Special Issue "Recent Advances in Biocatalysis and Metabolic Engineering for Biomanufacturing"

A special issue of Catalysts (ISSN 2073-4344). This special issue belongs to the section "Biocatalysis".

Deadline for manuscript submissions: 31 March 2019

Special Issue Editor

Guest Editor
Prof. Eun Yeol Lee

Department of Chemical Engineering, College of Engineering, Kyung Hee University, Gyeonggi-do17104, Korea
Website | E-Mail
Phone: 82312013839
Interests: biocatalysis; protein and metabolic engineering; biorefinery

Special Issue Information

Dear Colleagues,

The use of biocatalysts, including enzyme and metabolically engineered cells, has attracted a great deal of attention in chemical and bioindustry because biocatalytic reactions can be conducted under environmentally-benign conditions and in sustainable ways. The catalytic efficiency and chemo-, regio-, stereoselectivity of enzyme can be readily enhanced and modulated using protein engineering. Metabolic engineering seeks to enhance cellular biosynthetic productivity of target metabolites via controlling and redesigning metabolic pathways using multi-omics analysis, genome-scale modeling, metabolic flux control and reconstruction of novel pathways.

The aim of this Special Issue is to deal with the recent advances in biocatalysis and metabolic engineering for biomanufacturing of fuels, chemicals, biomaterials, and pharmaceuticals. Reviews and original research papers on the development of new strategies to improve the catalytic efficiency of enzyme, biosynthetic capability of cell factory and their applications in production of various bioproducts and chemicals are welcome.

Prof. Eun Yeol Lee
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Catalysts is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Biocatalysis
  • Biocatalytic reaction
  • Protein Engineering
  • Metabolic Engineering
  • Molecular Biotechnology
  • Biomass
  • Biorefinery
  • Immobilization
  • Cell factory
  • Biomanufacturing

Published Papers (14 papers)

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Research

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Open AccessFeature PaperArticle Enzymatic Synthesis of ω-Hydroxydodecanoic Acid By Employing a Cytochrome P450 from Limnobacter sp. 105 MED
Catalysts 2019, 9(1), 54; https://doi.org/10.3390/catal9010054
Received: 12 December 2018 / Revised: 2 January 2019 / Accepted: 3 January 2019 / Published: 8 January 2019
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Abstract
ω-Hydroxylated fatty acids are valuable and versatile building blocks for the production of various adhesives, lubricants, cosmetic intermediates, etc. The biosynthesis of ω-hydroxydodecanoic acid from vegetable oils is one of the important green pathways for their chemical-based synthesis. In the present study, the [...] Read more.
ω-Hydroxylated fatty acids are valuable and versatile building blocks for the production of various adhesives, lubricants, cosmetic intermediates, etc. The biosynthesis of ω-hydroxydodecanoic acid from vegetable oils is one of the important green pathways for their chemical-based synthesis. In the present study, the novel monooxygenase CYP153AL.m from Limnobacter sp. 105 MED was used for the whole-cell biotransformations. We constructed three-component system that was comprised of CYP153AL.m, putidaredoxin and putidaredoxin reductase from Pseudomonas putida. This in vivo study demonstrated that CYP153AL.m is a powerful catalyst for the biosynthesis of ω-hydroxydodecanoic acid. Under optimized conditions, the application of a solid-state powdered substrate rather than a substrate dissolved in DMSO significantly enhanced the overall reaction titer of the process. By employing this efficient system, 2 g/L of 12-hydroxydodecanoic acid (12-OHDDA) was produced from 4 g/L of its corresponding fatty acid, which was namely dodecanoic acid. Furthermore, the system was extended to produce 3.28 g/L of 12-OHDDA using 4 g/L of substrate by introducing native redox partners. These results demonstrate the utility of CYP153AL.m-catalyzed biotransformations in the industrial production of 12-OHDDA and other valuable building blocks. Full article
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Open AccessArticle Specific Immobilization of Escherichia coli Expressing Recombinant Glycerol Dehydrogenase on Mannose-Functionalized Magnetic Nanoparticles
Catalysts 2019, 9(1), 7; https://doi.org/10.3390/catal9010007
Received: 16 November 2018 / Revised: 9 December 2018 / Accepted: 13 December 2018 / Published: 24 December 2018
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Abstract
Mannose-functionalized magnetic nanoparticles were prepared for the immobilization of Escherichia coli cells harboring the recombinant glycerol dehydrogenase gene. Immobilization of whole E. coli cells on the carrier was carried out through specific binding between mannose on the nanoparticles and the FimH lectin on [...] Read more.
Mannose-functionalized magnetic nanoparticles were prepared for the immobilization of Escherichia coli cells harboring the recombinant glycerol dehydrogenase gene. Immobilization of whole E. coli cells on the carrier was carried out through specific binding between mannose on the nanoparticles and the FimH lectin on the E. coli cell surface via hydrogen bonds and hydrophobic interactions. The effects of various factors including cell concentration, pH, temperature, and buffer concentration were investigated. High degrees of immobilization (84%) and recovery of activity (82%) were obtained under the following conditions: cell/support 1.3 mg/mL, immobilization time 2 h, pH 8.0, temperature 4°C, and buffer concentration 50 mM. Compared with the free cells, the thermostability of the immobilized cells was improved 2.56-fold at 37 °C. More than 50% of the initial activity of the immobilized cells remained after 10 cycles. The immobilized cells were evaluated functionally by monitoring the catalytic conversion of glycerol to 1,3-dihydroxyacetone (DHA). After a 12 h reaction, the DHA produced by the immobilized cells was two-fold higher than that produced by the free cells. These results indicate that mannose-functionalized magnetic nanoparticles can be used for the specific recognition of gram-negative bacteria, which gives them great potential in applications such as the preparation of biocatalysts and biosensors and clinical diagnosis. Full article
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Open AccessArticle Biocatalytic Oxidations of Substrates through Soluble Methane Monooxygenase from Methylosinus sporium 5
Catalysts 2018, 8(12), 582; https://doi.org/10.3390/catal8120582
Received: 27 October 2018 / Revised: 13 November 2018 / Accepted: 21 November 2018 / Published: 26 November 2018
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Abstract
Methane, an important greenhouse gas, has a 20-fold higher heat capacity than carbon dioxide. Earlier, through advanced spectroscopy and structural studies, the mechanisms underlying the extremely stable C–H activation of soluble methane monooxygenase (sMMO) have been elucidated in Methylosinus trichosporium OB3b and Methylococcus [...] Read more.
Methane, an important greenhouse gas, has a 20-fold higher heat capacity than carbon dioxide. Earlier, through advanced spectroscopy and structural studies, the mechanisms underlying the extremely stable C–H activation of soluble methane monooxygenase (sMMO) have been elucidated in Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. Here, sMMO components—including hydroxylase (MMOH), regulatory (MMOB), and reductase (MMOR)—were expressed and purified from a type II methanotroph, Methylosinus sporium strain 5 (M. sporium 5), to characterize its hydroxylation mechanism. Two molar equivalents of MMOB are necessary to achieve catalytic activities and oxidized a broad range of substrates including alkanes, alkenes, halogens, and aromatics. Optimal activities were observed at pH 7.5 for most substrates possibly because of the electron transfer environment in MMOR. Substitution of MMOB or MMOR from another type II methanotroph, Methylocystis species M, retained specific enzyme activities, demonstrating the successful cross-reactivity of M. sporium 5. These results will provide fundamental information for further enzymatic studies to elucidate sMMO mechanisms. Full article
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Open AccessArticle Construction of a Vitreoscilla Hemoglobin Promoter-Based Tunable Expression System for Corynebacterium glutamicum
Catalysts 2018, 8(11), 561; https://doi.org/10.3390/catal8110561
Received: 31 October 2018 / Revised: 15 November 2018 / Accepted: 15 November 2018 / Published: 19 November 2018
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Abstract
Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, [...] Read more.
Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L). Full article
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Open AccessArticle Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from E. coli
Catalysts 2018, 8(11), 530; https://doi.org/10.3390/catal8110530
Received: 16 October 2018 / Revised: 31 October 2018 / Accepted: 6 November 2018 / Published: 8 November 2018
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Abstract
[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds [...] Read more.
[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional “O2-sensitive” [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., “O2-tolerant” [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the “unready” Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis. Full article
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Open AccessFeature PaperArticle Efficient Conversion of Acetate to 3-Hydroxypropionic Acid by Engineered Escherichia coli
Catalysts 2018, 8(11), 525; https://doi.org/10.3390/catal8110525
Received: 17 October 2018 / Revised: 1 November 2018 / Accepted: 3 November 2018 / Published: 7 November 2018
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Abstract
Acetate, which is an abundant carbon source, is a potential feedstock for microbial processes that produce diverse value-added chemicals. In this study, we produced 3-hydroxypropionic acid (3-HP) from acetate with engineered Escherichia coli. For the efficient conversion of acetate to 3-HP, we [...] Read more.
Acetate, which is an abundant carbon source, is a potential feedstock for microbial processes that produce diverse value-added chemicals. In this study, we produced 3-hydroxypropionic acid (3-HP) from acetate with engineered Escherichia coli. For the efficient conversion of acetate to 3-HP, we initially introduced heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus. Then, the acetate assimilating pathway and glyoxylate shunt pathway were activated by overexpressing acs (encoding acetyl-CoA synthetase) and deleting iclR (encoding the glyoxylate shunt pathway repressor). Because a key precursor malonyl-CoA is also consumed for fatty acid synthesis, we decreased carbon flux to fatty acid synthesis by adding cerulenin. Subsequently, we found that inhibiting fatty acid synthesis dramatically improved 3-HP production (3.00 g/L of 3-HP from 8.98 g/L of acetate). The results indicated that acetate can be used as a promising carbon source for microbial processes and that 3-HP can be produced from acetate with a high yield (44.6% of the theoretical maximum yield). Full article
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Open AccessArticle Immobilization of Eversa Lipase on Octyl Agarose Beads and Preliminary Characterization of Stability and Activity Features
Catalysts 2018, 8(11), 511; https://doi.org/10.3390/catal8110511
Received: 11 October 2018 / Revised: 22 October 2018 / Accepted: 31 October 2018 / Published: 2 November 2018
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Abstract
Eversa is an enzyme recently launched by Novozymes to be used in a free form as biocatalyst in biodiesel production. This paper shows for first time the immobilization of Eversa (a commercial lipase) on octyl and aminated agarose beads and the comparison of [...] Read more.
Eversa is an enzyme recently launched by Novozymes to be used in a free form as biocatalyst in biodiesel production. This paper shows for first time the immobilization of Eversa (a commercial lipase) on octyl and aminated agarose beads and the comparison of the enzyme properties to those of the most used lipase, the isoform B from Candida antarctica (CALB) immobilized on octyl agarose beads. Immobilization on octyl and aminated supports of Eversa has not had a significant effect on enzyme activity versus p-nitrophenyl butyrate (pNPB) under standard conditions (pH 7), but immobilization on octyl agarose beads greatly enhanced the stability of the enzyme under all studied conditions, much more than immobilization on aminated support. Octyl-Eversa was much more stable than octyl-CALB at pH 9, but it was less stable at pH 5. In the presence of 90% acetonitrile or dioxane, octyl-Eversa maintained the activity (even increased the activity) after 45 days of incubation in a similar way to octyl-CALB, but in 90% of methanol, results are much worse, and octyl-CALB became much more stable than Eversa. Coating with PEI has not a clear effect on octyl-Eversa stability, although it affected enzyme specificity and activity response to the changes in the pH. Eversa immobilized octyl supports was more active than CALB versus triacetin or pNPB, but much less active versus methyl mandelate esters. On the other hand, Eversa specificity and response to changes in the medium were greatly modulated by the immobilization protocol or by the coating of the immobilized enzyme with PEI. Thus, Eversa may be a promising biocatalyst for many processes different to the biodiesel production and its properties may be greatly improved following a suitable immobilization protocol, and in some cases is more stable and active than CALB. Full article
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Open AccessArticle Preparation of Magnetic Cross-Linked Amyloglucosidase Aggregates: Solving Some Activity Problems
Catalysts 2018, 8(11), 496; https://doi.org/10.3390/catal8110496
Received: 8 October 2018 / Revised: 23 October 2018 / Accepted: 24 October 2018 / Published: 26 October 2018
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Abstract
The preparation of Cross-Linked Enzyme Aggregates (CLEAs) is a simple and cost-effective technique capable of generating insoluble biocatalysts with high volumetric activity and improved stability. The standard CLEA preparation consists of the aggregation of the enzyme and its further crosslinking, usually with glutaraldehyde. [...] Read more.
The preparation of Cross-Linked Enzyme Aggregates (CLEAs) is a simple and cost-effective technique capable of generating insoluble biocatalysts with high volumetric activity and improved stability. The standard CLEA preparation consists of the aggregation of the enzyme and its further crosslinking, usually with glutaraldehyde. However, some enzymes have too low a content of surface lysine groups to permit effective crosslinking with glutaraldehyde, requiring co-aggregation with feeders rich in amino groups to aid the formation of CLEAs. The co-aggregation with magnetic particles makes their handling easier. In this work, CLEAs of a commercial amyloglucosidase (AMG) produced by Aspergillus niger were prepared by co-aggregation in the presence of polyethyleneimine (PEI) or starch with aminated magnetic nanoparticles (MNPs) or bovine serum albumin (BSA). First, CLEAs were prepared only with MNPs at different glutaraldehyde concentrations, yielding a recovered activity of around 20%. The addition of starch during the precipitation and crosslinking steps nearly doubled the recovered activity. Similar recovered activity (around 40%) was achieved when changing starch by PEI. Moreover, under the same conditions, AMG co-aggregated with BSA was also synthesized, yielding CLEAs with very similar recovered activity. Both CLEAs (co-aggregated with MNPs or BSA) were four times more stable than the soluble enzyme. These CLEAs were evaluated in the hydrolysis of starch at typical industrial conditions, achieving more than 95% starch-to-glucose conversion, measured as Dextrose Equivalent (DE). Moreover, both CLEAS could be reused for five cycles, maintaining a DE of around 90%. Although both CLEAs had good properties, magnetic CLEAs could be more attractive for industrial purposes because of their easy separation by an external magnetic field, avoiding the formation of clusters during the filtration or centrifugation recovery methods usually used. Full article
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Open AccessArticle Mass Transfer Performance of a String Film Reactor: A Bioreactor Design for Aerobic Methane Bioconversion
Catalysts 2018, 8(11), 490; https://doi.org/10.3390/catal8110490
Received: 25 September 2018 / Revised: 17 October 2018 / Accepted: 22 October 2018 / Published: 24 October 2018
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Abstract
The mass transfer performance of a string film reactor (SFR)—a bioreactor design for the aerobic bioconversion of methane—was investigated. The results showed that the SFR could achieve high mass transfer performance of gases, and the highest values of the mass transfer coefficients for [...] Read more.
The mass transfer performance of a string film reactor (SFR)—a bioreactor design for the aerobic bioconversion of methane—was investigated. The results showed that the SFR could achieve high mass transfer performance of gases, and the highest values of the mass transfer coefficients for oxygen and methane were 877.1 h−1 and 408.0 h−1, respectively. There were similar mass transfer coefficients for oxygen and methane in absorption experiments using air, methane, and air–methane mixed gas under the same liquid flow rate conditions, implying that each gas is delivered into the liquid without mutual interaction. The mass transfer performance of the SFR was significantly influenced by the liquid flow rate and the hydrophilicity of the string material, whereas the magnitude of the gas flow rate effect on the mass transfer performance depended on both the tested liquid flow rate and the gas flow rate. Furthermore, the mass transfer performance of the SFR was compared with those of other types of bioreactors. Full article
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Open AccessArticle Characterization of a New Glyoxal Oxidase from the Thermophilic Fungus Myceliophthora thermophila M77: Hydrogen Peroxide Production Retained in 5-Hydroxymethylfurfural Oxidation
Catalysts 2018, 8(10), 476; https://doi.org/10.3390/catal8100476
Received: 2 October 2018 / Revised: 15 October 2018 / Accepted: 16 October 2018 / Published: 19 October 2018
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Abstract
Myceliophthora thermophyla is a thermophilic industrially relevant fungus that secretes an assortment of hydrolytic and oxidative enzymes for lignocellulose degradation. Among them is glyoxal oxidase (MtGLOx), an extracellular oxidoreductase that oxidizes several aldehydes and α-hydroxy carbonyl substrates coupled to the reduction [...] Read more.
Myceliophthora thermophyla is a thermophilic industrially relevant fungus that secretes an assortment of hydrolytic and oxidative enzymes for lignocellulose degradation. Among them is glyoxal oxidase (MtGLOx), an extracellular oxidoreductase that oxidizes several aldehydes and α-hydroxy carbonyl substrates coupled to the reduction of O2 to H2O2. This copper metalloprotein belongs to a class of enzymes called radical copper oxidases (CRO) and to the “auxiliary activities” subfamily AA5_1 that is based on the Carbohydrate-Active enZYmes (CAZy) database. Only a few members of this family have been characterized to date. Here, we report the recombinant production, characterization, and structure-function analysis of MtGLOx. Electron Paramagnetic Resonance (EPR) spectroscopy confirmed MtGLOx to be a radical-coupled copper complex and small angle X-ray scattering (SAXS) revealed an extended spatial arrangement of the catalytic and four N-terminal WSC domains. Furthermore, we demonstrate that methylglyoxal and 5-hydroxymethylfurfural (HMF), a fermentation inhibitor, are substrates for the enzyme. Full article
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Open AccessArticle Fluorescein Diacetate Hydrolysis Using the Whole Biofilm as a Sensitive Tool to Evaluate the Physiological State of Immobilized Bacterial Cells
Catalysts 2018, 8(10), 434; https://doi.org/10.3390/catal8100434
Received: 16 August 2018 / Revised: 18 September 2018 / Accepted: 30 September 2018 / Published: 2 October 2018
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Abstract
Due to the increasing interest and the use of immobilized biocatalysts in bioremediation studies, there is a need for the development of an assay for quick and reliable measurements of their overall enzymatic activity. Fluorescein diacetate (FDA) hydrolysis is a widely used assay [...] Read more.
Due to the increasing interest and the use of immobilized biocatalysts in bioremediation studies, there is a need for the development of an assay for quick and reliable measurements of their overall enzymatic activity. Fluorescein diacetate (FDA) hydrolysis is a widely used assay for measuring total enzymatic activity (TEA) in various environmental samples or in monoculture researches. However, standard FDA assays for TEA measurements in immobilized samples include performing an assay on cells detached from the carrier. This causes an error, because it is not possible to release all cells from the carrier without affecting their metabolic activity. In this study, we developed and optimized a procedure for TEA quantification in the whole biofilm formed on the carrier without disturbing it. The optimized method involves pre-incubation of immobilized carrier in phosphate buffer (pH 7.6) on the orbital shaker for 15 min, slow injection of FDA directly into the middle of the immobilized carrier, and incubation on the orbital shaker (130 rpm, 30 °C) for 1 h. Biofilm dry mass was obtained by comparing the dried weight of the immobilized carrier with that of the unimmobilized carrier. The improved protocol provides a simple, quick, and more reliable quantification of TEA during the development of immobilized biocatalysts compared to the original method. Full article
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Open AccessArticle Biosynthesis of Nylon 12 Monomer, ω-Aminododecanoic Acid Using Artificial Self-Sufficient P450, AlkJ and ω-TA
Catalysts 2018, 8(9), 400; https://doi.org/10.3390/catal8090400
Received: 25 August 2018 / Revised: 14 September 2018 / Accepted: 15 September 2018 / Published: 18 September 2018
Cited by 2 | PDF Full-text (1745 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
ω-Aminododecanoic acid is considered as one of the potential monomers of Nylon 12, a high-performance member of the bioplastic family. The biosynthesis of ω-aminododecanoic acid from renewable sources is an attractive process in the polymer industry. Here, we constructed three artificial self-sufficient P450s [...] Read more.
ω-Aminododecanoic acid is considered as one of the potential monomers of Nylon 12, a high-performance member of the bioplastic family. The biosynthesis of ω-aminododecanoic acid from renewable sources is an attractive process in the polymer industry. Here, we constructed three artificial self-sufficient P450s (ArtssP450s) using CYP153A13 from Alcanivorax borkumensis and cytochrome P450 reductase (CPR) domains of natural self-sufficient P450s (CYP102A1, CYP102A5, and 102D1). Among them, artificial self-sufficient P450 (CYP153A13BM3CPR) with CYP102A1 CPR showed the highest catalytically activity for dodecanoic acid (DDA) substrate. This form of ArtssP450 was further co-expressed with ω-TA from Silicobacter pomeroyi and AlkJ from Pseudomonas putida GPo1. This single-cell system was used for the biotransformation of dodecanoic acid (DDA) to ω-aminododecanoic acid (ω-AmDDA), wherein we could successfully biosynthesize 1.48 mM ω-AmDDA from 10 mM DDA substrate in a one-pot reaction. The productivity achieved in the present study was five times higher than that achieved in our previously reported multistep biosynthesis method (0.3 mM). Full article
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Review

Jump to: Research

Open AccessReview Advancement of Metabolic Engineering Assisted by Synthetic Biology
Catalysts 2018, 8(12), 619; https://doi.org/10.3390/catal8120619
Received: 14 November 2018 / Revised: 26 November 2018 / Accepted: 27 November 2018 / Published: 4 December 2018
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Abstract
Synthetic biology has undergone dramatic advancements for over a decade, during which it has expanded our understanding on the systems of life and opened new avenues for microbial engineering. Many biotechnological and computational methods have been developed for the construction of synthetic systems. [...] Read more.
Synthetic biology has undergone dramatic advancements for over a decade, during which it has expanded our understanding on the systems of life and opened new avenues for microbial engineering. Many biotechnological and computational methods have been developed for the construction of synthetic systems. Achievements in synthetic biology have been widely adopted in metabolic engineering, a field aimed at engineering micro-organisms to produce substances of interest. However, the engineering of metabolic systems requires dynamic redistribution of cellular resources, the creation of novel metabolic pathways, and optimal regulation of the pathways to achieve higher production titers. Thus, the design principles and tools developed in synthetic biology have been employed to create novel and flexible metabolic pathways and to optimize metabolic fluxes to increase the cells’ capability to act as production factories. In this review, we introduce synthetic biology tools and their applications to microbial cell factory constructions. Full article
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Open AccessReview Combined Cross-Linked Enzyme Aggregates as Biocatalysts
Catalysts 2018, 8(10), 460; https://doi.org/10.3390/catal8100460
Received: 17 September 2018 / Revised: 11 October 2018 / Accepted: 14 October 2018 / Published: 17 October 2018
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Abstract
Enzymes are efficient biocatalysts providing an important tool in many industrial biocatalytic processes. Currently, the immobilized enzymes prepared by the cross-linked enzyme aggregates (CLEAs) have drawn much attention due to their simple preparation and high catalytic efficiency. Combined cross-linked enzyme aggregates (combi-CLEAs) including [...] Read more.
Enzymes are efficient biocatalysts providing an important tool in many industrial biocatalytic processes. Currently, the immobilized enzymes prepared by the cross-linked enzyme aggregates (CLEAs) have drawn much attention due to their simple preparation and high catalytic efficiency. Combined cross-linked enzyme aggregates (combi-CLEAs) including multiple enzymes have significant advantages for practical applications. In this review, the conditions or factors for the preparation of combi-CLEAs such as the proportion of enzymes, the type of cross-linker, and coupling temperature were discussed based on the reaction mechanism. The recent applications of combi-CLEAs were also reviewed. Full article
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: Exploring metagenomic enzymes: a novel esterase useful for short-chain ester synthesis
Authors: Thaís Carvalho Maester1,2,a, Mariana Rangel Pereira1,2,b, Aliandra Maura Gibertoni Malaman1; Janaina Pires Borges3; Eleni Gomes4; Eliana Gertrudes de Macedo Lemos1*
Affiliation: 1Department of Technology, São Paulo State University (UNESP), Jaboticabal, SP, Brazil;
2University of São Paulo (USP), São Paulo, SP, Brazil;
3Institute of Biosciences, Languages and Exact Sciences, Department of Chemistry and Environmental Sciences, São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil;
4Institute of Biosciences, Languages and Exact Sciences, Department of Biology, São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil;
Present address: aSupera Innovation and Technology Park, Ecobiotech company, Ribeirão Preto, SP, Brazil;
b Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom;
Corresponding author: Eliana G. M. Lemos: [email protected]
Abstract Lipolytic enzymes have gained attention due to their remarkable ability to carry out a variety of reactions. Enzyme-mediated esterification reactions can be a promising alternative to produce esters of commercial interest, replacing conventional chemical processes. In this study, the novel esterase-encoding gene est5 from a metagenomic library isolated from a microbial consortium specialized in diesel oil degradation was cloned and expressed in Escherichia coli. Sequence analysis revealed a 912 bp ORF encoding a 340-amino acid polypeptide with predicted molecular weight of 37 kDa that belongs to family V of the lipolytic enzymes. Recombinant EST5 was purified and presented higher activity at pH 7.5, 45°C, with a Tm of 47°C, although remained at least 50% active at low temperatures. EST5 exhibited broad substrate specificity toward p-nitrophenol esters with highest activity for p-nitrophenyl valerate (Kcat/Km rate of 1533.27 s-1.mM-1) and remained stable in the presence of up to 10% methanol and 20% dimethyl sulfoxide. Preliminary studies of EST5 in the synthesis of methyl butyrate led to a specific activity of 127.04 U.mg-1. The enzyme showed higher esterification activity than other literature results, including commercial enzymes. EST5 has potential for synthesis of flavor esters, providing a concept for its application in biotechnological processes.
Keywords: Lipolytic enzymes; Metagenome; Family V; Esterification; Flavor esters

 

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