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Special Issue "Enzyme Optimization and Immobilization"

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry, Molecular Biology and Biophysics".

Deadline for manuscript submissions: closed (31 August 2012)

Special Issue Editors

Guest Editor
Prof. Dr. Jose M. Guisan

Department of Biocatalysis, Institute of Catalysis, Spanish Research Council, ICP-CSIC, Campus UAM, 28049 Madrid, Spain
Website1 | Website2 | E-Mail
Phone: +34 91 585 48 09
Interests: enzyme engineering: purification, immobilization, stabilization, reactivation; hyperactivation; main enzymes: lipases, penicillin G acylase, endoxylanases, beta-xylosidases, etc.; enzyme processes: fine chemistry, food chemistry, analytical chemistry, green energy; enzyme reactors: stirred tanks, packed beds, etc.
Guest Editor
Dr. Fernando Lopez-Gallego

Department of Biocatalysis, Institute of Catalysis, Spanish Research Council, ICP-CSIC, Campus UAM, 28049 Madrid, Spain
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Special Issue Information

Dear Colleagues,

Enzymes are able to catalyze the most complex chemical processes under the most benign experimental and environmental conditions. However, enzymes, because of their biological origin, have some characteristics that are nor very useful for industrial implementation: they are soluble and unstable, they may be poorly active and selective towards non-natural substrates, they are inhibited by very high concentrations of substrates and products, etc. The proposed open-issue will bring together a number of interesting topics to improve the functional properties of industrial enzymes.

It is anticipated that the following topics will be included in the special issue:

  • industrial enzymes from extremophiles
  • improvement of enzyme properties by molecular biology
  • improvement of enzyme properties by immobilization techniques
  • new immobilization techniques
  • enzymes in non-conventional media

Prof. Dr. Jose M. Guisan
Dr. Fernando Lopez-Gallego
Guest Editors

Keywords

  • immobilization of industrial enzymes
  • enzyme stabilization
  • enzyme selectivity
  • enzyme inhibition
  • enzyme activity
  • novel enzyme processes
  • enzyme reactivation

Published Papers (24 papers)

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Research

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Open AccessArticle Effects of Heme Oxygenase-1 Upregulation on Blood Pressure and Cardiac Function in an Animal Model of Hypertensive Myocardial Infarction
Int. J. Mol. Sci. 2013, 14(2), 2684-2706; doi:10.3390/ijms14022684
Received: 19 November 2012 / Revised: 6 January 2013 / Accepted: 21 January 2013 / Published: 28 January 2013
Cited by 17 | PDF Full-text (2209 KB) | HTML Full-text | XML Full-text
Abstract
In this study, we evaluate the effect of HO-1 upregulation on blood pressure and cardiac function in the new model of infarct spontaneous hypertensive rats (ISHR). Male spontaneous hypertensive rats (SHR) at 13 weeks (n = 40) and age-matched male Wistar (WT)
[...] Read more.
In this study, we evaluate the effect of HO-1 upregulation on blood pressure and cardiac function in the new model of infarct spontaneous hypertensive rats (ISHR). Male spontaneous hypertensive rats (SHR) at 13 weeks (n = 40) and age-matched male Wistar (WT) rats (n = 20) were divided into six groups: WT (sham + normal saline (NS)), WT (sham + Co(III) Protoporphyrin IX Chloride (CoPP)), SHR (myocardial infarction (MI) + NS), SHR (MI + CoPP), SHR (MI + CoPP + Tin Mesoporphyrin IX Dichloride (SnMP)), SHR (sham + NS); CoPP 4.5 mg/kg, SnMP 15 mg/kg, for six weeks, one/week, i.p., n = 10/group. At the sixth week, echocardiography (UCG) and hemodynamics were performed. Then, blood samples and heart tissue were collected. Copp treatment in the SHR (MI + CoPP) group lowered blood pressure, decreased infarcted area, restored cardiac function (left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), +dp/dtmax, (−dp/dtmax)/left ventricular systolic pressure (LVSP)), inhibited cardiac hypertrophy and ventricular enlargement (downregulating left ventricular end-systolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and heart weight/body weight (HW/BW)), lowered serum CRP, IL-6 and Glu levels and increased serum TB, NO and PGI2 levels. Western blot and immunohistochemistry showed that HO-1 expression was elevated in the SHR (MI + CoPP) group, while co-administration with SnMP suppressed the benefit functions mentioned above. In conclusion, HO-1 upregulation can lower blood pressure and improve post-infarct cardiac function in the ISHR model. These functions may be involved in the inhibition of inflammation and the ventricular remodeling process and in the amelioration of glucose metabolism and endothelial dysfunction. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Characterization of a Polyamine Microsphere and Its Adsorption for Protein
Int. J. Mol. Sci. 2013, 14(1), 17-29; doi:10.3390/ijms14010017
Received: 31 August 2012 / Revised: 28 November 2012 / Accepted: 10 December 2012 / Published: 20 December 2012
Cited by 18 | PDF Full-text (474 KB) | HTML Full-text | XML Full-text
Abstract
A novel polyamine microsphere, prepared from the water-in-oil emulsion of polyethylenimine, was characterized. The investigation of scanning electron microscopy showed that the polyamine microsphere is a regular ball with a smooth surface. The diameter distribution of the microsphere is 0.37–4.29 μm. The isoelectric
[...] Read more.
A novel polyamine microsphere, prepared from the water-in-oil emulsion of polyethylenimine, was characterized. The investigation of scanning electron microscopy showed that the polyamine microsphere is a regular ball with a smooth surface. The diameter distribution of the microsphere is 0.37–4.29 μm. The isoelectric point of the microsphere is 10.6. The microsphere can adsorb proteins through the co-effect of electrostatic and hydrophobic interactions. Among the proteins tested, the highest value of adsorption of microsphere, 127.8 mg·g−1 microsphere, was obtained with lipase. In comparison with other proteins, the hydrophobic force is more important in promoting the adsorption of lipase. The microsphere can preferentially adsorb lipase from an even mixture of proteins. The optimum temperature and pH for the selective adsorption of lipase by the microsphere was 35 °C and pH 7.0. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Optimization of Lipase Production by Burkholderia sp. Using Response Surface Methodology
Int. J. Mol. Sci. 2012, 13(11), 14889-14897; doi:10.3390/ijms131114889
Received: 31 August 2012 / Revised: 23 October 2012 / Accepted: 8 November 2012 / Published: 13 November 2012
Cited by 6 | PDF Full-text (481 KB) | HTML Full-text | XML Full-text
Abstract
Response surface methodology (RSM) was employed to optimize the extracellular lipase production by Burkholderia sp. HL-10. Preliminary tests showed that olive oil, tryptone and Tween-80 exhibited significant effects on the lipase production. The optimum concentrations of these three components were determined using a
[...] Read more.
Response surface methodology (RSM) was employed to optimize the extracellular lipase production by Burkholderia sp. HL-10. Preliminary tests showed that olive oil, tryptone and Tween-80 exhibited significant effects on the lipase production. The optimum concentrations of these three components were determined using a faced-centered central composite design (FCCCD). The analysis of variance revealed that the established model was significant (p < 0.01). The optimized medium containing 0.65% olive oil (v/v), 2.42% tryptone (w/v) and 0.15% Tween-80 (v/v) resulted in a maximum activity of 122.3 U/mL, about three fold higher than that in basal medium. Approximately 99% of validity of the predicted value was achieved. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Preparation of Coaxial-Electrospun Poly[bis(p-methylphenoxy)]phosphazene Nanofiber Membrane for Enzyme Immobilization
Int. J. Mol. Sci. 2012, 13(11), 14136-14148; doi:10.3390/ijms131114136
Received: 1 August 2012 / Revised: 16 October 2012 / Accepted: 23 October 2012 / Published: 2 November 2012
Cited by 12 | PDF Full-text (1459 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
A core/sheath nanofiber membrane with poly[bis(p-methylphenoxy)]phospha-zene (PMPPh) as the sheath and easily spinnable polyacrylonitrile (PAN) as the core was prepared via a coaxial electrospinning process. Field-emission scanning electron microscopy and transmission electron microscopy were used to characterize the morphology of the
[...] Read more.
A core/sheath nanofiber membrane with poly[bis(p-methylphenoxy)]phospha-zene (PMPPh) as the sheath and easily spinnable polyacrylonitrile (PAN) as the core was prepared via a coaxial electrospinning process. Field-emission scanning electron microscopy and transmission electron microscopy were used to characterize the morphology of the nanofiber membrane. It was found that the concentration of the PAN spinning solution and the ratio of the core/sheath solution flow rates played a decisive role in the coaxial electrospinning process. In addition, the stabilized core/sheath PMPPh nanofiber membrane was investigated as a support for enzyme immobilization because of its excellent biocompatibility, high surface/volume ratio, and large porosity. Lipase from Candida rugosa was immobilized on the nanofiber membrane by adsorption. The properties of the immobilized lipase on the polyphosphazene nanofiber membrane were studied and compared with those of a PAN nanofiber membrane. The results showed that the adsorption capacity (20.4 ± 2.7 mg/g) and activity retention (63.7%) of the immobilized lipase on the polyphosphazene nanofiber membrane were higher than those on the PAN membrane. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle A Molecular Dynamics (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) Study on Ornithine Cyclodeaminase (OCD): A Tale of Two Iminiums
Int. J. Mol. Sci. 2012, 13(10), 12994-13011; doi:10.3390/ijms131012994
Received: 10 September 2012 / Revised: 27 September 2012 / Accepted: 27 September 2012 / Published: 11 October 2012
Cited by 7 | PDF Full-text (4638 KB) | HTML Full-text | XML Full-text
Abstract
Ornithine cyclodeaminase (OCD) is an NAD+-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD) and a hybrid quantum mechanics/molecular mechanics (QM/MM)
[...] Read more.
Ornithine cyclodeaminase (OCD) is an NAD+-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD) and a hybrid quantum mechanics/molecular mechanics (QM/MM) method in the ONIOM formalism, the catalytic mechanism of OCD has been examined. The rate limiting step is calculated to be the initial step in the overall mechanism: hydride transfer from the L-ornithine’s Cα–H group to the NAD+ cofactor with concomitant formation of a Cα=NH2+ Schiff base with a barrier of 90.6 kJ mol−1. Importantly, no water is observed within the active site during the MD simulations suitably positioned to hydrolyze the Cα=NH2+ intermediate to form the corresponding carbonyl. Instead, the reaction proceeds via a non-hydrolytic mechanism involving direct nucleophilic attack of the δ-amine at the Cα-position. This is then followed by cleavage and loss of the α-NH2 group to give the Δ1-pyrroline-2-carboxylate that is subsequently reduced to L-proline. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
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Open AccessArticle Biocatalytic Synthesis of Poly(δ-Valerolactone) Using a Thermophilic Esterase from Archaeoglobus fulgidus as Catalyst
Int. J. Mol. Sci. 2012, 13(10), 12232-12241; doi:10.3390/ijms131012232
Received: 26 July 2012 / Revised: 24 August 2012 / Accepted: 7 September 2012 / Published: 25 September 2012
Cited by 11 | PDF Full-text (218 KB) | HTML Full-text | XML Full-text
Abstract
The ring-opening polymerization of δ-valerolactone catalyzed by a thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically evaluated. Through
[...] Read more.
The ring-opening polymerization of δ-valerolactone catalyzed by a thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically evaluated. Through the optimization of reaction conditions, poly(δ-valerolactone) was produced in 97% monomer conversion, with a number-average molecular weight of 2225 g/mol, in toluene at 70 °C for 72 h. This paper has produced a new biocatalyst for the synthesis of poly(δ-valerolactone), and also deeper insight has been gained into the mechanism of thermophilic esterase-catalyzed ring-opening polymerization. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Purification and Characterization of a Ginsenoside Rb1-Hydrolyzing β-Glucosidase from Aspergillus niger KCCM 11239
Int. J. Mol. Sci. 2012, 13(9), 12140-12152; doi:10.3390/ijms130912140
Received: 13 July 2012 / Revised: 24 August 2012 / Accepted: 7 September 2012 / Published: 24 September 2012
Cited by 8 | PDF Full-text (854 KB) | HTML Full-text | XML Full-text
Abstract
Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme
[...] Read more.
Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme was estimated to be approximately 123 kDa. The optimal pH of the purified enzyme was pH 4.0, and the enzyme proved highly stable over a pH range of 5.0–10.0. The optimal temperature was 70 °C, and the enzyme became unstable at temperatures above 60 °C. The enzyme was inhibited by Cu2+, Mg2+, Co2+, and acetic acid (10 mM). In the specificity tests, the enzyme was found to be active against ginsenoside Rb1, but showed very low levels of activity against Rb2, Rc, Rd, Re, and Rg1. The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb1 to generate ginsenoside Rd and Rg3, and hydrolyzed 3-C,β-(1→2)-glucoside to generate F2. The properties of the enzyme indicate that it could be a useful tool in biotransformation applications in the ginseng industry, as well as in the development of novel drug compounds. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Polystyrene Attached Pt(IV)–Azomethine, Synthesis and Immobilization of Glucose Oxidase Enzyme
Int. J. Mol. Sci. 2012, 13(9), 11870-11880; doi:10.3390/ijms130911870
Received: 16 July 2012 / Revised: 27 August 2012 / Accepted: 3 September 2012 / Published: 19 September 2012
Cited by 9 | PDF Full-text (379 KB) | HTML Full-text | XML Full-text
Abstract
Modified polystyrene with Pt(IV)–azomethine (APS–Sch–Pt) was synthesized by means of condensation and demonstrated to be a promising enzyme support by studying the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on it. The characteristics of the immobilized glucose oxidase (APS–Sch–Pt–GOx) enzyme showed two
[...] Read more.
Modified polystyrene with Pt(IV)–azomethine (APS–Sch–Pt) was synthesized by means of condensation and demonstrated to be a promising enzyme support by studying the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on it. The characteristics of the immobilized glucose oxidase (APS–Sch–Pt–GOx) enzyme showed two optimum pH values that were pH = 4.0 and pH = 7. The insertion of stable Pt(IV)–azomethine spacers between the polystyrene backbone and the immobilized GOx, (APS–Sch–Pt–GOx), increases the enzymes’ activity and improves their affinity towards the substrate even at pH = 4. The influence of temperature, reusability and storage capacity on the free and immobilized glucose oxidase enzyme was investigated. The storage stability of the immobilized glucose oxidase was shown to be eleven months in dry conditions at +4 °C. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle High Yield of Wax Ester Synthesized from Cetyl Alcohol and Octanoic Acid by Lipozyme RMIM and Novozym 435
Int. J. Mol. Sci. 2012, 13(9), 11694-11704; doi:10.3390/ijms130911694
Received: 23 July 2012 / Revised: 3 September 2012 / Accepted: 3 September 2012 / Published: 17 September 2012
Cited by 12 | PDF Full-text (972 KB) | HTML Full-text | XML Full-text
Abstract
Wax esters are long-chain esters that have been widely applied in premium lubricants, parting agents, antifoaming agents and cosmetics. In this study, the biocatalytic preparation of a specific wax ester, cetyl octanoate, is performed in n-hexane using two commercial immobilized lipases, i.e.
[...] Read more.
Wax esters are long-chain esters that have been widely applied in premium lubricants, parting agents, antifoaming agents and cosmetics. In this study, the biocatalytic preparation of a specific wax ester, cetyl octanoate, is performed in n-hexane using two commercial immobilized lipases, i.e., Lipozyme® RMIM (Rhizomucor miehei) and Novozym® 435 (Candida antarctica). Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) are employed to evaluate the effects of reaction time (1–5 h), reaction temperature (45–65 °C), substrate molar ratio (1–3:1), and enzyme amount (10%–50%) on the yield of cetyl octanoate. Using RSM to optimize the reaction, the maximum yields reached 94% and 98% using Lipozyme® RMIM and Novozym® 435, respectively. The optimum conditions for synthesis of cetyl octanoate by both lipases are established and compared. Novozym® 435 proves to be a more efficient biocatalyst than Lipozyme® RMIM. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Optimization of Ligninolytic Enzyme Activity and Production Rate with Ceriporiopsis subvermispora for Application in Bioremediation by Varying Submerged Media Composition and Growth Immobilization Support
Int. J. Mol. Sci. 2012, 13(9), 11365-11384; doi:10.3390/ijms130911365
Received: 21 June 2012 / Revised: 23 August 2012 / Accepted: 5 September 2012 / Published: 12 September 2012
Cited by 2 | PDF Full-text (1107 KB) | HTML Full-text | XML Full-text
Abstract
Response surface methodology (central composite design of experiments) was employed to simultaneously optimize enzyme production and productivities of two ligninolytic enzymes produced by Ceriporiopsis subvermispora. Concentrations of glucose, ammonium tartrate and Polysorbate 80 were varied to establish the optimal composition of liquid
[...] Read more.
Response surface methodology (central composite design of experiments) was employed to simultaneously optimize enzyme production and productivities of two ligninolytic enzymes produced by Ceriporiopsis subvermispora. Concentrations of glucose, ammonium tartrate and Polysorbate 80 were varied to establish the optimal composition of liquid media (OLM), where the highest experimentally obtained activities and productivities were 41 U L−1 and 16 U L−1 day−1 for laccase (Lac), and 193 U L−1 and 80 U L−1 day−1 for manganese peroxidase (MnP). Considering culture growth in OLM on various types of immobilization support, the best results were obtained with 1 cm beech wood cubes (BWCM). Enzyme activities in culture filtrate were 152 U L−1 for Lac and 58 U L−1 for MnP, since the chemical composition of this immobilization material induced higher Lac activity. Lower enzyme activities were obtained with polyurethane foam. Culture filtrates of OLM and BWCM were applied for dye decolorization. Remazol Brilliant Blue R (RBBR) was decolorized faster and more efficiently than Copper(II)phthalocyanine (CuP) with BWCM (80% and 60%), since Lac played a crucial role. Decolorization of CuP was initially faster than that of RBBR, due to higher MnP activities in OLM. The extent of decolorization after 14 h was 60% for both dyes. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
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Open AccessArticle Combining the Physical Adsorption Approach and the Covalent Attachment Method to Prepare a Bifunctional Bioreactor
Int. J. Mol. Sci. 2012, 13(9), 11443-11454; doi:10.3390/ijms130911443
Received: 30 July 2012 / Revised: 28 August 2012 / Accepted: 5 September 2012 / Published: 12 September 2012
Cited by 3 | PDF Full-text (508 KB) | HTML Full-text | XML Full-text
Abstract
Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The
[...] Read more.
Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The property of amino-functionalized mesoporous silica was characterized by N2 adsorption-desorption and thermogravimetric (TG) analysis. The feature of the silica-based matrix before and after myoglobin adsorption was identified by fourier transform infrared (FTIR) and UV/VIS measurement. With o-dianisidine and H2O2 as the substrate, the peroxidase activity of adsorbed myoglobin was determined. With Micrococus lysodeilicus as the substrate, the antibacterial activity of covalently tethered lysozyme was measured. Results demonstrated that the final product not only presented peroxidase activity of the myoglobin but yielded antibacterial activity of the lysozyme. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle An Imprinted Cross-Linked Enzyme Aggregate (iCLEA) of Sucrose Phosphorylase: Combining Improved Stability with Altered Specificity
Int. J. Mol. Sci. 2012, 13(9), 11333-11342; doi:10.3390/ijms130911333
Received: 28 August 2012 / Revised: 5 September 2012 / Accepted: 5 September 2012 / Published: 11 September 2012
Cited by 11 | PDF Full-text (366 KB) | HTML Full-text | XML Full-text
Abstract
The industrial use of sucrose phosphorylase (SP), an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability
[...] Read more.
The industrial use of sucrose phosphorylase (SP), an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA). In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme’s specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C. This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
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Open AccessArticle Optimization of Xylanase Production from Penicillium sp.WX-Z1 by a Two-Step Statistical Strategy: Plackett-Burman and Box-Behnken Experimental Design
Int. J. Mol. Sci. 2012, 13(8), 10630-10646; doi:10.3390/ijms130810630
Received: 6 June 2012 / Revised: 23 July 2012 / Accepted: 2 August 2012 / Published: 23 August 2012
Cited by 8 | PDF Full-text (330 KB) | HTML Full-text | XML Full-text
Abstract
The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in
[...] Read more.
The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO3, MgSO4, and CaCl2. The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO3, 12.71; MgSO4, 0.96; and CaCl2, 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle New Biofuel Integrating Glycerol into Its Composition Through the Use of Covalent Immobilized Pig Pancreatic Lipase
Int. J. Mol. Sci. 2012, 13(8), 10091-10112; doi:10.3390/ijms130810091
Received: 9 July 2012 / Revised: 26 July 2012 / Accepted: 2 August 2012 / Published: 13 August 2012
Cited by 15 | PDF Full-text (383 KB) | HTML Full-text | XML Full-text
Abstract
By using 1,3-specific Pig Pancreatic lipase (EC 3.1.1.3 or PPL), covalently immobilized on AlPO4/Sepiolite support as biocatalyst, a new second-generation biodiesel was obtained in the transesterification reaction of sunflower oil with ethanol and other alcohols of low molecular weight. The resulting
[...] Read more.
By using 1,3-specific Pig Pancreatic lipase (EC 3.1.1.3 or PPL), covalently immobilized on AlPO4/Sepiolite support as biocatalyst, a new second-generation biodiesel was obtained in the transesterification reaction of sunflower oil with ethanol and other alcohols of low molecular weight. The resulting biofuel is composed of fatty acid ethyl esters and monoglycerides (FAEE/MG) blended in a molar relation 2/1. This novel product, which integrates glycerol as monoacylglycerols (MG) into the biofuel composition, has similar physicochemical properties compared to those of conventional biodiesel and also avoids the removal step of this by-product. The biocatalyst was found to be strongly fixed to the inorganic support (75%). Nevertheless, the efficiency of the immobilized enzyme was reduced to half (49.1%) compared to that of the free PPL. The immobilized enzyme showed a remarkable stability as well as a great reusability (more than 40 successive reuses) without a significant loss of its initial catalytic activity. Immobilized and free enzymes exhibited different reaction mechanisms, according to the different results in the Arrhenius parameters (Ln A and Ea). However, the use of supported PPL was found to be very suitable for the repetitive production of biofuel due to its facile recyclability from the reaction mixture. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
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Open AccessArticle Synthesis of Triptorelin Lactate Catalyzed by Lipase in Organic Media
Int. J. Mol. Sci. 2012, 13(8), 9971-9979; doi:10.3390/ijms13089971
Received: 23 May 2012 / Revised: 30 June 2012 / Accepted: 2 August 2012 / Published: 10 August 2012
PDF Full-text (129 KB) | HTML Full-text | XML Full-text
Abstract
Triptorelin lactate was successfully synthesized by porcine pancreatic lipase (PPL) in organic solvents. The effects of acyl donor, substrate ratio, organic solvent, temperature, and water activity were investigated. Under the optimum conditions, a yield of 30% for its ester could be achieved in
[...] Read more.
Triptorelin lactate was successfully synthesized by porcine pancreatic lipase (PPL) in organic solvents. The effects of acyl donor, substrate ratio, organic solvent, temperature, and water activity were investigated. Under the optimum conditions, a yield of 30% for its ester could be achieved in the reaction for about 48 h. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Enzymatic Properties and Mutational Studies of Chalcone Synthase from Physcomitrella patens
Int. J. Mol. Sci. 2012, 13(8), 9673-9691; doi:10.3390/ijms13089673
Received: 2 May 2012 / Revised: 6 July 2012 / Accepted: 9 July 2012 / Published: 3 August 2012
Cited by 6 | PDF Full-text (722 KB) | HTML Full-text | XML Full-text
Abstract
PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn
[...] Read more.
PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant’s active site. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Comparative Studies on the Induction of Trichoderma harzianum Mutanase by α-(1→3)-Glucan-Rich Fruiting Bodies and Mycelia of Laetiporus sulphureus
Int. J. Mol. Sci. 2012, 13(8), 9584-9598; doi:10.3390/ijms13089584
Received: 10 July 2012 / Revised: 24 July 2012 / Accepted: 24 July 2012 / Published: 31 July 2012
Cited by 4 | PDF Full-text (325 KB) | HTML Full-text | XML Full-text
Abstract
Mutanase (α-(1→3)-glucanase) is a little-known inductive enzyme that is potentially useful in dentistry. Here, it was shown that the cell wall preparation (CWP) obtained from the fruiting body or vegetative mycelium of polypore fungus Laetiporus sulphureus is rich in α-(1→3)-glucan and can be
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Mutanase (α-(1→3)-glucanase) is a little-known inductive enzyme that is potentially useful in dentistry. Here, it was shown that the cell wall preparation (CWP) obtained from the fruiting body or vegetative mycelium of polypore fungus Laetiporus sulphureus is rich in α-(1→3)-glucan and can be successfully used for mutanase induction in Trichoderma harzianum. The content of this biopolymer in the CWP depended on the age of fruiting bodies and increased along with their maturation. In the case of CWP prepared from vegetative mycelia, the amount of α-(1→3)-glucan depended on the mycelium age and also on the kind of medium used for its cultivation. All CWPs prepared from the individually harvested fruiting body specimens induced high mutanase activity (0.53–0.82 U/mL) in T. harzianum after 3 days of cultivation. As for the CWPs obtained from the hyphal mycelia of L. sulpureus, the maximal enzyme productivity (0.34 U/mL after 3 days of incubation) was recorded for CWP prepared from the 3 week-old mycelium cultivated in Sabouraud medium. Statistically, a high positive correlation was found between the total percentage content of α-(1→3)-glucan in the CWP and the mutanase activity. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Optimization of Glutamine Peptide Production from Soybean Meal and Analysis of Molecular Weight Distribution of Hydrolysates
Int. J. Mol. Sci. 2012, 13(6), 7483-7495; doi:10.3390/ijms13067483
Received: 5 May 2012 / Revised: 5 June 2012 / Accepted: 8 June 2012 / Published: 18 June 2012
PDF Full-text (1250 KB) | HTML Full-text | XML Full-text
Abstract
The process parameters of enzymatic hydrolysis and molecular weight distribution of glutamine (Gln) peptides from soybean meal were investigated. The Protamex® hydrolysis pH of 6.10, temperature of 56.78 °C, enzyme to substrate ratio (E/S) of 1.90 and hydrolysis time of 10.72 h
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The process parameters of enzymatic hydrolysis and molecular weight distribution of glutamine (Gln) peptides from soybean meal were investigated. The Protamex® hydrolysis pH of 6.10, temperature of 56.78 °C, enzyme to substrate ratio (E/S) of 1.90 and hydrolysis time of 10.72 h were found to be the optimal conditions by response surface methodology (RSM) for a maximal degree of hydrolysis (DH) value of 16.63% and Gln peptides content at 5.95 mmol/L. The soybean meal was hydrolyzed by a combination of Protamex® and trypsinase so that DH and Gln peptides would reach 22.02% and 6.05 mmol/mL, respectively. The results of size exclusion chromatography indicated that the relative proportion of the molecular weight < 1000 Da fraction increased with DH values from 6.76%, 11.13%, 17.89% to 22.02%, most notably the 132–500 Da fractions of hydrolysates were 42.14%, 46.57%, 58.44% and 69.65%. High DH values did not lead to high Gln peptides content of the hydrolysate but to the low molecular weight Gln peptides. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessArticle Immobilization of Laccase for Oxidative Coupling of Trans-Resveratrol and Its Derivatives
Int. J. Mol. Sci. 2012, 13(5), 5998-6008; doi:10.3390/ijms13055998
Received: 5 April 2012 / Revised: 4 May 2012 / Accepted: 9 May 2012 / Published: 18 May 2012
Cited by 12 | PDF Full-text (228 KB) | HTML Full-text | XML Full-text
Abstract
Trametes villosa Laccase (TVL) was immobilized through physical adsorption on SBA-15 mesoporous silica and the immobilized TVL was used in the oxidative coupling of trans-resveratrol. Higher loading and activity of the immobilized enzyme on SBA-15 were obtained when compared with the free enzyme.
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Trametes villosa Laccase (TVL) was immobilized through physical adsorption on SBA-15 mesoporous silica and the immobilized TVL was used in the oxidative coupling of trans-resveratrol. Higher loading and activity of the immobilized enzyme on SBA-15 were obtained when compared with the free enzyme. The effects of reaction conditions, such as buffer type, pH, temperature and substrate concentration were investigated, and the optimum conditions were screened and resulted in enzyme activity of up to 10.3 μmol/g·h. Furthermore, the oxidative couplings of the derivatives of trans-resveratrol were also catalyzed by immobilized TVL. The immobilized TVL was recyclable and could maintain 78% of its initial activity after reusing it four times. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)

Review

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Open AccessReview Potential Applications of Carbohydrases Immobilization in the Food Industry
Int. J. Mol. Sci. 2013, 14(1), 1335-1369; doi:10.3390/ijms14011335
Received: 30 October 2012 / Revised: 17 December 2012 / Accepted: 18 December 2012 / Published: 11 January 2013
Cited by 14 | PDF Full-text (328 KB) | HTML Full-text | XML Full-text
Abstract
Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and
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Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessReview From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes
Int. J. Mol. Sci. 2013, 14(1), 1232-1277; doi:10.3390/ijms14011232
Received: 3 September 2012 / Revised: 14 November 2012 / Accepted: 24 December 2012 / Published: 10 January 2013
Cited by 86 | PDF Full-text (1151 KB) | HTML Full-text | XML Full-text
Abstract
Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments
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Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Figures

Open AccessReview Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites
Int. J. Mol. Sci. 2012, 13(12), 15901-15924; doi:10.3390/ijms131215901
Received: 27 September 2012 / Revised: 1 November 2012 / Accepted: 13 November 2012 / Published: 28 November 2012
Cited by 23 | PDF Full-text (461 KB) | HTML Full-text | XML Full-text
Abstract
Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may
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Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessReview Computational Protein Engineering: Bridging the Gap between Rational Design and Laboratory Evolution
Int. J. Mol. Sci. 2012, 13(10), 12428-12460; doi:10.3390/ijms131012428
Received: 20 August 2012 / Revised: 16 September 2012 / Accepted: 17 September 2012 / Published: 28 September 2012
Cited by 17 | PDF Full-text (2055 KB) | HTML Full-text | XML Full-text
Abstract
Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary
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Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation “hotspots” with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)
Open AccessReview Optimization to Low Temperature Activity in Psychrophilic Enzymes
Int. J. Mol. Sci. 2012, 13(9), 11643-11665; doi:10.3390/ijms130911643
Received: 24 August 2012 / Revised: 7 September 2012 / Accepted: 10 September 2012 / Published: 17 September 2012
Cited by 58 | PDF Full-text (314 KB) | HTML Full-text | XML Full-text
Abstract
Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity
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Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts. Full article
(This article belongs to the Special Issue Enzyme Optimization and Immobilization)

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