Bo Mattiasson

The potential of polymeric cryogels in bioseparation

This is a review discussing the production and properties of cryogels (from the Greek κριoσ (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point.Cryogels can be obtained through the formation of both physically and covalently cross-linked heterogeneous polymer networks. Interconnected systems of macropores and sponge-like morphology are typical for cryogels, allowing unhindered diffusion of solutes of practically any size. Most of the water present in spongy cryogels is capillary bound and can be removed mechanically by squeezing. The properties of cryogels can be regulated by the temperature of cryogelation, the time the sample is kept in a frozen state and freezing/thawing rates, by the nature of the solvent and by the use of soluble and insoluble additives. The unique macroporous morphology of cryogels, in combination with osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of large entities such as protein aggregates, membrane fragments, viruses, cell organells and even whole cells. Special attention is given to immunosorption of viruses on cryogel-based sorbents. As chromatographic materials, cryogels can be used both in bead form and as spongy cylindrical blocks (monoliths) synthesized inside the chromatographic column. The macroporous nature of cryogels is also advantageous for their application as matrices in the immobilization of biocatalysts operating in both aqueous and organic solvents. New potential applications of cryogels are discussed.

Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.

Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples:

The principal goal of this work was to establish the feasibility of two biosensor technologies with enhanced specificity and selectivity for the detection of several bioavailable heavy metals in environmental samples. Two parallel strategies have been followed. The first approach was to construct whole cell bacterial biosensors that emit a bioluminescent or fluorescent signal in the presence of a biologically available heavy metal. The molecular basis of σ-54 promoters as sensing elements of environmental pollutants has been determined and a number of metal-induced promoter regions have been identified, sequenced and cloned as promoter cassettes. The specificity of the promoter cassettes has been determined using luxCDABE reporter systems. Whole cell-biosensors containing metal-induced lux reporter systems have been incorporated into different matrices for their later immobilisation on optic fibres and characterised in terms of their sensitivity and storage capacity. The second type of sensors was based on the direct interaction between metal-binding proteins and heavy metal ions. In this case, the capacitance changes of the proteins, such as synechoccocal metallothionein (as a GST-SmtA fusion protein) and the mercury regulatory protein, MerR, were detected in the presence of femtomolar to millimolar metal ion concentrations.

Microbial BOD sensors for wastewater analysis

The field of biosensors for measuring biochemical oxygen demand (BOD) is reviewed. Particularly, BOD sensors constructed on the biofilm configuration are discussed regarding performance characteristics like linearity, response time, precision, agreement between BOD values obtained from the biosensors and the conventional 5-days test, as well as toxic resistance to various compounds and operational stability. The techniques for improving the agreement between the sensor BOD and BOD5 are described. Information provided also includes BOD biosensors based on respirometers and other measuring principles, the commercial BOD instruments, as well as the current limitations of BOD biosensor development.

Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather

Two alkaline protease producing alkaliphilic bacterial strains, designated as AL-20 and AL-89, were isolated from a naturally occurring alkaline habitat. The two strains were identified as Nesternkonia sp. and Bacillus pseudofirmus, respectively. Both strains grew and produced alkaline protease using feather as the sole source of carbon and nitrogen. Addition of 0.5% glucose to the feather medium increased protease production by B. pseudofirmus AL-89 and suppressed enzyme production by Nesternkonia sp. AL-20. The enzymes from both organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight, determined using SDS–PAGE, was 23 kDa for protease AL-20 and 24 kDa for protease AL-89. Protease AL-20 was active in a broad pH range displaying over 90% of its maximum activity between pH 7.5 and 11.5 with a peak at pH 10. The enzyme is unique in that unlike all other microbial serine proteases known so far, it did not require Ca2+ for activity and thermal stability. Its optimum temperature for activity was at 70 °C and was stable after 1 h incubation at 65 °C both in the presence and absence of Ca2+. These properties make protease AL-20 an ideal candidate for detergent application. Protease AL-89 on the other hand require Ca2+ for activity and stability at temperature values above 50 °C. Its optimum activity was at 60 and 70 °C in the absence and presence of Ca2+, respectively. It displayed a pH optimum of 11 and retained about 70% or more of its original activity between pH 6.5 and 11. B. pseudofirmus AL-89, and the protease it produce offers an interesting potential for the enzymatic and/or microbiological hydrolysis of feather to be used as animal feed supplement.

Organic solvents for bioorganic synthesis

SummaryThe influence of solvents on enzymatic activity and stability was investigated. As a model reaction the α-chymotrypsin-catalyzed esterification of N-acetyl-l-phenylalanine with ethanol was used. The enzyme was adsorbed on porous glass beads and used in various solvents. Small amounts of water were added to increase the enzymatic activity. These enzyme preparations obeyed. Michaelis-Menten kinetics. Km,app decreased slightly with the log P value of the solvent while Vapp increased markedly with the log P value. Log P values were also useful for generalizing the influence of solvents on enzyme stability. The enzyme preparations showed a markedly higher thermostability in dry solvents having log P values >0.7 than in less hydrophobic solvents.Also the operational stability was better in the more hydrophobic solvents. The amount of water added to the enzyme preparations greatly influenced the initial reaction rates. For some solvents optimal water contents were determined. The thermostability decreased with increasing water content.The observations are summarized in the conclusion that more hydrophobic solvents are preferable to less hydrophobic ones. The log P value gives a good guidance when selecting an organic solvent for enzymatic conversions.

Studies on a matrix-bound three-enzyme system

Abstract The kinetic behaviour of a matrix-bound three-enzyme system has been studied and compared with that of an analogous system consisting of the three enzymes unbound and in solution. The enzymes chosen were β-galactosidase, hexokinase and glucose-6-phosphate dehydrogenase which carry out three consecutive reactions. It was found that the efficiency of the coupled reaction catalyzed by the matrix-bound three-enzyme system was higher prior to reaching steady state than that catalyzed by the corresponding soluble system. Although a higher efficiency than that of the corresponding soluble system was also found for the coupled reaction when only the two last enzymes of the matrix-bound system were implicated, the measured increase in efficiency between the complete three-enzyme systems was even more pronounced, indicating a cumulative efficiency effect.

Evaluation of parameters for monitoring an anaerobic co-digestion process

Abstract The system investigated in this study is an anaerobic digester at a municipal wastewater treatment plant operating on sludge from the wastewater treatment, co-digested with carbohydrate-rich food-processing waste. The digester is run below maximum capacity to prevent overload. Process monitoring at present is not extensive, even for the measurement of on-line gas production rate and off-line pH. Much could be gained if a better program for monitoring and control was developed, so that the full capacity of the system could be utilised without the risk of overload. The only limit presently set for correct process operation is that the pH should be above 6.8. In the present investigation, the pH was compared with alkalinity, gas production rate, gas composition and the concentration of volatile fatty acids (VFA). Changes in organic load were monitored in the full-scale anaerobic digester and in laboratory-scale models of the plant. Gas-phase parameters showed a slow response to changes in load. The VFA concentrations were superior for indicating overload of the microbial system, but alkalinity and pH also proved to be good monitoring parameters. The possibility of using pH as a process indicator is, however, strongly dependent on the buffering capacity. In this study, a minor change in the amount of carbohydrates in the substrate had drastic effects on the buffering effect of the system.

Triglyceride interesterification by lipases. 1. Cocoa butter equivalents from a fraction of palm oil

Twelve commercially available triacylglycerol lipase preparations were screened for their suitability as catalysts in the interesterification of palm oil mid fraction and ethyl stearate to form a cocoa butter equivalent. Five fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipase preparations was tested with sunflower seed oil and was independent of their interesterification activity. The operational stability of three of the preparations most suited for production of cocoa butter equivalents was examined. The amount of a commercial lipase preparation loaded onto a support was surveyed for optimum short-term catalytic activity.The influence of solvent concentration on the reaction rate and the purity of the product was examined at two temperatures. The optimum solvent concentration at 40°C was 1–1.5 grams of solvent/gram of substrate; at 60°C, the rate of interesterification diminished and the purity of the product decreased with increasing amounts of solvent.Four of the commercial lipase preparations found to be suitable interesterification catalysts were immobilized on five supports and their ability to catalyze the interesterification of a triglyceride and palmitic acid or ethyl palmitate was measured. The choice of support and substrate form (esterified or free fatty acid) greatly affected the catalytic activity. Some preparations were more affected by the choice of support, others by the form of the substrate. No preparation yielded maximum activity on all supports, and no support was found which produced an immobilized enzyme preparation of high activity with every commercial lipase preparation. Caution is advised in transferring observations about the suitability of a support from tests on one commerical enzyme preparation to others; individual testing is required.

Immobilised activated sludge based biosensor for biochemical oxygen demand measurement

A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4-8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD5 analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below +/- 5.6%, standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. +/- 15% standard deviation.