Biochemical Engineering Journal (v.48, #1)

BEJ Keywords (II).

Recovery of protein from brewer's spent grain by ultrafiltration by De-Song Tang; Gang-Ming Yin; Yuan-Zhe He; Song-Qing Hu; Bing Li; Lin Li; Hui-Ling Liang; Devajit Borthakur (1-5).
Application of ultrafiltration in recovery of protein from brewer's spent grain (BSG) was studied in this work. The effectiveness in removing water and salts was evaluated. Results indicated that increasing of cross-flow rate could improve the limiting flux. More than 92% of the protein was retained by the membranes with both MWCO of 5 and 30 kDa. The protein contents in the final product were 20.09 ± 1.40% and 15.98 ± 0.58%, respectively by 5 and 30 kDa membranes compared with that of 4.86 ± 0.61% concentrated by rotary evaporation. It indicated that ultrafiltration had good ability in the removal of salts in the extract solution and improved the quality of final products. The 5 kDa membrane had a little higher protein retention capacity than that of 30 kDa.
Keywords: Brewers’ spent grain; Protein; Membrane; Ultrafiltration;

Enzyme immobilization on amphiphilic polymer particles having grafted polyionic polymer chains by Masahiro Yasuda; Hibiki Nikaido; Wilhelm R. Glomm; Hiroyasu Ogino; Kosaku Ishimi; Haruo Ishikawa (6-12).
Previously, we have shown that amphiphilic polymer particles functionalized with both hydrophilic guanidino groups and hydrophobic acyl groups have been shown to immobilize a large amount of lipase with the immobilized lipase retaining high transesterification activity in organic solvent. However, the stability of immobilized lipase in organic solvent was found to be insufficient. In the present study, the chemical environment surrounding the immobilized enzyme was made more hydrophilic in order to enhance the stability of immobilized lipase in organic solvent. For this purpose, polyionic polymer chains containing amino group in addition to amphiphilic groups were introduced. Monodisperse acrylic polymer particles (∼10 μm) were synthesized by seed polymerization in the presence of pore forming agent, and subsequently modified with poly(allylamine) and various reagents to introduce amphiphilic functional groups. The half-life of Rhizopus delemar lipase immobilized on these macroporous amphiphilic polymer particles in hexane was 2.46 times high compared to lipase immobilized on amphiphilic polymer particles with guanidino and stearoyl groups.
Keywords: Enzyme immobilization; Polyionic graft chain; Amphiphilic polymer particle; Enzymatic reaction in organic phase;

In the present work, we study the covalent immobilization of l-lactate dehydrogenase (LDH) on silica-coated magnetic nanoclusters (SiMag). Immobilization of LDH on magnetic nanoclusters provides several advantages. First, it can ease the enzyme recovery through magnetic separation, rendering the immobilized LDH applicable for batch reactor which configuration can minimize the product inhibition. Second, the immobilization resulted in the increase of product inhibition constants of LDH which can further minimize the product inhibition. A 5-fold and a 1.6-fold increase of apparent competitive product inhibition constant of NAD+ (K iq ) and of lactate (K iq K p /K q ) were observed, respectively. Third, the immobilized LDH showed improvement on thermal stability, with 5–7-fold reduced deactivation rate constant in second phase (A) of biphasic deactivation behavior, but with no significant difference in the first phase (B). Present study also develops a method to improve the immobilization process by understanding the surface interaction involved during the covalent immobilization of enzyme on magnetic nanoclusters. We found that the addition of sodium chloride during the immobilization could improve the relative activity of immobilized LDH from 50% to about 75%. This was due to the possibility of hydrophobic interaction in controlling a better LDH orientation on particle surface, and thus avoiding any severe deactivation. Protein representation using Jmol software also confirmed this possibility.
Keywords: Dehydrogenase; Immobilized enzyme; Magnetic nanoclusters; Oriented immobilization; Stability;

Biodiesel production from biomass of an oleaginous fungus by Gemma Vicente; L. Fernando Bautista; Rosalía Rodríguez; F. Javier Gutiérrez; Irantzu Sádaba; Rosa M. Ruiz-Vázquez; Santiago Torres-Martínez; Victoriano Garre (22-27).
The present paper introduces the filamentous fungus Mucor circinelloides as a potential feedstock for biodiesel production. These microbial lipids showed a high content (>85%) of saponifiable matter and a suitable fatty acid profile for biodiesel production. The effectiveness of the lipid extraction process was studied for three different solvent systems: chloroform:methanol, chloroform:methanol:water and n-hexane. Biodiesel was produced by acid-catalysed transesterification/esterification following two different approaches: transformation of extracted microbial lipids and direct transformation of dry microbial biomass. After 8 h of reaction at 65 °C in the presence of BF3, H2SO4 or HCl as acid catalysts, the direct process produced fatty acid methyl esters (FAMEs) with higher purities (>99% for all catalysts) than those from the two-step process (91.4–98.0%). In addition, the yield was also significantly higher in the direct transformation due to a more efficient lipid extraction when the acid catalyst was present.
Keywords: Biodiesel; Fatty acid methyl esters; Microorganism; Fungi; Mucor circinelloides; Microbial oil;

This study was undertaken to find out the optimum reaction conditions, i.e. pH, temperature, enzyme and substrate concentration for the thermostable cellulase enzyme activity of Aspergillus heteromorphus using response surface methodology (RSM). Estimated optimum conditions for cellulase enzyme were found as: pH 4.8, temperature 60 °C, crude enzyme/substrate concentration ratio was 4:6. The maximum enzymatic activity calculated was observed 13.05 U/ml against the predicted value 11.26 U/ml. Cellulase system of A. heteromorphus is thermostable retaining 60.0% of residual activity after 1 h of pre-incubation at 90 °C when assay is carried out at same temperature. Value of correlation coefficient (R 2 0.9977) and significant value for model p  < 0.0001 indicated validity of model fitness and adequate for optimization.
Keywords: Aspergillus heteromorphus; Cellulase; Enzyme activity; Optimization; Response surface methodology (RSM); Cellulose;

Introduction of carbonyl group to sugars using pyranose 2-oxidase-immobilized membrane by Hirokazu Seto; Hidetaka Kawakita; Keisuke Ohto (36-41).
To introduce the carbonyl group to sugars in a continuous process, pyranose 2-oxidase was immobilized on a poly(vinylidene fluoride) membrane by hydrophobic interaction, and sugar solutions were passed through the membrane. The amount of immobilized pyranose 2-oxidase reached 14 mg g−1 in 1 h in permeation mode. With increase in the space velocity of the solution of one of the sugars used (sucrose), the reactivity of the membrane decreased, indicating that the space velocity of the system should be less than 10 h−1. Glucose, sucrose, and dextran solutions were circulated through the enzyme-immobilized membrane. Glucose and sucrose were completely converted to keto-glucose and keto-sucrose in 75 and 225 h, respectively, whereas dextran was not converted. The operational and storage stabilities were investigated using glucose as substrate, resulting that relative activity of pyranose 2-oxidase immobilized onto Durapore was kept for 7 days. The membrane system immobilizing pyranose 2-oxidase is thus effective for conversion of monosaccharides or disaccharides by introduction of carbonyl groups.
Keywords: Pyranose 2-oxidase; Keto-sugar; Enzyme immobilization; Poly(vinylidene fluoride); Membrane; Circulation system;

A lab scale bubble column (BC) was utilized to treat waste gas containing toluene by free microorganism of Pseudomonas putida WQ-03. The bioreactor had a height to diameter ratio of 10 and a working volume of 17.3 L. The gas stream was injected from the bottom of the BC and then the pollutant was degraded by the bacteria. A complex three-dimensional transient computational fluid dynamic (CFD) model was established for detailed description of toluene treatment in the bioreactor. The model coupled three simultaneous aspects of gas–liquid multiphase flow, interphase mass transfer and intrinsic microbial kinetics. The Haldane's model was adopted for the inhibition of toluene on the growth of microbe and the kinetic constants were obtained experimentally. The simulation was validated by experimental data of the dissolved oxygen and toluene concentrations as well as the toluene removing efficiency. Good agreement between the experiments and the simulation was achieved. Mass transfer and bioreaction were compared to determine the rate-limiting step. The effects of operating parameters were discussed in simulation results and further predictions of the transient dispersions of toluene, oxygen and cell were also provided by the developed model.
Keywords: Waste gas treatment; 3D transient CFD model; Toluene; Bioreactor; Hydrodynamics; Mass transfer;

Enhanced production of xylanase by a newly isolated Aspergillus terreus under solid state fermentation using palm industrial waste: A statistical optimization by Garapati Suvarna Lakshmi; Chaganti Subba Rao; Ravella Sreenivas Rao; Phil J. Hobbs; Reddy Shetty Prakasham (51-57).
Xylanase production by a newly isolated Aspergillus terreus MTCC 8661 was optimized using palm fiber in solid state fermentation (SSF). Different fermentation parameters such as incubation temperature, moisture content, medium pH, particle size, incubation time, inoculum, xylose and sodium nitrate concentrations were investigated at the individual and interactive level by the Taguchi methodology. All selected fermentation parameters influenced xylanase production. Moisture content, incubation time and inoculum concentration were the major [∼85%] influential parameters on xylanase production at the individual level. At the interactive level, inoculum concentration was important and accounted for more than 50% of the severity index with particle size and incubation temperature. Xylanase production improved from 41,000 to 115,000 U/g indicating 227% improvement after optimization suggesting that this fungal strain, A. terreus MTCC 8661, has the commercial potential for hemicellulosic enzyme production.
Keywords: Xylanase; Hemicellulosic material hydrolyzing enzyme; Aspergillus terreus; Optimization; Solid state fermentation; Taguchi methodology;

Highly soluble and stable recombinant holo-phycocyanin alpha subunit expressed in Escherichia coli by Shaofang Liu; Huaxin Chen; Song Qin; Weijie Zhang; Xiangyu Guan; Yandu Lu (58-64).
C-phycocyanin (Cpc) is one of the phycobiliproteins with highly fluorescent and various pharmacological activities. Holo-Cpc-α subunit (holo-CpcA) expressed in Escherichia coli resulted in low yield and tended to aggregate after purification. In this study, we constructed a new plasmid coding holo-CpcA fused with hexahistidine and maltose-binding protein tag, which designated as HMCpcA, to improve its solubility and stability without the impairment of its spectra and fluorescent properties. HMCpcA was significantly more stable over time and a wider range of pH as compared to holo-CpcA. In addition, both the solubility and yields of HMCpcA increase significantly. We here provided an example to demonstrate that MBP could also improve the stability of the protein it fused while it has been reported as a soluble fusion partner before. This novel fluorescent protein will facilitate the large-scale production and be potentially applicable for the development of fluorescent probes, as well as antioxidant agents.
Keywords: Maltose-binding protein; Phycocyanin-α subunit; Protein engineering; Recombinant protein; Chromatography; Recombinant protein production;

Artificial chaperone consists of a detergent (cetyltrimethylammonium bromide, CTAB) for binding denatured protein and a stripper (β-cyclodextrin) for the release of the denatured protein from the detergent–protein complex. The authors have proposed artificial chaperone (AC)-assisted immobilized metal affinity chromatography (AC-IMAC) for protein refolding and purification, and have herein studied the effect of various operating conditions on the refolding efficiency of enhanced green fluorescent protein (EGFP) from solubilized inclusion bodies. It was found that the addition of CTAB to denatured EGFP at a molar ratio of six was favorable for effective refolding, and the binding time of CTAB–EGFP mixture should be at 20–40 min. There was an optimal flow rate of on-column refolding buffer, and optimum urea concentrations in both the on-column refolding buffer as well as in elution buffer that maximized refolding efficiency. Under optimized conditions, over 90% refolding was achieved at 0.5 mg/mL loading, and by loading 4 mL of 1.35 mg/mL denatured protein solution, 92% EGFP was recovered with a fluorescence recovery of 87% and concentration of about 1 mg/mL. The total recovery was increased by over 20% after the condition optimization. The effects of the operating conditions are expected to provide insight into the better understanding of the process for applications to other recombinant proteins expressed as inclusion bodies.
Keywords: Bioseparations; Protein; Refolding; Chromatography; Operating condition; Enhanced green fluorescent protein;

Improving power production in acetate-fed microbial fuel cells via enrichment of exoelectrogenic organisms in flow-through systems by Abhijeet P. Borole; Choo Y. Hamilton; Tatiana Vishnivetskaya; David Leak; Calin Andras (71-80).
An exoelectrogenic, biofilm-forming microbial consortium was enriched in an acetate-fed microbial fuel cell (MFC) using a flow-through anode coupled to an air-cathode. An MFC design with low electrode spacing, high specific electrode surface area with minimal dead volume and control of external resistance was used. In addition, continuous feeding of carbon source was employed and the MFC was operated at intermittent high flows to enable removal of non-biofilm-forming organisms over a period of 6 months. The consortium enriched using the modified design and operating conditions resulted in a power density of 345 W m−3 of net anode volume (3650 mW m−2), when coupled to a ferricyanide cathode. The enriched consortium included β-, δ-, γ-Proteobacteria, Bacteroidetes and Firmicutes. Members of the order Rhodocyclaceae and Burkholderiaceae (Azospira sp. (49%), Acidovorax sp. (11%) and Comamonas sp. (7%)), dominated the microbial consortium. Denaturing gradient gel electrophoresis (DGGE) analysis based on primers selective for archaea indicated presence of very few methanogens. Limiting the delivery of the carbon source via continuous feeding corresponding to the maximum cathodic oxidation rates permitted in the flow-through, air-cathode MFC resulted in coulombic efficiencies reaching 88 ± 5.7%.
Keywords: Microbial fuel cell; Biocatalyst; Enrichment; Exoelectrogenic; Biofilm-forming; Direct electron transfer; Diversity;

Optimization of inulin hydrolysis by inulinase accounting for enzyme time- and temperature-dependent deactivation by Emanuele Ricca; Vincenza Calabrò; Stefano Curcio; Gabriele Iorio (81-86).
In the present work a deactivation model for an inulinase from Aspergillus niger is presented; a first order kinetic is found and the deactivation constant k d is related to temperature through the Arrhenius model. The deactivation model was satisfactorily validated and implemented into a kinetic model for inulin hydrolysis predictions; the result is a complete model that is able to predict reaction performances for substrate concentrations ranging between 10 and 40 g/l and reaction temperature up to 60 °C, even on a long time scale. The model is also shown to be a powerful tool to understand reaction paths and to choose the optimal reaction conditions for long time scale processes. A relevant problem for enzymatic process temperature optimization is formulated and solved by means of the predictive model determined.
Keywords: Kinetic model; Enzyme deactivation; Fructose production; Inulin hydrolysis; Process optimization; Thermal effects;

The hybrid xylanase gene Xyn2-A2 was previously constructed and expressed in Pichia pastoris. In this study, the hybrid recombinant enzyme (PTXC2) was prepared from a 2-L bioreactor and subsequently characterized. The produced xylanase was estimated by SDS-PAGE to be 26 kDa and practically free of cellulolytic activity. The optimal temperature and pH of PTXC2 was 65 °C and pH 6.0, respectively. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30 min incubation at this temperature. The specificity of PTXC2 towards different natural substrates was evaluated. PTXC2 was highly specific towards xylans tested but exhibited low activities towards cellulosic substrates, such as gellan gum (9.7%), Avicel (1.5%) and carboxymethylcellulose (CMC, 1.2%). The apparent K m values of oat-spelt xylan and birchwood xylan was 1.6 and 1.8 mg/mL, respectively. Analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as an endo-xylanase with xylotriose as the main hydrolysis products. These attributes should make the enzyme an attractive applicant for various applications, such as large-scale production of xylooligosaccharides.
Keywords: Xylanase; Expression; Characterization; Thermostability; Xylooligosaccharides;

Immobilization of enzymatic extract from Penicillium camemberti with lipoxygenase activity onto a hybrid layered double hydroxide by Rogelio Morales Borges; Gregorio Guadalupe Carbajal Arizaga; Fernando Wypych (93-98).
A Zn/Al layered double hydroxide was synthesized by alkaline co-precipitation with azelate ions (OOC(CH2)7COO). The interlayer space of the layered material is occupied by organic ions as confirmed by X-ray diffraction and FTIR spectroscopy. The resulting hybrid material was tested as support for Penicillium camemberti enzymatic extract, containing lipoxygenase (LOX) activity. The optimal condition for LOX immobilization is done with 0.6 mol L−1 potassium phosphate buffer and pH 6.0. The affinity for the substrate is the same after immobilization, however the specific activity slightly decreases. The immobilization enhanced the thermal stability, which was evident with incubation at 60 °C where the immobilized enzyme retains 68% of specific activity while the free enzyme is inhibited. Recycling assays showed that after eight reaction cycles, the immobilized enzyme retains 60% of the activity.
Keywords: Enzyme; Microbial; Immobilized; Lipoxygenase; Composite;

Adsorption equilibria of bio-based butanol solutions using zeolite by Arjan Oudshoorn; Luuk A.M. van der Wielen; Adrie J.J. Straathof (99-103).
1-Butanol can be produced by clostridial fermentations with acetone and ethanol as by-products. The butanol can be present up to ∼20 g L−1 depending on process conditions and microbial strain. The high-silica zeolite CBV28014 has been proven to adsorb butanol selective over water, while showing higher affinity for butanol than for acetone and ethanol. Multi-component acetone–butanol–ethanol (ABE) adsorption on CBV28014 has been modeled using a single site extended Langmuir adsorption model and the ideal adsorbed solution (IAS) theory model. The IAS model describes multi-component adsorption of ABE in synthetic mixtures and ABE in filtered fermentation broth by CBV28014 more accurately than the single site extended Langmuir model.
Keywords: Bioseparations; Adsorption; Bioresources; Biofuels; Butanol; Zeolite;

Influence of pH and acid solutes on the phase behaviour of aqueous solutions containing poly(ethylene glycol) and poly(ethyleneimine) by Dragomir S. Yankov; J.P. Martin Trusler; Roumiana P. Stateva; Georgi St. Cholakov (104-110).
In this paper, which forms part of a study on aqueous two-phase systems (ATPS) for separation of low-molar-mass organic acids in the biotechnology industry, we examine the phase behaviour of ATPSs containing poly(ethylene glycol) (PEG) and poly(ethyleneimine) (PEI) as a function of pH, the nature of the acid used for titrating PEI, and the addition of lactic acid. We show that increasing the pH leads to contraction of the two-phase regions and that titrating PEI with a higher polyvalent acid results in a larger two-phase region. We propose a mechanism explaining the experimentally observed phase behaviour. Finally, we demonstrate that the lactic acid partition coefficient is very favourable (the acid partitions preferentially to the PEI-rich phase), which confirms that the (PEG + PEI) ATPSs could be used to advantage as a medium for lactic acid bio-transformation.
Keywords: ATPS; Lactic acid; Partition coefficient;

Two chitosanases (CHSB1 and CHSB2) were purified from the culture supernatant of Bacillus cereus TKU018 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of CHSB1 and CHSB2 determined by SDS–PAGE were approximately 44 kDa and 22 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSB1 and CHSB2 were (pH 5, 60 °C; pH 5–7, <40 °C) and (pH 7, 50 °C; pH 4–7, <50 °C), respectively. CHSB1 and CHSB2 were both inhibited by EDTA and CHSB1 was inhibited completely by 5 mM Zn2+. CHSB1 and CHSB2 degraded chitosan with DD ranging from 60% to 95%, but did not degrade chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU018 culture supernatant (1.5% SPP) incubated for 3–4 days has 75% relative antioxidant activity (DPPH scavenging ability). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.
Keywords: Chitosanase; Antioxidant; Shrimp shell wastes; Squid pen wastes; Bacillus cereus; Reducing sugar;

Increasing the substrate specificity of Bacillus stearothermophillus lactate dehydrogenase by DNA shuffling by Baris Binay; Debbie K. Shoemark; Richard B. Sessions; Anthony R. Clarke; Nevin Gul Karaguler (118-123).
Previously it is suggested that a single mutation (Q102R) in the lactate dehydrogenase (LDH) gene from Bacillus stearothermophillus (bs) could switch the substrate specificity by 3 orders of magnitude from lactate to malate and produce a highly efficient malate dehydrogenase (MDH). In order to examine if random mutation and screening could improve this, a DNA-shuffling method would be used to generate a mutant LDH and recombinant LDH genes which will be later used for transforming Escherichia coli. The recombinant colonies are blotted and screened for their ability to catalyse the oxidation of malate. The most active MDH produced by this method is only slightly more efficient than the rationally designed Q102R variant. In addition to this mutation, the shuffled version incorporates further seven residue changes which are chemically conservative. These experiments demonstrate that the blind shuffling can achieve a huge shift in specificity which was a known, highly effective single-site mutation designed using structural knowledge.
Keywords: Molecular modelling; NAD+-dependent lactate dehydrogenase enzyme; Protein engineering; Recombinant protein; Steady-state kinetic; Substrate specificity;

A novel cyclodextrin glycosyltransferase from Bacillus sphaericus strain 41: Production, characterization and catalytic properties by Cristiane Moriwaki; Lívia Rosas Ferreira; Júlia Regina Tedesco Rodella; Graciette Matioli (124-131).
The alkalophilic Bacillus sphaericus strain 41 was isolated from soybean-soil culture, using a highly alkaline pH medium containing 1% Na2CO3. The cyclodextrin glycosyltransferase (CGTase) from this microorganism was purified up to 315-fold with a yield of 31%, by biospecific affinity-column chromatography using Sepharose 6B gel and β-cyclodextrin (β-CD) as the ligand. The molecular weight of the purified enzyme was estimated to be 59 kDa by SDS-PAGE. In addition to the cyclization, the CGTase showed disproportionation and coupling activities. For cyclization activity, the optimal pH was 6.0 and the temperature was 65 °C. The enzyme showed pH stability in the range of 6.0–7.0. Thermal deactivation was noticeable above 70 °C, and the enzyme was highly stable below 65 °C. The activation and deactivation energy for the production of β-CD were 9.4 kcal/mol and 28.0 kcal/mol, respectively. The influence of substrate concentration on the initial rate of CD production was studied, and the kinetic parameters were determined. The K m was 0.0008 mol/L and V max was 0.0631 mol of β-CD/(L h), using maltodextrin as substrate. The CGTase was strongly inhibited by the products, and produced a level of CDs reaching 22 g/L with a β-CD ratio of 54%. This enzyme produced α-, β- and γ-CD in the ratio of 0.40:1:0.45.
Keywords: Bacillus sphaericus; Cyclodextrin glycosyltransferase; Enzyme; Microbial; Kinetics; Optimization;

A new salt inducible expression system for Lactococcus lactis by Noora Sirén; Kalle Salonen; Matti Leisola; Antti Nyyssölä (132-135).
A new expression system for Lactococcus lactis based on the salt inducible BusA promoter and the BusR repressor gene of L. lactis MG1363 was developed. To achieve salt induction, the expression of BusR was modulated by introducing mutations to its promoter sequence. An activity of 6.0 μkat l−1 of the model enzyme Lactobacillus amylovorus α-amylase was achieved in the bioreactor cultivation. The major advantage of the current expression system is that no additions of inducing agents are needed into bioreactor cultivations.
Keywords: Bioreactor systems; BusR; Lactococcus lactis; Salt induction; Recombinant proteins; Expression systems;

Pressure-induced covalent immobilization of enzymes onto solid surface by Dileep Kumar Kannoujia; Shakir Ali; Pradip Nahar (136-140).
Here in, we demonstrate a rapid method for covalent immobilization of enzymes onto activated polystyrene microtiter plate by application of pressure. Thus, when HRP, taken in an activated well of a polystyrene microtiter plate, is subjected to an optimum pressure of 2.0 × 105  Pa for an optimum time of 25 min in a closed chamber, it shows more than 5-fold increase in absorbance value compared to control experiment carried out without applying pressure. Pressure-induced immobilized HRP shows better thermal and storage stability with respect to free enzyme. Reusability study shows that immobilized enzyme retained almost full activity after three successive uses; however activity falls to half after ten cycles of repeated use. The Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax) for pressure-induced immobilized HRP are found to be 55.3 μM and 0.957 mM/min, respectively, whereas for free enzyme the corresponding values are 34.7 μM and 1.089 mM/min. The method is found to be equally effective for immobilization of other enzymes including glucose oxidase, alkaline phosphatase and invertase. As no thermal incubation is required, pressure-induced immobilization could be used for immobilization of heat sensitive biomolecules useful for preparation of biosensors, biochips and protein-microarrays.
Keywords: Covalent immobilization; Activated surface; Enzyme immobilization; Horseradish peroxidase; Polystyrene; Pressure;