides was realized using α-chymotrypsin as a catalyst in ethanol or acetonitrile containing small amounts of water. In these reaction systems, the precipitates of phosphate salt, which was used as a component of buffer solution, are considered to act as carriers of chymotrypsin. It was found that peptide formation is competitive with hydrolysis of the substrate ester, but the secondary synthesis of the peptide from the hydrolysate was also considered to proceed. The yield of the peptide after 24 h reaction was strongly dependent on the water concentration; maximum yields of the peptide were obtained at water concentrations below 10% (v/v). The addition of tertiary amines, such as triethyl amine, markedly increased the peptide yield, probably due to the increase in the concentration of the nucleophilic amine components by neutralization of hydrohalides of amino acid amides. The effect of reaction temperature and the reactions with CT immobilized on PVA, chitosan, or TEAE-cellulose are also described.     

Domain cooperativity in multidomain proteins: what can we learn from molecular alignment in anisotropic media?

Many proteins have modular design with multiple globular domains connected via flexible linkers. As a simple model of such system, we study a tandem construct consisting of two identical SH3 domains and a variable-length Gly/Ser linker. When the linker is short, this construct represents a dumbbell-shaped molecule with limited amount of domain-domain mobility. Due to its elongated shape, this molecule efficiently aligns in steric alignment media. As the length of the linker increases, the two domains become effectively uncoupled and begin to behave as independent entities. Consequently, their degree of alignment drops, approaching that found in the (near-spherical) isolated SH3 domains. To model the dependence of alignment parameters on the length of the interdomain linker, we have generated in silico a series of conformational ensembles representing SH3 tandems with different linker length. These ensembles were subsequently used as input for alignment prediction software PALES. The predicted alignment tensors were compared with the results of experimental measurements using a series of tandem-SH3 samples in PEG/hexanol alignment media. This comparison broadly confirmed the expected trends. At the same time, it has been found that the isolated SH3 domain aligns much stronger than expected. This finding can be attributed to complex morphology of the PEG/hexanol media and/or to weak site-specific interactions between the protein and the media. In the latter case, there are strong indications that electrostatic interactions may play a role. The fact that PEG/hexanol does not behave as a simple steric media should serve as a caution for studies that use PALES as a quantitative prediction tool (especially for disordered proteins). Further progress in this area depends on our ability to accurately model the anisotropic media and its site-specific interactions with protein molecules. Once this ability is improved, it should be possible to use the alignment parameters as a measure of domain-domain cooperativity, thus identifying the situations where two domains transiently interact with each other or become coupled through a partially structured linker.     

What if cell culture media do not mimic in vivo redox settings?

Here, I address the topic of suitability for redox research of common settings in cell cultures. This is done through the prism of in vitro anticancer effects of vitamin C. Cell culture media show lower concentrations of iron and a higher level of oxygen compared to interstitial fluid. Such a setup promotes ascorbate-mediated production and accumulation of hydrogen peroxide, which efficiently kills a variety of cancer cell lines. However, the anticancer effects are annihilated if the iron level is corrected to mimic in vivo concentrations. It appears that the potential benefits of application of vitamin C in cancer treatment have been significantly overestimated. This might be true for other pro-oxidative agents as well, such as some (poly)phenols. We urgently need to establish medium formula and culture maintenance settings that are optimal for redox research.     

Two-step enzymatic selective synthesis of water-soluble ketoprofen–saccharide conjugates in organic media

Ketoprofen–saccharide conjugates were synthesized by selectively enzymatic hydrolysis and acylation. Firstly, the (S)-ketoprofen vinyl ester was prepared by enzymatic hydrolysis of (R,S)-ketoprofen vinyl ester. Then enzymatic transesterification of (S)-ketoprofen vinyl ester with a series of saccharides were performed by the catalysis of a commercial protease from Bacillus licheniformis (BLP) in organic medium mixture of pyridine and tert-butanol. The ketoprofen was selectively conjugated onto the primary hydroxyl group of saccharides and with high yield after 72 h. Partition coefficient determination showed that all the products have better water solubility than parent ketoprofen. Chemical hydrolysis experiment indicated that 50% ketoprofen could be release from ketoprofen glucoside and maltoside in aqueous buffer (pH 7.4) within 48 h.     

FIA for down-stream processing — determination of β-galactosidase in viscous extraction media

A Flow Injection Analysis method for determination of the enzyme -galactosidase produced in a fermentation process has been developed. The analytical range was 10–1000 U/cm³ and the sample throughput was 40 h^–1. The FIA set up was connected directly to the down-stream process and the enzyme activity in the extraction step was measured on-line. The viscosity of the samples from the process is high causing certain physical effects on the FIA system.     

Effects of emulsion viscosity during surfactant‐enhanced soil flushing in porous media

Surfactants can potentially improve the efficiency of pump‐and‐treat technology for remediation of aquifers contaminated by nonaqueous phase liquids (NAPLs). However, the formation of emulsions during the removal process can Increase the viscosity in the system. This can result in pore clogging and reduction of flow, which inhibits the contaminant removal process. Formation of viscous emulsions has been identified in previous research as one of the probable causes for in situ field test failures using surfactant‐enhanced soil‐flushing technology. However, the effects of in situ emulsification and viscosity increases have not been quantified previously. The purpose of this article is to investigate effects of in situ emulsification on the remediation process. Laboratory column studies examined the mobilization of m‐xylene from porous media using a 1% alcohol ethoxylate surfactant solution (Witconol® SN90). Effects of in situ emulsification were determined. Glass columns (1.1 cm i.d. × 30 cm) were packed with 0.2‐mm glass beads to model soil media. Viscosities of emulsion solutions prepared with 1 % SN90 and various concentrations of m‐xylene were measured and compared with effluent collected during column‐flushing experiments. It was determined that as m‐xylene concentration in the emulsion solution Increases, viscosity increases. Viscosity increases caused a decrease in relative permeability within the soil column. As a result, the hydraulic gradient required to maintain a constant flowrate of 1.1 ml/min (using a syringe pump) through the soil column increased. Results show that a relatively small increase in viscosity could have a noticeable effect on the mobilization process. It is suggested that the surfactant/contaminant systems be screened to determine emulsion theology and the potential effects on the remediation process. The use of low‐concentration alcohol cosurfactants to reduce system viscosity was evaluated and was shown to be ineffective.     

High frequency androgenesis in indica × Basmati rice hybrids using liquid culture media

An improved procedure has been developed for high frequency androgenesis in indica × Basmati rice hybrids using a liquid culture medium. Anthers from fourteen genotypes comprising of indica × Basmati rice F₁ hybrids, F₂ plants and the parental rice cultivars, were floated in liquid RZM, N6M, and Heh5M media. Anther culture frequencies (percentage of anthers forming calluses) in most of the genotypes were significantly higher in RZM medium (16–75%) compared to those obtained in N6M (7–29%) and Heh5M (7–41%) media. Agarose (1.0% w/v)-solidified MSR1 medium containing 3.0% (w/v) maltose, 1 mg l⁻¹ kinetin, 1 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹α-naphthalene acetic acid (NAA) induced green shoot regeneration at high frequencies compared to the medium (MSR2) lacking BA. In all the genotypes, microspore calluses initiated in RZM medium regenerated green shoots with over tenfold higher frequencies compared to the calluses initiated in other two media. High plant regeneration frequencies (up to 270 green plants/1000 anthers) were obtained from microspore-derived calluses of some of the F₁ hybrids (Gobind × Basmati 370, Gobind × Taraori Basmati) and F₂ plants (Gobind × Basmati 370, Gobind × Taraori Basmati, HKR86-3 × Taraori Basmati) as compared to their actual parents. Cytological analysis of the root tips of the progeny seedlings of the microspore-derived plants revealed haploids at a frequency of about 50%; 22% of the microspore- derived plants had > 5% spikelet fertility and were diploid. Use of RZM liquid and MSR1 media, respectively for anther culture and plant regeneration resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice F₁ hybrids/F₂ plants which were comparable to those reported for japonica rice varieties/hybrids leading to the improved feasibility of using doubled haploids in genetic, breeding and mapping research with indica rice. This revised version was published online in June 2006 with corrections to the Cover Date.