Immobilized Enzyme Bioreactor Research Articles

Establishment of a functional and sustainable bio-system engineering process for the saccharification of starch with industrial focus

Enzyme immobilization provides an excellent opportunity to use the enzymes for several times with greater productivity. The main aim of the present study is the establishment of a functional and sustainable bio-system engineering process based on immobilized α-amylase enzyme for the saccharification of starch with industrial focus as well as cost effective sustainable bioprocess system by using indigenous industrial waste materials as carrier agents. In this study, α-amylase was immobilized in different types of support matrices like alginate, sawdust, sugarcane bagasse and the activity of immobilized enzymes were analyzed for the breakdown of starch. The experimental results showed that the productivity of immobilized enzyme was around 2.3 times higher than the free enzyme. Conjugates of bagasse-alginate showed the highest result. A fixed batch immobilized enzyme bioreactor had been developed which could be used for the production of various valuable products in the industrial scale.
 Jahangirnagar University J. Biol. Sci. 8(1): 45-55, 2019 (June)

Performance and Applications of Semifluidized Bioreactors – A Review

AbstractThis paper reviews the design features and performance characteristics of semifluidized bed bioreactors. Both biofilm reactors and immobilized enzyme bioreactors have been analysed. Also, both two phase (liquid – solid) and three phase (gas – liquid – solid) operations are considered. Apart from the hydrodynamic parameters (semifluidization velocity, bed expansion ratio, height of packed section formed, fractional fluid holdups in both fluidized and packed sections), all other operating/system parameters affecting reactor performance such as axial dispersion, intrinsic kinetics of bioconversion (mostly nonlinear), the effectiveness factor and Thiele – type modulus have also been surveyed. The specific advantages and attractive features of these bioreactors have been highlighted based on data from successful case studies such as bioplastic synthesis, biodiesel manufacture, Xanthan gum production and aerobic waste water treatment with and without LPO (Liquid Phase Oxygen) utilization.

Determination of total phenolic content of Prunella L. by immobilized enzyme bioreactor

A new method for determination of total phenolic content of four Prunella L. species was developed by immobilizing horseradish peroxidase (HRP) on styrene-divinylbenzene-polygluteraldehyde (STY-DVB-PGA) polymer. The method is based on the spectrophotometric measurement of the final quinone-imine colored product, absorbing at 510 nm, by horseradish peroxidase oxidation in presence of hydrogen peroxide. The effects of solvents were investigated for the extraction of phenolic compounds in Prunella L. plants. HRP was covalently attached on the STY-DVB-PGA with 90% efficiency at optimum immobilization conditions. The total phenol contents ranged from 4 to 152 mg GAE per g of dried sample for pure solvents and ranged from 19 to 763 mg GAE per g of dried sample for acidic extracts using the immobilized enzymatic method. STY-DVB-PGA beads are a good potential support for the immobilization of HRP enzyme and can be used for determination of the total phenolic content of plants. In the analysis of four Prunella L. species, high correlations were obtained between enzymatic and Folin methods, supporting the validity of the enzymatic method proposed. The enzymatic (free/immobilized) methods are more specific and rapid than the Folin method. Acidic solvents are more effective than pure solvents for extraction of phenolic compounds in Prunella L. species.

제초제 검출 키트 개발을 위한 식물 해독효소 고정화 연구

Glutathione S-transferase는 식물의 해독작용에 중추적인 역할을 하는 화학 효소이다. 본 연구에서는 제초제 검출 키트 개발에 응용을 위하여 식물 해독작용에 중추적인 역할을 하는 glutathione Stransferase의 고정화 방법을 연구하였다. Chloroacetanilide계 제초제에 높은 효소 활성을 보이는 벼 유래 OsGSTF3에 공유결합을 통한 polystyrene-alkylamine 비드와 리간드결합을 통한 agarose-aminoalkyl 비드,포괄법을 통한 Na-alginate 비드를 이용하여 고정화를 실시하였다. 정제된 OsGSTF3 10 mg을 사용하여 고정화 하였을 때 0.62 mg/g 비드로 polystyrene-alkylamine에 가장 효율적으로 고정화 되었다. 고정화 된 OsGSTF3의 효소 활성은 야생형의 30%를 나타내었으며, 재사용에 의한 효소활성 측정시 3회 까지 처음 활성의 80% 이상을 유지하였다. Glutathione S-transferase is known to play a crucial role in detoxification in many cases. To develop a herbicide detection biosensor, we in this study attempted to immobilize glutathione S-transferase enzyme on solid supports, polystyrene and agarose, and Na-alginate. These matrixes were attractive materials for the construction of biosensors and might also have utility for the production of immobilized enzyme bioreactors. We also compared the activities of glutathione-S-transferase immobilized OsGSTF3 and free OsGSTF3. The specific activity of the free enzyme in solution was 3.3 higher than the immobilized enzyme. These results suggest that 50% of the enzyme was bound with the catalytic site in polystyrene-alkylamine bead and immobilized enzymes showed 80% remaining activity until 3 times reuse.

Characterization of the dynamic behavior of nonlinear biosystems in the presence of model uncertainty using singular invariance PDEs: Application to immobilized enzyme and cell bioreactors

A new approach to the problem of characterizing the dynamic behavior of nonlinear biosystems in the presence of model uncertainty using the notion of slow invariant manifold is proposed. The problem of interest is addressed within the context of singular partial differential equations (PDE) theory, and in particular, through a system of singular quasi-linear invariance PDEs for which a general set of conditions for solvability is provided. Within the class of analytic solutions, this set of conditions guarantees the existence and uniqueness of a locally analytic solution which represents the system's slow invariant manifold exponentially attracting all dynamic trajectories in the absence of model uncertainty. An exact reduced-order model is then obtained through the restriction of the original biosystem dynamics on the slow manifold. The analyticity property of the solution to the invariance PDEs enables the development of a series solution method that can be easily implemented using MAPLE leading to polynomial approximations up to the desired degree of accuracy. Furthermore, the aforementioned attractivity property and the transition towards the above manifold is analyzed and characterized in the presence of model uncertainty. Finally, examples of certain immobilized enzyme bioreactors are considered to elucidate aspects of the proposed context of analysis.

Effect of biocatalyst swelling on the operation of packed-bed immobilized enzyme bioreactor

A general mathematical model was developed for predicting the performance and simulation of a packed-bed immobilized enzyme reactor performing a reaction that follows Michaelis–Menten kinetics with competitive product inhibition. The performance of a packed-bed immobilized enzyme reactor was analyzed taking into account the effect of bed swelling on various diffusional phenomena such as axial dispersion, internal and external mass transfer limitations. The numerical solutions were compared with experimental data obtained for a packed-bed reactor operating with β-galactosidase entrapped in Ca-alginate-K-κ-carrageenan gels for lactose hydrolysis.

Production of chitosan oligosaccharides by chitosanase directly immobilized on an agar gel-coated multidisk impeller

An immobilized enzyme bioreactor consisting of an agar gel-coated multidisk impeller was developed for the hydrolysis of highly viscous chitosan solutions, and the operating conditions for the production of physiologically active chitosan oligosaccharides (pentamers and hexamers) were investigated. Chitosanase was directly immobilized on the agar gel-coated multidisk impeller by a multipoint attachment method. The high stability of the immobilized enzyme was confirmed by means of five repetitions of a batch hydrolysis reaction. When the enzyme activity at the support surface was relatively high, the yield of the target products was higher at an impeller speed of 2 s −1 than at a speed of 1 s −1. However, no significant increase in yield was observed at impeller speeds higher than 2 s −1 in reactions at either of the two substrate concentrations tested (5 and 20 kg/m 3). When the surface enzyme activity was low, the impeller speed did not affect the yield of the target products. The maximum yield of pentamers and hexamers increased as the surface enzyme activity decreased, and high yields (>30%) were obtained at activities below 160 U/m 2. From the viewpoint of productivity, the optimal surface-enzyme activity was about 340 U/m 2, and at that activity, the yield of target products was 22%. This yield was higher than that reported for conventional acid hydrolysis. To maximize both the productivity and the yield of the target products, the surface area for the immobilized enzyme should be increased. Our results suggest that it may be possible to obtain high yields of pentamers and hexamers of chitosan oligosaccharides from highly viscous chitosan solutions with this reactor.

Enterococcus faecalis phosphomevalonate kinase

The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

Enzyme Immobilization in Porous Silicon: Quantitative Analysis of the Kinetic Parameters for Glutathione-S-transferases

Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (approximately 6.5 microm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is approximately 4x higher than the immobilized enzyme, for which we measured an apparent K'(m)(GSH) value of 1.0 +/- 0.3. The maximum velocity, V'(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is approximately 20 times lower than that in solution. Results suggest approximately 25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.

Cascade of bioreactors in series for conversion of 3-phospho- d-glycerate into d-ribulose-1,5-bisphosphate: kinetic parameters of enzymes and operation variables

A novel scheme employing enzymatic catalysts is described enabling conversion of d-ribulose-1,5-bisphosphate (RuBP) from 3-phospho- d-glycerate (3-PGA) without loss of carbon. Bioreactors harboring immobilized enzymes namely, phosphoglycerate kinase (PGK), glycerate phosphate dehydrogenase, triose phosphate isomerase (TIM), aldolase, transketolase (TKL), phosphatase (PTASE/FP), epimerase (EMR) and phosphoribulokinase (PRK), in accordance with this novel scheme were employed. These reactors were designed and constructed based on simulations carried out to study their performance under various operational conditions and allowed production of about 56 ± 3% RuBP from 3-PGA. This method of synthesis of RuBP from 3-PGA employing immobilized enzyme bioreactors may be used for continuous regeneration of RuBP in biocatalytic carbon dioxide fixation processes from emissions where RuBP acts as acceptor of carbon dioxide to produce 3-PGA, rendering the fixation process continuous.

Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro

To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1–4 h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.

Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro

To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1–4 h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.

Development of novel microscale system as immobilized enzyme bioreactor.

This study involves a novel method for immobilized enzyme catalysis. The focus of the work was to design and construct a microscale bioreactor using microfabrication techniques traditionally employed within the semiconductor industry. Enzymes have been immobilized on the microreactor walls by incorporating them directly into the wall material. Fabricated microchannels have cross-sectional dimensions on the order of hundreds of micrometers, constructed using polydimethylsiloxane cast on silicon/SU-8 molds. The resulting ratio of high surface area to volume creates an efficient, continuous-flow reaction system. Transverse features also containing enzymes were molded directly into the channels.

Modeling of glucose isomerization in a fluidized bed immobilized enzyme bioreactor

A heterogeneous model describing the glucose isomerization process in an immobilized enzyme, fluidized-bed bioreactor was developed. The model without any adjustable parameters was used to predict the isomerization process. An excellent agreement between model predictions and experimental data for two isomerases (PG-GI Complex and Sweetase) obtained from the literature was found. The key parameters which measured the extent of external and internal mass-transport resistances, as well as the degree of back-mixing were quantified and discussed. The effects of some operating and design parameters on the performance of the fluidized-bed bioreactor were also analyzed.

Biomaterials and biomedical engineering

The current status and future prospects of biomaterials, in the context of chemical engineering science and biomedical engineering, is examined. Research on artificial organs, controlled drug delivery systems, tissue engineering, materials for minimally invasive surgery, medical contrast imaging agents, gene therapy, immobilized enzyme bioreactors, and biosensors is evaluated. Techniques for assessing the performance of biomaterials are considered. The role of new types of materials, not yet used in medicine, such as electrically conducting polymers, is discussed. Finally, an analysis of future educational and training issues in biomaterials in the context of chemical engineering is provided.

Optimization of immobilized enzyme hydrolysis combined with high-performance liquid chromatography/thermospray mass spectrometry for the determination of neuropeptides

Peptidases, including chymotrypsin, thermolysin, trypsin, V 8 protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with HPLC/thermospray MS for the analysis of neuropeptides. The optimal operating conditions for each immobilized enzyme bioreactor were determined. Optimal hydrolysis usually occurred at the highest percentage of aqueous solution in the mobile phase at pH 7–8 and 40–50°C. Often post-HPLC column addition of aqueous solutions before the bioreactor could improve activity and thermospray sensitivity without changing the HPLC separation. Enzymatic hydrolysis requirements were compatible under conditions for HPLC separation and thermospray MS detection of the selected neuropeptides. Synthetic α-, β-, and γ-endorphins were the primary neuropeptides used to evaluate on-line immobilized enzyme bioreactor/MS. HPLC followed by peptidase hydrolysis produced characteristic hydrolysis products for confirming the peptides' identity using thermospray MS detection. Furthermore, the peptide formed from enzymatic hydrolysis resulted in a MS ion current 10–40 times higher than that of the [M + 2H] 2+ ion for unhydrolyzed β-endorphin. The increased sensitivity achieved for detecting the hydrolysis products permits detection and quantitation of synthetic peptides down to 800 fmol.

Synthesis of branched cyclodextrin with immobilized enzyme bioreactor

Synthesis of branched cyclodextrin with immobilized enzyme bioreactor

Medical reactors

Four examples illustrating the application of reaction principles in medical therapies are discussed: 1) drug delivery systems controlled by reaction only, 2) drug delivery systems controlled by reaction and diffusion, 3) immobilized enzyme bioreactors to remove toxic substances from blood, and 4) mammalian cell reactors to address organ donor problems. These examples serve not only to examine how reactor design can be applied to medical problems, but to address both the similarities and differences of medical reactors compared to other reactors.

Maltotetraose, A New Saccharide of Tertiary Property

AbstractMaltotetraose, a linear tetramer of α‐D‐glucose, has many potential uses in the food and pharmaceutical industries because of its unique characteristics. A consortium of Nihon Shokuhin Kako Co., Ltd., Kirin Brewery Co., Ltd., Chiyoda Corp. and Yokogawa Electric Corp. has developed a continuous maltotetraose production process which uses an immobilized enzyme bioreactor system. Because maltotetraose syrup is a brand‐new saccharide having low sweetness and resistance to retrogradation, it has begun to be used as a property enhancer for various foods, powdering materials, saccharide for dry milk, liquid diets for the sick and a body reinforcement agent for refreshing drinks. In addition to the properties of nutrition and taste, the tertiary property of organism control has been discovered; that is, feeding with a maltotetraose‐rich corn syrup inhibits the growth of intestinal putrefactive bacteria such as C. perfringens and Enterobacteriaceae.