Editorial

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4 pages, 178 KiB

Open AccessEditorial

Editorial Catalysts: Special Issue on Novel Enzyme and Whole-Cell Biocatalysts

by Anwar Sunna and Richard Daniellou

Catalysts 2020, 10(9), 1088; https://doi.org/10.3390/catal10091088 - 20 Sep 2020

Cited by 1 | Viewed by 1778

Abstract

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(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

Research

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12 pages, 1980 KiB

Open AccessArticle

Response Surface Methodology Approach for Optimized Biodiesel Production from Waste Chicken Fat Oil

by Fatima Shafiq, Muhammad Waseem Mumtaz, Hamid Mukhtar, Tooba Touqeer, Syed Ali Raza, Umer Rashid, Imededdine Arbi Nehdi and Thomas Shean Yaw Choong

Catalysts 2020, 10(6), 633; https://doi.org/10.3390/catal10060633 - 06 Jun 2020

Cited by 19 | Viewed by 3654

Abstract

Biodiesel is gaining acceptance as an alternative fuel in a scenario where fossil fuel reserves are being depleted rapidly. Therefore, it is considered as the fuel of the future due to its sustainability, renewable nature and environment friendly attributes. The optimal yield of [...] Read more.

Biodiesel is gaining acceptance as an alternative fuel in a scenario where fossil fuel reserves are being depleted rapidly. Therefore, it is considered as the fuel of the future due to its sustainability, renewable nature and environment friendly attributes. The optimal yield of biodiesel from cheap feed stock oils is a challenge to add cost effectiveness without compromising the fuel quality. In the current experiment, waste chicken fat oil was taken as the feedstock oil to produce biodiesel through the chemical and enzymatic route of transesterification. The process of chemical transesterification was performed using KOH and sodium methoxide, while enzymatic transesterification was done by using free Aspergillus terreus lipase and Aspergillus terreus lipase immobilized on functionalized Fe₃O₄ nanoparticles (Fe₃O₄_PDA_Lipase) as biocatalysts. The physico-chemical properties of the understudy feedstock oil were analyzed to check the feasibility as a feedstock for the biodiesel synthesis. The feedstock oil was found suitable for biodiesel production based upon quality assessment. Optimization of various reaction parameters (the temperature and time of reaction, catalyst concentration and methanol-to-oil mole ratio) was performed based on the response surface methodology (RSM). The maximum yield of biodiesel (90.6%) was obtained from waste chicken fat oil by using Fe₃O₄_PDA_Lipase as an immobilized nano-biocatalyst. Moreover, the above said optimum yield was obtained when transesterification was done using 6% Fe₃O₄_PDA_Lipase with a methanol-to-oil ratio of 6:1 at 42 °C for 36 h. Biodiesel production was monitored by FTIR spectroscopic analysis, whereas compositional profiling was done by GC–MS. The measured fuel properties—cloud point, pour point, flash point, fire point and kinematic viscosity—met the biodiesel specifications by American Society for Testing and Materials (ASTM). Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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17 pages, 2710 KiB

Open AccessArticle

Bacillus subtilis Lipase A—Lipase or Esterase?

by Paula Bracco, Nelleke van Midden, Epifanía Arango, Guzman Torrelo, Valerio Ferrario, Lucia Gardossi and Ulf Hanefeld

Catalysts 2020, 10(3), 308; https://doi.org/10.3390/catal10030308 - 07 Mar 2020

Cited by 23 | Viewed by 4857

Abstract

The question of how to distinguish between lipases and esterases is about as old as the definition of the subclassification is. Many different criteria have been proposed to this end, all indicative but not decisive. Here, the activity of lipases in dry organic [...] Read more.

The question of how to distinguish between lipases and esterases is about as old as the definition of the subclassification is. Many different criteria have been proposed to this end, all indicative but not decisive. Here, the activity of lipases in dry organic solvents as a criterion is probed on a minimal α/β hydrolase fold enzyme, the Bacillus subtilis lipase A (BSLA), and compared to Candida antarctica lipase B (CALB), a proven lipase. Both hydrolases show activity in dry solvents and this proves BSLA to be a lipase. Overall, this demonstrates the value of this additional parameter to distinguish between lipases and esterases. Lipases tend to be active in dry organic solvents, while esterases are not active under these circumstances. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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24 pages, 4466 KiB

Open AccessArticle

A Machine Learning Approach for Efficient Selection of Enzyme Concentrations and Its Application for Flux Optimization

by Anamya Ajjolli Nagaraja, Philippe Charton, Xavier F. Cadet, Nicolas Fontaine, Mathieu Delsaut, Birgit Wiltschi, Alena Voit, Bernard Offmann, Cedric Damour, Brigitte Grondin-Perez and Frederic Cadet

Catalysts 2020, 10(3), 291; https://doi.org/10.3390/catal10030291 - 04 Mar 2020

Cited by 17 | Viewed by 4931

Abstract

The metabolic engineering of pathways has been used extensively to produce molecules of interest on an industrial scale. Methods like gene regulation or substrate channeling helped to improve the desired product yield. Cell-free systems are used to overcome the weaknesses of engineered strains. [...] Read more.

The metabolic engineering of pathways has been used extensively to produce molecules of interest on an industrial scale. Methods like gene regulation or substrate channeling helped to improve the desired product yield. Cell-free systems are used to overcome the weaknesses of engineered strains. One of the challenges in a cell-free system is selecting the optimized enzyme concentration for optimal yield. Here, a machine learning approach is used to select the enzyme concentration for the upper part of glycolysis. The artificial neural network approach (ANN) is known to be inefficient in extrapolating predictions outside the box: high predicted values will bump into a sort of “glass ceiling”. In order to explore this “glass ceiling” space, we developed a new methodology named glass ceiling ANN (GC-ANN). Principal component analysis (PCA) and data classification methods are used to derive a rule for a high flux, and ANN to predict the flux through the pathway using the input data of 121 balances of four enzymes in the upper part of glycolysis. The outcomes of this study are i. in silico selection of optimum enzyme concentrations for a maximum flux through the pathway and ii. experimental in vitro validation of the “out-of-the-box” fluxes predicted using this new approach. Surprisingly, flux improvements of up to 63% were obtained. Gratifyingly, these improvements are coupled with a cost decrease of up to 25% for the assay. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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14 pages, 5675 KiB

Open AccessArticle

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

by David Aregger, Christin Peters and Rebecca M. Buller

Catalysts 2020, 10(2), 254; https://doi.org/10.3390/catal10020254 - 19 Feb 2020

Cited by 7 | Viewed by 4279

Abstract

Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene [...] Read more.

Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene reductases are of high academic and industrial interest. Here, we present the characterization of a novel ene reductase from Paenibacillus polymyxa, named Ppo-Er1, belonging to the recently identified subgroup III of the old yellow enzyme family. The determination of substrate scope, solvent stability, temperature, and pH range of Ppo-Er1 is one of the first examples of a detailed biophysical characterization of a subgroup III enzyme. Notably, Ppo-Er1 possesses a wide temperature optimum (T_opt: 20–45 °C) and retains high conversion rates of at least 70% even at 10 °C reaction temperature making it an interesting biocatalyst for the conversion of temperature-labile substrates. When assaying a set of different organic solvents to determine Ppo-Er1′s solvent tolerance, the ene reductase exhibited good performance in up to 40% cyclohexane as well as 20 vol% DMSO and ethanol. In summary, Ppo-Er1 exhibited activity for thirteen out of the nineteen investigated compounds, for ten of which Michaelis–Menten kinetics could be determined. The enzyme exhibited the highest specificity constant for maleimide with a k_cat/K_M value of 287 mM⁻¹ s⁻¹. In addition, Ppo-Er1 proved to be highly enantioselective for selected substrates with measured enantiomeric excess values of 92% or higher for 2-methyl-2-cyclohexenone, citral, and carvone. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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19 pages, 4327 KiB

Open AccessArticle

Expression, Characterisation and Homology Modelling of a Novel Hormone-Sensitive Lipase (HSL)-Like Esterase from Glaciozyma antarctica

by Hiryahafira Mohamad Tahir, Raja Noor Zaliha Raja Abd Rahman, Adam Thean Chor Leow and Mohd Shukuri Mohamad Ali

Catalysts 2020, 10(1), 58; https://doi.org/10.3390/catal10010058 - 01 Jan 2020

Cited by 16 | Viewed by 3450

Abstract

Microorganisms, especially those that survive in extremely cold places such as Antarctica, have gained research attention since they produce a unique feature of the protein, such as being able to withstand at extreme temperature, salinity, and pressure, that make them desired for biotechnological [...] Read more.

Microorganisms, especially those that survive in extremely cold places such as Antarctica, have gained research attention since they produce a unique feature of the protein, such as being able to withstand at extreme temperature, salinity, and pressure, that make them desired for biotechnological application. Here, we report the first hormone-sensitive lipase (HSL)-like esterase from a Glaciozyma species, a psychrophilic yeast designated as GlaEst12-like esterase. In this study, the putative lipolytic enzyme was cloned, expressed in E. coli, purified, and characterised for its biochemical properties. Protein sequences analysis showed that GlaEst12 shared about 30% sequence identity with chain A of the bacterial hormone-sensitive lipase of E40. It belongs to the H group since it has the conserved motifs of Histidine-Glycine-Glycine-Glycine (HGGG)and Glycine-Aspartate-Serine-Alanine-Glycine (GDSAG) at the amino acid sequences. The recombinant GlaEst12 was successfully purified via one-step Ni-Sepharose affinity chromatography. Interestingly, GlaEst12 showed unusual properties with other enzymes from psychrophilic origin since it showed an optimal temperature ranged between 50–60 °C and was stable at alkaline pH conditions. Unlike other HSL-like esterase, this esterase showed higher activity towards medium-chain ester substrates rather than shorter chain ester. The 3D structure of GlaEst12, predicted by homology modelling using Robetta software, showed a secondary structure composed of mainly α/β hydrolase fold, with the catalytic residues being found at Ser²³², Glu³⁴¹, and His³⁷¹. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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11 pages, 1997 KiB

Open AccessArticle

Low-Level Organic Solvents Improve Multienzyme Whole-Cell Catalytic Synthesis of Myricetin-7-O-Glucuronide

by Yan Yang, Min-Zhi Liu, Yun-Song Cao, Chang-Kun Li and Wei Wang

Catalysts 2019, 9(11), 970; https://doi.org/10.3390/catal9110970 - 18 Nov 2019

Cited by 5 | Viewed by 3196

Abstract

Multienzyme whole-cell biocatalysts are preferred in industrial applications, and two major concerns regarding the use of these biocatalysts, cell viability and cell membrane integrity, must be addressed. In this work, the transformation of myricetin to myricetin-7-O-glucuronide catalyzed by an engineered Escherichia [...] Read more.

Multienzyme whole-cell biocatalysts are preferred in industrial applications, and two major concerns regarding the use of these biocatalysts, cell viability and cell membrane integrity, must be addressed. In this work, the transformation of myricetin to myricetin-7-O-glucuronide catalyzed by an engineered Escherichia coli strain was taken as the model reaction to examine the impacts of low-level organic solvents on whole-cell biocatalysis. Low-level organic solvents (2%, v/v) showed a significant increase (roughly 13-fold) in myricetin-7-O-glucuronide yields. No obvious compromises of cellular viability and integrity were observed by a flow cytometry assay or in the determination of extracellular protein leakage, suggesting the addition of low-level organic solvents accommodates whole E. coli cells. Furthermore, a scaled-up reaction was conducted to test the capability and efficiency of whole-cell catalysis in the presence of organic solvents. This study presents a promising and simple means to enhance the productivity of multienzyme whole-cell catalysis without losing the barrier functions of the cell membrane. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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16 pages, 5475 KiB

Open AccessArticle

Decoding Essential Amino Acid Residues in the Substrate Groove of a Non-Specific Nuclease from Pseudomonas syringae

by Lynn Sophie Schwardmann, Sarah Schmitz, Volker Nölle and Skander Elleuche

Catalysts 2019, 9(11), 941; https://doi.org/10.3390/catal9110941 - 09 Nov 2019

Cited by 3 | Viewed by 2783

Abstract

Non-specific nucleases (NSN) are of interest for biotechnological applications, including industrial downstream processing of crude protein extracts or cell-sorting approaches in microfabricated channels. Bacterial nucleases belonging to the superfamily of phospholipase D (PLD) are featured for their ability to catalyze the hydrolysis of [...] Read more.

Non-specific nucleases (NSN) are of interest for biotechnological applications, including industrial downstream processing of crude protein extracts or cell-sorting approaches in microfabricated channels. Bacterial nucleases belonging to the superfamily of phospholipase D (PLD) are featured for their ability to catalyze the hydrolysis of nucleic acids in a metal-ion-independent manner. In order to gain a deeper insight into the composition of the substrate groove of a NSN from Pseudomonas syringae, semi-rational mutagenesis based on a structure homology model was applied to identify amino acid residues on the protein’s surface adjacent to the catalytic region. A collection of 12 mutant enzymes each with a substitution to a positively charged amino acid (arginine or lysine) was produced in recombinant form and biochemically characterized. Mutations in close proximity to the catalytic region (inner ring) either dramatically impaired or completely abolished the enzymatic performance, while amino acid residues located at the border of the substrate groove (outer ring) only had limited or no effects. A K119R substitution mutant displayed a relative turnover rate of 112% compared to the original nuclease. In conclusion, the well-defined outer ring of the substrate groove is a potential target for modulation of the enzymatic performance of NSNs belonging to the PLD superfamily. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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13 pages, 3456 KiB

Open AccessArticle

Surfactant Imprinting Hyperactivated Immobilized Lipase as Efficient Biocatalyst for Biodiesel Production from Waste Cooking Oil

by Huixia Yang and Weiwei Zhang

Catalysts 2019, 9(11), 914; https://doi.org/10.3390/catal9110914 - 01 Nov 2019

Cited by 28 | Viewed by 3273

Abstract

Enzymatic production of biodiesel from waste cooking oil (WCO) could contribute to resolving the problems of energy demand and environment pollutions.In the present work, Burkholderia cepacia lipase (BCL) was activated by surfactant imprinting, and subsequently immobilized in magnetic cross-linked enzyme aggregates (mCLEAs) with [...] Read more.

Enzymatic production of biodiesel from waste cooking oil (WCO) could contribute to resolving the problems of energy demand and environment pollutions.In the present work, Burkholderia cepacia lipase (BCL) was activated by surfactant imprinting, and subsequently immobilized in magnetic cross-linked enzyme aggregates (mCLEAs) with hydroxyapatite coated magnetic nanoparticles (HAP-coated MNPs). The maximum hyperactivation of BCL mCLEAs was observed in the pretreatment of BCL with 0.1 mM Triton X-100. The optimized Triton-activated BCL mCLEAs was used as a highly active and robust biocatalyst for biodiesel production from WCO, exhibiting significant increase in biodiesel yield and tolerance to methanol. The results indicated that surfactant imprinting integrating mCLEAs could fix BCL in their active (open) form, experiencing a boost in activity and allowing biodiesel production performed in solvent without further addition of water. A maximal biodiesel yield of 98% was achieved under optimized conditions with molar ratio of methanol-to-WCO 7:1 in one-time addition in hexane at 40 °C. Therefore, the present study displays a versatile method for lipase immobilization and shows great practical latency in renewable biodiesel production. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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13 pages, 16142 KiB

Open AccessArticle

Screening and Comparative Characterization of Microorganisms from Iranian Soil Samples Showing ω-Transaminase Activity toward a Plethora of Substrates

by Najme Gord Noshahri, Jamshid Fooladi, Christoph Syldatk, Ulrike Engel, Majid M. Heravi, Mohammad Zare Mehrjerdi and Jens Rudat

Catalysts 2019, 9(10), 874; https://doi.org/10.3390/catal9100874 - 22 Oct 2019

Cited by 8 | Viewed by 3882

Abstract

In this study, soil microorganisms from Iran were screened for ω-transaminase (ω-TA) activity based on growth on minimal media containing (rac)-α-methylbenzylamine (rac-α-MBA) as a sole nitrogen source. Then, for the selection of strains with high enzyme activity, a colorimetric o-xylylendiamine assay [...] Read more.

In this study, soil microorganisms from Iran were screened for ω-transaminase (ω-TA) activity based on growth on minimal media containing (rac)-α-methylbenzylamine (rac-α-MBA) as a sole nitrogen source. Then, for the selection of strains with high enzyme activity, a colorimetric o-xylylendiamine assay was conducted. The most promising strains were identified by 16S rDNA sequencing. Five microorganisms showing high ω-TA activity were subjected to determine optimal conditions for ω-TA activity, including pH, temperature, co-solvent, and the specificity of the ω-TA toward different amine donors and acceptors. Among the five screened microorganisms, Bacillus halotolerans turned out to be the most promising strain: Its cell-free extract showed a highly versatile amino donor spectrum toward aliphatic, aromatic chiral amines and a broad range of pH activity. Transaminase activity also exhibited excellent solvent tolerance, with maximum turnover in the presence of 30% (v/v) DMSO. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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15 pages, 1469 KiB

Open AccessArticle

Comparative Analysis and Biochemical Characterization of Two Endo-β-1,3-Glucanases from the Thermophilic Bacterium Fervidobacterium sp.

by Christin Burkhardt, Christian Schäfers, Jörg Claren, Georg Schirrmacher and Garabed Antranikian

Catalysts 2019, 9(10), 830; https://doi.org/10.3390/catal9100830 - 01 Oct 2019

Cited by 7 | Viewed by 2855

Abstract

Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in [...] Read more.

Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in the Azores. Sequence comparison revealed that both genes had high similarities to genes from Fervidobacterium nodosum Rt17-B1. The two proteins showed sequence similarities of 62% to each other and belong to the glycoside hydrolase (GH) family 16. For biochemical characterization, both laminarinases were heterologously produced in Escherichia coli and purified to homogeneity. FLamA and FLamB exhibited similar properties and both showed highest activity towards laminarin at 90 °C and pH 6.5. The two enzymes were thermostable but differed in their half-life at 80 °C with 5 h and 1 h for FLamA and FLamB, respectively. In contrast to other laminarinases, both enzymes prefer β-1,3-glucans and mixed-linked glucans as substrates. However, FLamA and FLamB differ in their catalytic efficiency towards laminarin. Structure predictions were made and showed minor differences particularly in a kink adjacent to the active site cleft. The high specific activities and resistance to elevated temperatures and various additives make both enzymes suitable candidates for application in biomass conversion. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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11 pages, 1433 KiB

Open AccessArticle

Hydrolysis of Glycosyl Thioimidates by Glycoside Hydrolase Requires Remote Activation for Efficient Activity

by Laure Guillotin, Zeinab Assaf, Salvatore G. Pistorio, Pierre Lafite, Alexei V. Demchenko and Richard Daniellou

Catalysts 2019, 9(10), 826; https://doi.org/10.3390/catal9100826 - 01 Oct 2019

Cited by 5 | Viewed by 3097

Abstract

Chemoenzymatic synthesis of glycosides relies on efficient glycosyl donor substrates able to react rapidly and efficiently, yet with increased stability towards chemical or enzymatic hydrolysis. In this context, glycosyl thioimidates have previously been used as efficient donors, in the case of hydrolysis or [...] Read more.

Chemoenzymatic synthesis of glycosides relies on efficient glycosyl donor substrates able to react rapidly and efficiently, yet with increased stability towards chemical or enzymatic hydrolysis. In this context, glycosyl thioimidates have previously been used as efficient donors, in the case of hydrolysis or thioglycoligation. In both cases, the release of the thioimidoyl aglycone was remotely activated through a protonation driven by a carboxylic residue in the active site of the corresponding enzymes. A recombinant glucosidase (DtGly) from Dictyoglomus themophilum, previously used in biocatalysis, was also able to use such glycosyl thioimidates as substrates. Yet, enzymatic kinetic values analysis, coupled to mutagenesis and in silico modelling of DtGly/substrate complexes demonstrated that the release of the thioimidoyl moiety during catalysis is only driven by its leaving group ability, without the activation of a remote protonation. In the search of efficient glycosyl donors, glycosyl thioimidates are attractive and efficient. Their utility, however, is limited to enzymes able to promote leaving group release by remote activation. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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11 pages, 1812 KiB

Open AccessArticle

Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase

by Zhihai Liu, Alessandra Piccirilli, Dejun Liu, Wan Li, Yang Wang and Jianzhong Shen

Catalysts 2019, 9(9), 744; https://doi.org/10.3390/catal9090744 - 02 Sep 2019

Cited by 10 | Viewed by 3719

Abstract

The New Delhi metallo-β-lactamase-1 (NDM-1) is a typical carbapenemase and plays a crucial role in antibiotic-resistance bacterial infection. Phylogenetic analysis, performed on known NDM-variants, classified NDM enzymes in seven clusters. Three of them include a major number of NDM-variants. In this study, we [...] Read more.

The New Delhi metallo-β-lactamase-1 (NDM-1) is a typical carbapenemase and plays a crucial role in antibiotic-resistance bacterial infection. Phylogenetic analysis, performed on known NDM-variants, classified NDM enzymes in seven clusters. Three of them include a major number of NDM-variants. In this study, we evaluated the role of the V88L substitution in NDM-24 by kinetical and structural analysis. Functional results showed that V88L did not significantly increase the resistance level in the NDM-24 transformant toward penicillins, cephalosporins, meropenem, and imipenem. Concerning ertapenem, E. coli DH5α/NDM-24 showed a MIC value 4-fold higher than that of E. coli DH5α/NDM-1. The determination of the k_cat, K_m, and k_cat/K_m values for NDM-24, compared with NDM-1 and NDM-5, demonstrated an increase of the substrate hydrolysis compared to all the β-lactams tested, except penicillins. The thermostability testing revealed that V88L generated a destabilized effect on NDM-24. The V88L substitution occurred in the β-strand and low β-sheet content in the secondary structure, as evidenced by the CD analysis data. In conclusion, the V88L substitution increases the enzyme activity and decreases the protein stability. This study characterizes the role of the V88L substitution in NDM-24 and provides insight about the NDM variants evolution. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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15 pages, 2248 KiB

Open AccessArticle

Highly Selective Oxidation of 5-Hydroxymethylfurfural to 5-Hydroxymethyl-2-Furancarboxylic Acid by a Robust Whole-Cell Biocatalyst

by Ran Cang, Li-Qun Shen, Guang Yang, Zhi-Dong Zhang, He Huang and Zhi-Gang Zhang

Catalysts 2019, 9(6), 526; https://doi.org/10.3390/catal9060526 - 12 Jun 2019

Cited by 27 | Viewed by 5110

Abstract

Value-added utilization of biomass-derived 5-hydroxymethylfurfural (HMF) to produce useful derivatives is of great interest. In this work, extremely radiation resistant Deinococcus wulumuqiensis R12 was explored for the first time as a new robust biocatalyst for selective oxidation of HMF to 5-hydroxymethylfuroic acid (HMFCA). [...] Read more.

Value-added utilization of biomass-derived 5-hydroxymethylfurfural (HMF) to produce useful derivatives is of great interest. In this work, extremely radiation resistant Deinococcus wulumuqiensis R12 was explored for the first time as a new robust biocatalyst for selective oxidation of HMF to 5-hydroxymethylfuroic acid (HMFCA). Its resting cells exhibited excellent catalytic performance in a broad range of pH and temperature values, and extremely high tolerance to HMF and the HMFCA product. An excellent yield of HMFCA (up to 90%) was achieved when the substrate concentration was set to 300 mM under the optimized reaction conditions. In addition, 511 mM of product was obtained within 20 h by employing a fed-batch strategy, affording a productivity of 44 g/L per day. Of significant synthetic interest was the finding that the D. wulumuqiensis R12 cells were able to catalyze the selective oxidation of other structurally diverse aldehydes to their corresponding acids with good yield and high selectivity, indicating broad substrate scope and potential widespread applications in biotechnology and organic chemistry. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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18 pages, 2332 KiB

Open AccessArticle

Immobilization of β-Galactosidases on the Lactobacillus Cell Surface Using the Peptidoglycan-Binding Motif LysM

by Mai-Lan Pham, Anh-Minh Tran, Suwapat Kittibunchakul, Tien-Thanh Nguyen, Geir Mathiesen and Thu-Ha Nguyen

Catalysts 2019, 9(5), 443; https://doi.org/10.3390/catal9050443 - 12 May 2019

Cited by 11 | Viewed by 3756

Abstract

Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from [...] Read more.

Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from a putative extracellular transglycosylase Lp_3014 of Lactobacillus plantarum WCFS1 to display two different lactobacillal β-galactosidases, the heterodimeric LacLM-type from Lactobacillus reuteri and the homodimeric LacZ-type from Lactobacillus delbrueckii subsp. bulgaricus, on the cell surface of different Lactobacillus spp. The β-galactosidases were fused with the LysM domain and the fusion proteins, LysM-LacLMLreu and LysM-LacZLbul, were successfully expressed in Escherichia coli and subsequently displayed on the cell surface of L. plantarum WCFS1. β-Galactosidase activities obtained for L. plantarum displaying cells were 179 and 1153 U per g dry cell weight, or the amounts of active surface-anchored β-galactosidase were 0.99 and 4.61 mg per g dry cell weight for LysM-LacLMLreu and LysM-LacZLbul, respectively. LysM-LacZLbul was also displayed on the cell surface of other Lactobacillus spp. including L. delbrueckii subsp. bulgaricus, L. casei and L. helveticus, however L. plantarum is shown to be the best among Lactobacillus spp. tested for surface display of fusion LysM-LacZLbul, both with respect to the immobilization yield as well as the amount of active surface-anchored enzyme. The immobilized fusion LysM-β-galactosidases are catalytically efficient and can be reused for several repeated rounds of lactose conversion. This approach, with the β-galactosidases being displayed on the cell surface of non-genetically modified food-grade organisms, shows potential for applications of these immobilized enzymes in the synthesis of prebiotic galacto-oligosaccharides. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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15 pages, 2296 KiB

Open AccessArticle

Effects of Random Mutagenesis and In Vivo Selection on the Specificity and Stability of a Thermozyme

by Giuseppe Perugino, Andrea Strazzulli, Marialuisa Mazzone, Mosè Rossi and Marco Moracci

Catalysts 2019, 9(5), 440; https://doi.org/10.3390/catal9050440 - 11 May 2019

Cited by 4 | Viewed by 2741

Abstract

Factors that give enzymes stability, activity, and substrate recognition result from the combination of few weak molecular interactions, which can be difficult to study through rational protein engineering approaches. We used irrational random mutagenesis and in vivo selection to test if a β-glycosidase [...] Read more.

Factors that give enzymes stability, activity, and substrate recognition result from the combination of few weak molecular interactions, which can be difficult to study through rational protein engineering approaches. We used irrational random mutagenesis and in vivo selection to test if a β-glycosidase from the thermoacidophile Saccharolobus solfataricus (Ssβ-gly) could complement an Escherichia coli strain unable to grow on lactose. The triple mutant of Ssβ-gly (S26L, P171L, and A235V) was more active than the wild type at 85 °C, inactivated at this temperature almost 300-fold quicker, and showed a 2-fold higher k_cat on galactosides. The three mutations, which were far from the active site, were analyzed to test their effect at the structural level. Improved activity on galactosides was induced by the mutations. The S26L and P171L mutations destabilized the enzyme through the removal of a hydrogen bond and increased flexibility of the peptide backbone, respectively. However, the flexibility added by S26L mutation improved the activity at T > 60 °C. This study shows that random mutagenesis and biological selection allowed the identification of residues that are critical in determining thermal activity, stability, and substrate recognition. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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12 pages, 2684 KiB

Open AccessArticle

Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins

by Christian Sonnendecker and Wolfgang Zimmermann

Catalysts 2019, 9(3), 242; https://doi.org/10.3390/catal9030242 - 06 Mar 2019

Cited by 16 | Viewed by 3552

Abstract

Cyclodextrin glucanotransferases (CGTases) convert starch to cyclodextrins (CD) of various sizes. To engineer a CGTase for the synthesis of large-ring CD composed of 9 to 12 glucose units, a loop structure of the protein involved in substrate binding was targeted for semi-rational mutagenesis. [...] Read more.

Cyclodextrin glucanotransferases (CGTases) convert starch to cyclodextrins (CD) of various sizes. To engineer a CGTase for the synthesis of large-ring CD composed of 9 to 12 glucose units, a loop structure of the protein involved in substrate binding was targeted for semi-rational mutagenesis. Based on multiple protein alignments and protein structure information, a mutagenic megaprimer was designed to encode a partial randomization of eight amino acid residues within the loop region. The library obtained encoding amino acid sequences occurring in wild type CGTases in combination with a screening procedure yielded sequences displaying a changed CD product specificity. As a result, variants of the CGTase from the alkaliphilic Bacillus sp. G825-6 synthesizing mainly CD9 to CD12 could be obtained. When the mutagenesis experiment was performed with the CGTase G825-6 variant Y183R, the same loop alterations that increased the total CD synthesis activity resulted in lower activities of the variant enzymes created. In the presence of the amino acid residue R183, the synthesis of CD8 was suppressed and larger CD were obtained as the main products. The alterations not only affected the product specificity, but also influenced the thermal stability of some of the CGTase variants indicating the importance of the loop structure for the stability of the CGTase. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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14 pages, 2980 KiB

Open AccessArticle

Characterisation of the First Archaeal Mannonate Dehydratase from Thermoplasma acidophilum and Its Potential Role in the Catabolism of D-Mannose

by Dominik Kopp, Robert Willows and Anwar Sunna

Catalysts 2019, 9(3), 234; https://doi.org/10.3390/catal9030234 - 03 Mar 2019

Cited by 6 | Viewed by 5753

Abstract

Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and [...] Read more.

Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and physiological studies. Mannonate dehydratases from archaea have not been studied in detail. Here, we identified and characterised the first archaeal mannonate dehydratase (TaManD) from the thermoacidophilic archaeon Thermoplasma acidophilum. The recombinant TaManD enzyme was optimally active at 65 °C and showed high specificity towards D-mannonate and its lactone, D-mannono-1,4-lactone. The gene encoding for TaManD is located adjacent to a previously studied mannose-specific aldohexose dehydrogenase (AldT) in the genome of T. acidophilum. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that the mannose-specific AldT produces the substrates for TaManD, demonstrating the possibility for an oxidative metabolism of mannose in T. acidophilum. Among previously studied mannonate dehydratases, TaManD showed closest homology to enzymes belonging to the xylose isomerase-like superfamily. Genetic analysis revealed that closely related mannonate dehydratases among archaea are not located in a hexuronate gene cluster like in bacteria, but next to putative aldohexose dehydrogenases, implying a different physiological role of mannonate dehydratases in those archaeal species. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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14 pages, 1226 KiB

Open AccessArticle

An Innovative Biocatalyst for Continuous 2G Ethanol Production from Xylo-Oligomers by Saccharomyces cerevisiae through Simultaneous Hydrolysis, Isomerization, and Fermentation (SHIF)

by Thais S. Milessi-Esteves, Felipe A.S. Corradini, Willian Kopp, Teresa C. Zangirolami, Paulo W. Tardioli, Roberto C. Giordano and Raquel L.C. Giordano

Catalysts 2019, 9(3), 225; https://doi.org/10.3390/catal9030225 - 01 Mar 2019

Cited by 12 | Viewed by 3147

Abstract

Many approaches have been considered aimed at ethanol production from the hemicellulosic fraction of biomass. However, the industrial implementation of this process has been hindered by some bottlenecks, one of the most important being the ease of contamination of the bioreactor by bacteria [...] Read more.

Many approaches have been considered aimed at ethanol production from the hemicellulosic fraction of biomass. However, the industrial implementation of this process has been hindered by some bottlenecks, one of the most important being the ease of contamination of the bioreactor by bacteria that metabolize xylose. This work focuses on overcoming this problem through the fermentation of xylulose (the xylose isomer) by native Saccharomyces cerevisiae using xylo-oligomers as substrate. A new concept of biocatalyst is proposed, containing xylanases and xylose isomerase (XI) covalently immobilized on chitosan, and co-encapsulated with industrial baker’s yeast in Ca-alginate gel spherical particles. Xylo-oligomers are hydrolyzed, xylose is isomerized, and finally xylulose is fermented to ethanol, all taking place simultaneously, in a process called simultaneous hydrolysis, isomerization, and fermentation (SHIF). Among several tested xylanases, Multifect CX XL A03139 was selected to compose the biocatalyst bead. Influences of pH, Ca²⁺, and Mg²⁺ concentrations on the isomerization step were assessed. Experiments of SHIF using birchwood xylan resulted in an ethanol yield of 0.39 g/g, (76% of the theoretical), selectivity of 3.12 g_ethanol/g_xylitol, and ethanol productivity of 0.26 g/L/h. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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23 pages, 1152 KiB

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Challenges and Opportunities in Identifying and Characterising Keratinases for Value-Added Peptide Production

by Juan Pinheiro De Oliveira Martinez, Guiqin Cai, Matthias Nachtschatt, Laura Navone, Zhanying Zhang, Karen Robins and Robert Speight

Catalysts 2020, 10(2), 184; https://doi.org/10.3390/catal10020184 - 03 Feb 2020

Cited by 39 | Viewed by 7137

Abstract

Keratins are important structural proteins produced by mammals, birds and reptiles. Keratins usually act as a protective barrier or a mechanical support. Millions of tonnes of keratin wastes and low value co-products are generated every year in the poultry, meat processing, leather and [...] Read more.

Keratins are important structural proteins produced by mammals, birds and reptiles. Keratins usually act as a protective barrier or a mechanical support. Millions of tonnes of keratin wastes and low value co-products are generated every year in the poultry, meat processing, leather and wool industries. Keratinases are proteases able to breakdown keratin providing a unique opportunity of hydrolysing keratin materials like mammalian hair, wool and feathers under mild conditions. These mild conditions ameliorate the problem of unwanted amino acid modification that usually occurs with thermochemical alternatives. Keratinase hydrolysis addresses the waste problem by producing valuable peptide mixes. Identifying keratinases is an inherent problem associated with the search for new enzymes due to the challenge of predicting protease substrate specificity. Here, we present a comprehensive review of twenty sequenced peptidases with keratinolytic activity from the serine protease and metalloprotease families. The review compares their biochemical activities and highlights the difficulties associated with the interpretation of these data. Potential applications of keratinases and keratin hydrolysates generated with these enzymes are also discussed. The review concludes with a critical discussion of the need for standardized assays and increased number of sequenced keratinases, which would allow a meaningful comparison of the biochemical traits, phylogeny and keratinase sequences. This deeper understanding would facilitate the search of the vast peptidase family sequence space for novel keratinases with industrial potential. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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18 pages, 3263 KiB

Open AccessReview

Cofactor F₄₂₀-Dependent Enzymes: An Under-Explored Resource for Asymmetric Redox Biocatalysis

by Mihir V. Shah, James Antoney, Suk Woo Kang, Andrew C. Warden, Carol J. Hartley, Hadi Nazem-Bokaee, Colin J. Jackson and Colin Scott

Catalysts 2019, 9(10), 868; https://doi.org/10.3390/catal9100868 - 20 Oct 2019

Cited by 26 | Viewed by 5853

Abstract

The asymmetric reduction of enoates, imines and ketones are among the most important reactions in biocatalysis. These reactions are routinely conducted using enzymes that use nicotinamide cofactors as reductants. The deazaflavin cofactor F₄₂₀ also has electrochemical properties that make it suitable as [...] Read more.

The asymmetric reduction of enoates, imines and ketones are among the most important reactions in biocatalysis. These reactions are routinely conducted using enzymes that use nicotinamide cofactors as reductants. The deazaflavin cofactor F₄₂₀ also has electrochemical properties that make it suitable as an alternative to nicotinamide cofactors for use in asymmetric reduction reactions. However, cofactor F₄₂₀-dependent enzymes remain under-explored as a resource for biocatalysis. This review considers the cofactor F₄₂₀-dependent enzyme families with the greatest potential for the discovery of new biocatalysts: the flavin/deazaflavin-dependent oxidoreductases (FDORs) and the luciferase-like hydride transferases (LLHTs). The characterized F₄₂₀-dependent reductions that have the potential for adaptation for biocatalysis are discussed, and the enzymes best suited for use in the reduction of oxidized cofactor F₄₂₀ to allow cofactor recycling in situ are considered. Further discussed are the recent advances in the production of cofactor F₄₂₀ and its functional analog F_O-5′-phosphate, which remains an impediment to the adoption of this family of enzymes for industrial biocatalytic processes. Finally, the prospects for the use of this cofactor and dependent enzymes as a resource for industrial biocatalysis are discussed. Full article

(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)

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