Special Issue "Biocatalytic Process Optimization"

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

Deadline for manuscript submissions: closed (30 June 2020).

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A printed edition of this Special Issue is available here.

Special Issue Editors

Prof. Dr. Chia-Hung Kuo
E-Mail Website1 Website2
Guest Editor
National Kaohsiung University of Science and Technology, Kaohsiung, Taiwan
Interests: cellulase; lipase esterification and trans esterification; amylase; enzymatic kinetics; ultrasound-assisted enzymatic reaction; enzyme extraction; biotransformation; saccharification; response surface methodology; artificial neural network; wine fermentation
Special Issues and Collections in MDPI journals
Prof. Dr. Chwen-Jen Shieh
E-Mail Website
Guest Editor
National Chung-Hsing University, Taichung, Taiwan
Interests: biodiesel; lipid biocatalysis; enzyme technology; bioprocess optimization; supercritical fluid technology; Chinese herb medicine biotechnology

Special Issue Information

Dear Colleagues,

Biocatalysis is very appealing to industry because it allows, in principle, the synthesis of products not accessible by chemical synthesis. Enzymes are very effective and precise biocatalysts as they are enantioselective, with mild reaction conditions and green chemistry. Biocatalysis is currently widely used in the pharmaceutical industry, food industry, cosmetic industry, and textile industry. This includes enzyme production, biocatalytic process development, biotransformation, enzyme engineering, immobilization, and recycling of biocatalysts. One of the most challenging problems in biocatalysis applications is process optimization. Experimental design combined with response surface methodology or artificial neural network are powerful tools for process optimization.

This Special Issue aims to cover recent progress and advances in the field of biocatalysis optimization using any methods, such as experimental design, response surface methodology or artificial neural networks. Other methodologies, even as one-factor-at-a-time experiments for optimization related to biocatalysis, are also welcome. We welcome both original research and review articles focused on optimizing the enzymatic process.

Assoc. Prof. Dr. Chia-Hung Kuo
Prof. Dr. Chwen-Jen Shieh
Guest Editors

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Keywords

  • enzyme
  • biocatalysis
  • optimization
  • lipase
  • cellulase
  • protease
  • enzyme immobilization
  • glucosidase
  • biotransformation
  • enzymatic kinetics
  • ultrasound-assisted enzymatic reaction
  • enzyme extraction
  • experimental design
  • response surface methodology (RSM)
  • artificial neural network (ANN)

Published Papers (19 papers)

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Editorial

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Open AccessEditorial
Biocatalytic Process Optimization
Catalysts 2020, 10(11), 1303; https://doi.org/10.3390/catal10111303 - 12 Nov 2020
Viewed by 470
Abstract
Biocatalysis refers to the use of microorganisms and enzymes in chemical reactions, has become increasingly popular and is frequently used in industrial applications due to the high efficiency and selectivity of biocatalysts [...] Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)

Research

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Open AccessArticle
Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants
Catalysts 2020, 10(8), 823; https://doi.org/10.3390/catal10080823 - 23 Jul 2020
Cited by 2 | Viewed by 723
Abstract
Naringin dihydrochalcone (DC) is originally derived from the flavonoid naringin, which occurs naturally in citrus fruits, especially in grapefruit. It is used as an artificial sweetener with a strong antioxidant activity with potential applications in food and pharmaceutical fields. At present, enzymatic and [...] Read more.
Naringin dihydrochalcone (DC) is originally derived from the flavonoid naringin, which occurs naturally in citrus fruits, especially in grapefruit. It is used as an artificial sweetener with a strong antioxidant activity with potential applications in food and pharmaceutical fields. At present, enzymatic and chemical methods to make products of naringin DC by hydroxylation reactions have not been developed. Here, an enzymatic strategy for the efficient synthesis of potentially valuable products from naringin DC, a glycoside of phloretin, was developed using Bacillus megaterium CYP102A1 monooxygenase. The major product was identified to be neoeriocitrin DC by NMR and LC-MS analyses. Sixty-seven mutants of CYP102A1 were tested for hydroxylation of naringin DC to produce neoeriocitrin DC. Six mutants with high activity were selected to determine the kinetic parameters and total turnover numbers (TTNs). The kcat value of the most active mutant was 11 min−1 and its TTN was 315. The productivity of neoeriocitrin DC production increased up to 1.1 mM h−1, which corresponds to 0.65 g L−1 h−1. In this study, we achieved a regioselective hydroxylation of naringin DC to produce neoeriocitrin DC. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Characterization of Electrode Performance in Enzymatic Biofuel Cells Using Cyclic Voltammetry and Electrochemical Impedance Spectroscopy
Catalysts 2020, 10(7), 782; https://doi.org/10.3390/catal10070782 - 13 Jul 2020
Cited by 5 | Viewed by 1238
Abstract
The main objective of this study was to examine the quantitative performance of the electrochemical redox reaction of glucose by glucosidase and oxygen with laccase in a phosphate buffer solution at pH 7.0. The characterization of electrode performance was performed by using electrochemical [...] Read more.
The main objective of this study was to examine the quantitative performance of the electrochemical redox reaction of glucose by glucosidase and oxygen with laccase in a phosphate buffer solution at pH 7.0. The characterization of electrode performance was performed by using electrochemical analysis such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The use of such electrochemical analysis (CV and EIS) enables a better understanding of the redox process, the charge transfer resistance, and, hence, the potential mass transfer among the electrode materials in phosphorus buffer solution. The experimental results show that the maximum power densities of the bioanode and the biocathode electrodes were 800 µA/cm2 and 600 µA/cm2, respectively. Both the bioanode and biocathode show high internal resistance. The occurrence of peak-separation shows an excellent mass-transfer mechanism and better chemical reactivity in the electrode. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessFeature PaperArticle
Continuous Production of 2-Phenylethyl Acetate in a Solvent-Free System Using a Packed-Bed Reactor with Novozym® 435
Catalysts 2020, 10(6), 714; https://doi.org/10.3390/catal10060714 - 26 Jun 2020
Cited by 2 | Viewed by 658
Abstract
2-Phenylethyl acetate (2-PEAc), a highly valued natural volatile ester, with a rose-like odor, is widely added in cosmetics, soaps, foods, and drinks to strengthen scent or flavour. Nowadays, 2-PEAc are commonly produced by chemical synthesis or extraction. Alternatively, biocatalysis is a potential method [...] Read more.
2-Phenylethyl acetate (2-PEAc), a highly valued natural volatile ester, with a rose-like odor, is widely added in cosmetics, soaps, foods, and drinks to strengthen scent or flavour. Nowadays, 2-PEAc are commonly produced by chemical synthesis or extraction. Alternatively, biocatalysis is a potential method to replace chemical synthesis or extraction for the production of natural flavour. Continuous synthesis of 2-PEAc in a solvent-free system using a packed bed bioreactor through immobilized lipase-catalyzed transesterification of ethyl acetate (EA) with 2-phenethyl alcohol was studied. A Box–Behnken experimental design with three-level-three-factor, including 2-phenethyl alcohol (2-PE) concentration (100–500 mM), flow rate (1–5 mL min−1) and reaction temperature (45–65 °C), was selected to investigate their influence on the molar conversion of 2-PEAc. Then, response surface methodology and ridge max analysis were used to discuss in detail the optimal reaction conditions for the synthesis of 2-PEAc. The results indicated both 2-PE concentration and flow rate are significant factors in the molar conversion of 2-PEAc. Based on the ridge max analysis, the maximum molar conversion was 99.01 ± 0.09% under optimal conditions at a 2-PE concentration of 62.07 mM, a flow rate of 2.75 mL min−1, and a temperature of 54.03 °C, respectively. The continuous packed bed bioreactor showed good stability for 2-PEAc production, enabling operation for at least 72 h without a significant decrease of conversion. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Whole-Cells of Yarrowia lipolytica Applied in “One Pot” Indolizine Biosynthesis
Catalysts 2020, 10(6), 629; https://doi.org/10.3390/catal10060629 - 05 Jun 2020
Cited by 1 | Viewed by 647
Abstract
A series of yeast strains was tested in order to evaluate their catalytic potential in biocatalysis of one-pot indolizine’s synthesis. Yeast cultivation was performed in a submerged system at 28 °C for 72 h at 180 rpm. An assessment of the reagents’ toxicity [...] Read more.
A series of yeast strains was tested in order to evaluate their catalytic potential in biocatalysis of one-pot indolizine’s synthesis. Yeast cultivation was performed in a submerged system at 28 °C for 72 h at 180 rpm. An assessment of the reagents’ toxicity on yeast viability and metabolic functionality concluded that the growth potential of three Yarrowia lipolytica strains were least affected by the reactants compared to the other yeast strains. Further, crude fermentation products (biomass and cell-free supernatant)—obtained by submerged cultivation of these yeasts—were used in multistep cascade reactions for the production of fluorescent indolizine compounds with important biologic activities. A whole–cell catalyzed multicomponent reaction of activated alkynes, α-bromo-carbonyl reagents and 4,4′-bipyridine, at room temperature in buffer solution led to the efficient synthesis of bis-indolizines 4a, 4b and 4c, in good-to-excellent yields (47%–77%). The metabolites of the selected Y. lipolytica strains can be considered effective biocatalysts in cycloaddition reactions and the high purity and bioconversion yields of the synthesized indolizines indicates a great potential of this type of “green” catalysts. Seeds of Triticum estivum L. were used to investigate the impact of the final products on the germination and seedling growth. The most sensitive physiological parameters suggest that indolizines, at the concentrations tested, have non-toxic effect on germination and seedling growth of wheat, fact also confirmed by confocal laser scanning microscopy images. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Hepatoprotective Effects of Pleurotus ostreatus Protein Hydrolysates Yielded by Pepsin Hydrolysis
Catalysts 2020, 10(6), 595; https://doi.org/10.3390/catal10060595 - 26 May 2020
Cited by 3 | Viewed by 581
Abstract
Pleurotus ostreatus protein extract (POPE) was prepared by the alkali precipitation method with 0.3% (w/v) NaOH. POPEP-III with a MW of 3000–5000 Da was acquired by pepsin enenzymatic hydrolysis. POPEP-III displayed noteworthy effects of 1,1-diphenyl-2-picrylhydrazyl DPPH and H2O2 [...] Read more.
Pleurotus ostreatus protein extract (POPE) was prepared by the alkali precipitation method with 0.3% (w/v) NaOH. POPEP-III with a MW of 3000–5000 Da was acquired by pepsin enenzymatic hydrolysis. POPEP-III displayed noteworthy effects of 1,1-diphenyl-2-picrylhydrazyl DPPH and H2O2 scavenging activities, Fe2+ chelating ability, lipid peroxidation inhibition capacity, and metal reducing power. The administration of POPEP-III in mice significantly prevented prior CCl4-induced strengthen serum ALT and AST activities, changing from 365.44 ± 36.87 IU/L to 220.23 ± 22.27 IU/L and 352.52 IU/L to 206.75 ± 17.26 IU/L, respectively (p < 0.001), and suppressed hepatic malondialdehyde (MDA) formation from 15.28 ± 3.47 nmol/mg prot to 10.04 ± 2.06 nmol/mg prot (p < 0.001). Mice treated with POPEP-III demonstrated augmented activities of superoxide dismutase (SOD) in the liver, from 187.49 ± 19.81 U/mg prot to 233.35 ± 34.23 U/mg prot, and of glutathione peroxidase (GSH-Px), from 84.01 ± 14.54 U/mg prot to 115.9 ± 16.57 U/mg prot (p < 0.05). POPEP-III also prevented CCl4-induced oxidative liver histological alteration. The results suggest that POPEP-III can protect the liver from CCl4-induced oxidative damage. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Development and Optimization of Lipase-Catalyzed Synthesis of Phospholipids Containing 3,4-Dimethoxycinnamic Acid by Response Surface Methodology
Catalysts 2020, 10(5), 588; https://doi.org/10.3390/catal10050588 - 24 May 2020
Cited by 2 | Viewed by 787
Abstract
The interesterification reaction of egg-yolk phosphatidylcholine (PC) with ethyl ester of 3,4-dimethoxycinnamic acid (E3,4DMCA) catalyzed by Novozym 435 in hexane as a reaction medium was shown to be an effective method for the synthesis of corresponding structured O-methylated phenophospholipids. The effects of [...] Read more.
The interesterification reaction of egg-yolk phosphatidylcholine (PC) with ethyl ester of 3,4-dimethoxycinnamic acid (E3,4DMCA) catalyzed by Novozym 435 in hexane as a reaction medium was shown to be an effective method for the synthesis of corresponding structured O-methylated phenophospholipids. The effects of substrate molar ratios, time of the reaction and enzyme load on the process of incorporation of 3,4DMCA into PC were evaluated by using the experimental factorial design of three factors and three levels. The results showed that a substrate molar ratio is a crucial variable for the maximization of the synthesis of 3,4-dimethoxycinnamoylated phospholipids. Under optimized parameters of 1/10 substrate molar ratio PC/E3,4DMCA, enzyme load 30% (w/w), hexane as a medium and incubation time of 3 days, the incorporation of aromatic acid into phospholipid fraction reached 21 mol%. The modified phosphatidylcholine (3,4DMCA-PC) and modified lysophosphatidylcholine (3,4DMCA-LPC) were obtained in isolated yields of 3.5% and 27.5% (w/w), respectively. The developed method of phosphatidylcholine interesterification is the first described in the literature dealing with 3,4DMCA and allows us to obtain new O-methylated phenophospholipids with potential applications as food additives or nutraceuticals with pro-health activity. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Effects of Long-Term Supplementation with Aluminum or Selenium on the Activities of Antioxidant Enzymes in Mouse Brain and Liver
Catalysts 2020, 10(5), 585; https://doi.org/10.3390/catal10050585 - 23 May 2020
Cited by 1 | Viewed by 610
Abstract
The aim of this study was to investigate the effects of aluminum (Al) or selenium (Se) on the “primary” antioxidant defense system enzymes (superoxide dismutase, catalase, and glutathione reductase) in cells of mouse brain and liver after long-term (8-week) exposure to drinking water [...] Read more.
The aim of this study was to investigate the effects of aluminum (Al) or selenium (Se) on the “primary” antioxidant defense system enzymes (superoxide dismutase, catalase, and glutathione reductase) in cells of mouse brain and liver after long-term (8-week) exposure to drinking water supplemented with AlCl3 (50 mg or 100 mg Al/L in drinking water) or Na2SeO3 (0.2 mg or 0.4 mg Se/L in drinking water). Results have shown that a high dose of Se increased the activities of superoxide dismutase and catalase in mouse brain and liver. Exposure to a low dose of Se resulted in an increase in catalase activity in mouse brain, but did not show any statistically significant changes in superoxide dismutase activity in both organs. Meanwhile, the administration of both doses of Al caused no changes in activities of these enzymes in mouse brain and liver. The greatest sensitivity to the effect of Al or Se was exhibited by glutathione reductase. Exposure to both doses of Al or Se resulted in statistically significant increase in glutathione reductase activity in both brain and liver. It was concluded that 8-week exposure to Se caused a statistically significant increase in superoxide dismutase, catalase and glutathione reductase activities in mouse brain and/or liver, however, these changes were dependent on the used dose. The exposure to both Al doses caused a statistically significant increase only in glutathione reductase activity of both organs. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
Open AccessCommunication
Enantioselective Epoxidation by Flavoprotein Monooxygenases Supported by Organic Solvents
Catalysts 2020, 10(5), 568; https://doi.org/10.3390/catal10050568 - 19 May 2020
Cited by 2 | Viewed by 1272
Abstract
Styrene and indole monooxygenases (SMO and IMO) are two-component flavoprotein monooxygenases composed of a nicotinamide adenine dinucleotide (NADH)-dependent flavin adenine dinucleotide (FAD)-reductase (StyB or IndB) and a monooxygenase (StyA or IndA). The latter uses reduced FAD to activate oxygen and to oxygenate the [...] Read more.
Styrene and indole monooxygenases (SMO and IMO) are two-component flavoprotein monooxygenases composed of a nicotinamide adenine dinucleotide (NADH)-dependent flavin adenine dinucleotide (FAD)-reductase (StyB or IndB) and a monooxygenase (StyA or IndA). The latter uses reduced FAD to activate oxygen and to oxygenate the substrate while releasing water. We circumvented the need for the reductase by direct FAD reduction in solution using the NAD(P)H-mimic 1-benzyl-1,4-dihydronicotinamide (BNAH) to fuel monooxygenases without NADH requirement. Herein, we report on the hitherto unknown solvent tolerance for the indole monooxygenase from Gemmobacter nectariphilus DSM15620 (GnIndA) and the styrene monooxygenase from Gordonia rubripertincta CWB2 (GrStyA). These enzymes were shown to convert bulky and rather hydrophobic styrene derivatives in the presence of organic cosolvents. Subsequently, BNAH-driven biotransformation was furthermore optimized with regard to the applied cosolvent and its concentration as well as FAD and BNAH concentration. We herein demonstrate that GnIndA and GrStyA enable selective epoxidations of allylic double bonds (up to 217 mU mg−1) in the presence of organic solvents such as tetrahydrofuran, acetonitrile, or several alcohols. Notably, GnIndA was found to resist methanol concentrations up to 25 vol.%. Furthermore, a diverse substrate preference was determined for both enzymes, making their distinct use very interesting. In general, our results seem representative for many IMOs as was corroborated by in silico mutagenetic studies. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessFeature PaperArticle
Synthesis of DHA/EPA Ethyl Esters via Lipase-Catalyzed Acidolysis Using Novozym® 435: A Kinetic Study
Catalysts 2020, 10(5), 565; https://doi.org/10.3390/catal10050565 - 19 May 2020
Cited by 4 | Viewed by 943
Abstract
DHA/EPA ethyl ester is mainly used in the treatment of arteriosclerosis and hyperlipidemia. In this study, DHA+EPA ethyl ester was synthesized via lipase-catalyzed acidolysis of ethyl acetate (EA) with DHA+EPA concentrate in n-hexane using Novozym® 435. The DHA+EPA concentrate (in free [...] Read more.
DHA/EPA ethyl ester is mainly used in the treatment of arteriosclerosis and hyperlipidemia. In this study, DHA+EPA ethyl ester was synthesized via lipase-catalyzed acidolysis of ethyl acetate (EA) with DHA+EPA concentrate in n-hexane using Novozym® 435. The DHA+EPA concentrate (in free fatty acid form), contained 54.4% DHA and 16.8% EPA, was used as raw material. A central composite design combined with response surface methodology (RSM) was used to evaluate the relationship between substrate concentrations and initial rate of DHA+EPA ethyl ester production. The results indicated that the reaction followed the ordered mechanism and as such, the ordered mechanism model was used to estimate the maximum reaction rate (Vmax) and kinetic constants. The ordered mechanism model was also combined with the batch reaction equation to simulate and predict the conversion of DHA+EPA ethyl ester in lipase-catalyzed acidolysis. The integral equation showed a good predictive relationship between the simulated and experimental results. 88–94% conversion yields were obtained from 100–400 mM DHA+EPA concentrate at a constant enzyme activity of 200 U, substrate ratio of 1:1 (DHA+EPA: EA), and reaction time of 300 min. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Biocatalysis of Industrial Kraft Pulps: Similarities and Differences between Hardwood and Softwood Pulps in Hydrolysis by Enzyme Complex of Penicillium verruculosum
Catalysts 2020, 10(5), 536; https://doi.org/10.3390/catal10050536 - 13 May 2020
Cited by 4 | Viewed by 809
Abstract
Kraft pulp enzymatic hydrolysis is a promising method of woody biomass bioconversion. The influence of composition and structure of kraft fibers on their hydrolysis efficiency was evaluated while using four substrates, unbleached hardwood pulp (UHP), unbleached softwood pulp (USP), bleached hardwood pulp (BHP), [...] Read more.
Kraft pulp enzymatic hydrolysis is a promising method of woody biomass bioconversion. The influence of composition and structure of kraft fibers on their hydrolysis efficiency was evaluated while using four substrates, unbleached hardwood pulp (UHP), unbleached softwood pulp (USP), bleached hardwood pulp (BHP), and bleached softwood pulp (BSP). Hydrolysis was carried out with Penicillium verruculosum enzyme complex at a dosage of 10 filter paper units (FPU)/g pulp. The changes in fiber morphology and structure were visualized while using optical and electron microscopy. Fiber cutting and swelling and quick xylan destruction were the main processes at the beginning of hydrolysis. The negative effect of lignin content was more pronounced for USP. Drying decreased the sugar yield of dissolved hydrolysis products for all kraft pulps. Fiber morphology, different xylan and mannan content, and hemicelluloses localization in kraft fibers deeply affected the hydrolyzability of bleached pulps. The introduction of additional xylobiase, mannanase, and cellobiohydrolase activities to enzyme mixture will further improve the hydrolysis of bleached pulps. A high efficiency of never-dried bleached pulp bioconversion was shown. At 10% substrate concentration, hydrolysates with more than 50 g/L sugar concentration were obtained. The bioconversion of never-dried BHP and BSP could be integrated into working kraft pulp mills. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessCommunication
Continuous-Flow Synthesis of β-Amino Acid Esters by Lipase-Catalyzed Michael Addition of Aromatic Amines
Catalysts 2020, 10(4), 432; https://doi.org/10.3390/catal10040432 - 16 Apr 2020
Cited by 2 | Viewed by 722
Abstract
A continuous-flow procedure for the synthesis of β-amino acid esters has been developed via lipase-catalyzed Michael reaction of various aromatic amines with acrylates. Lipase TL IM from Thermomyces lanuginosus was first used to catalyze Michael addition reaction of aromatic amines. Compared with other [...] Read more.
A continuous-flow procedure for the synthesis of β-amino acid esters has been developed via lipase-catalyzed Michael reaction of various aromatic amines with acrylates. Lipase TL IM from Thermomyces lanuginosus was first used to catalyze Michael addition reaction of aromatic amines. Compared with other methods, the salient features of this work include green reaction conditions (methanol as reaction medium), short residence time (30 min), readily available catalyst and a reaction process that is easy to control. This enzymatic synthesis of β-amino acid esters performed in continuous-flow microreactors is an innovation that provides a new strategy for the fast biotransformation of β-amino acid esters. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Shaking Rate during Production Affects the Activity of Escherichia coli Surface-Displayed Candida antarctica Lipase A
Catalysts 2020, 10(4), 382; https://doi.org/10.3390/catal10040382 - 01 Apr 2020
Cited by 2 | Viewed by 587
Abstract
In this study, a surface-display system was applied for the expression of lipase A in an E. coli expression system. Since the target protein was exposed on the cell membrane, the shaking rate during culturing might have increased the oxygen mass transfer rate [...] Read more.
In this study, a surface-display system was applied for the expression of lipase A in an E. coli expression system. Since the target protein was exposed on the cell membrane, the shaking rate during culturing might have increased the oxygen mass transfer rate and the shear stress, both of which would be detrimental to the surface-displayed protein. The shaking rate did indeed have an effect on the properties of the surface-displayed lipase A from Candida antarctica (sdCALA). When cultivated at a shaking rate of less than 50 rpm, the specific activity of sdCALA was low, which was due to the limited amount of dissolved oxygen. When the shaking rate was greater than 100 rpm, the specific activity decreased as a result of shear stress. When cultivating CALA and sdCALA at various temperatures and values of pH, both proteins displayed the same activity profile, with the optimum conditions being 60 °C and pH 6. A kinetic study revealed that the sdCALA cultivated at 100 rpm gave a higher value of νm (0.074 μmol/mL/min) and a lower value of Km (0.360 μmol/mL) relative to those obtained at 200 rpm and relative to those of the free CALA. sdCALA retained over 80% of its activity after treatment at 70 °C for 30 min, but its activity decreased rapidly when the temperature was above 80 °C. The specific activity of sdCALA decreased in the presence of acetonitrile and acetone relative to that of the control (50% ethanol), regardless of the solvent concentration. The highest activity (0.67 U/mL) was obtained when the ethanol concentration was 30%. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System
Catalysts 2020, 10(2), 258; https://doi.org/10.3390/catal10020258 - 20 Feb 2020
Cited by 4 | Viewed by 855
Abstract
Calycosin-7-O-β-D-glucoside (Cy7G) is one of the principal components of Radix astragali. This isoflavonoid glucoside is regarded as an indicator to assess the quality of R. astragali and exhibits diverse pharmacological activities. In this study, uridine diphosphate-dependent glucosyltransferase (UGT) UGT88E18 was [...] Read more.
Calycosin-7-O-β-D-glucoside (Cy7G) is one of the principal components of Radix astragali. This isoflavonoid glucoside is regarded as an indicator to assess the quality of R. astragali and exhibits diverse pharmacological activities. In this study, uridine diphosphate-dependent glucosyltransferase (UGT) UGT88E18 was isolated from Glycine max and expressed in Escherichia coli. Recombinant UGT88E18 could selectively and effectively glucosylate the C7 hydroxyl group of calycosin to synthesize Cy7G. A one-pot reaction by coupling UGT88E18 to sucrose synthase (SuSy) from G. max was developed. The UGT88E18–SuSy cascade reaction could recycle the costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The important factors for UGT88E18–SuSy cascade reaction, including UGT88E18/SuSy ratios, different temperatures, and pH values, different concentrations of dimethyl sulfoxide (DMSO), UDP, sucrose, and calycosin, were optimized. We produced 10.5 g L−1 Cy7G in the optimal reaction conditions by the stepwise addition of calycosin. The molar conversion of calycosin was 97.5%, with a space–time yield of 747 mg L−1 h−1 and a UDPG recycle of 78 times. The present study provides a new avenue for the efficient and cost-effective semisynthesis of Cy7G and other valuable isoflavonoid glucosides by UGT–SuSy cascade reaction. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
A Novel Route for Agarooligosaccharide Production with the Neoagarooligosaccharide-Producing β-Agarase as Catalyst
Catalysts 2020, 10(2), 214; https://doi.org/10.3390/catal10020214 - 10 Feb 2020
Cited by 2 | Viewed by 708
Abstract
Enzymes are catalysts with high specificity. Different compounds could be produced by different enzymes. In case of agaro-oligosaccharides, agarooligosaccharide (AOS) can be produced by α-agarase through cleaving the α-1,3-glycosidic linkages of agarose, while neoagarooligosaccharide (NAOS) can be produced by β-agarase through cleaving the [...] Read more.
Enzymes are catalysts with high specificity. Different compounds could be produced by different enzymes. In case of agaro-oligosaccharides, agarooligosaccharide (AOS) can be produced by α-agarase through cleaving the α-1,3-glycosidic linkages of agarose, while neoagarooligosaccharide (NAOS) can be produced by β-agarase through cleaving the β-1,4-glycosidic linkages of agarose. However, in this study, we showed that β-agarase could also be used to produce AOSs with high purity and yield. The feasibility of our route was confirmed by agarotriose (A3) and agaropentaose (A5) formation from agaroheptaose (A7) and agarononoses (A9) catalyzed by β-agarase. Agarose was firstly liquesced by citric acid into a mixture of AOSs. The AOSs mixture was further catalyzed by β-agarase. When using the neoagarotetraose-forming β-agarase AgWH50B, agarotriose could be produced with the yield of 48%. When using neoagarotetraose, neoagarohexaose-forming β-agarase DagA, both agarotriose and agaropentaose could be produced with the yield of 14% and 13%, respectively. Our method can be used to produce other value-added agaro-oligosaccharides from agarose by different agarolytic enzymes. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Characterization of Protein Hydrolysates from Eel (Anguilla marmorata) and Their Application in Herbal Eel Extracts
Catalysts 2020, 10(2), 205; https://doi.org/10.3390/catal10020205 - 08 Feb 2020
Cited by 3 | Viewed by 810
Abstract
The enzymatic hydrolysis of fish proteins is the principle method for converting under-utilized fish into valuable products for the pharmaceutical and health food industries. In this study, three commercial enzymes (alcalase, bromelain, and papain) were tested for their ability to create eel protein [...] Read more.
The enzymatic hydrolysis of fish proteins is the principle method for converting under-utilized fish into valuable products for the pharmaceutical and health food industries. In this study, three commercial enzymes (alcalase, bromelain, and papain) were tested for their ability to create eel protein hydrolysates (EPHs) from whole eel (Anguilla marmorata). Freeze-dried EPHs had almost more than 80% solubility (p < 0.05) in solutions ranging from pH 2–10. The amino acid profiles of the EPHs showed a high percentage of essential amino acids, including histidine, threonine, valine, isoleucine, and leucine. The emulsion activity index (EAI) of EPH resulted as follows: alcalase group (36.8 ± 2.00) > bromelain group (21.3 ± 1.30) > papain group (16.2 ± 1.22), and the emulsion stability index (ESI) of EPH was: alcalase group (4.00 ± 0.34) > bromelain group (2.62 ± 0.44) > papain group (1.44 ± 0.09). As such, EPH has a high nutritional value and could be used as a supplement to diets lacking protein. EPH showed excellent solubility and processed interfacial properties, which are governed by its concentration. Among of them the alcalase group had the best antioxidant effect at 1,1-diphenyl-2-pyridinohydrazinyl (DPPH) radical method, determination of reducing power and ABTS test compared with other groups. EPH may be useful in developing commercial products like herbal eel extracts that are beneficial to human health. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
DERA in Flow: Synthesis of a Statin Side Chain Precursor in Continuous Flow Employing Deoxyribose-5-Phosphate Aldolase Immobilized in Alginate-Luffa Matrix
Catalysts 2020, 10(1), 137; https://doi.org/10.3390/catal10010137 - 18 Jan 2020
Cited by 2 | Viewed by 994
Abstract
Statins, cholesterol-lowering drugs used for the treatment of coronary artery disease (CAD), are among the top 10 prescribed drugs worldwide. However, the synthesis of their characteristic side chain containing two chiral hydroxyl groups can be challenging. The application of deoxyribose-5-phosphate aldolase (DERA) is [...] Read more.
Statins, cholesterol-lowering drugs used for the treatment of coronary artery disease (CAD), are among the top 10 prescribed drugs worldwide. However, the synthesis of their characteristic side chain containing two chiral hydroxyl groups can be challenging. The application of deoxyribose-5-phosphate aldolase (DERA) is currently one of the most promising routes for the synthesis of this side chain. Herein, we describe the development of a continuous flow process for the biosynthesis of a side chain precursor. Design of experiments (DoE) was used to optimize the reaction conditions (pH value and temperature) in batch. A pH of 7.5 and a temperature of 32.5 °C were identified to be the optimal process settings within the reaction space considered. Additionally, an immobilization method was developed using the alginate-luffa matrix (ALM), which is a fast, simple, and inexpensive method for enzyme immobilization. Furthermore, it is non-toxic, biodegradable, and from renewable resources. The final continuous process was operated stable for 4 h and can produce up to 4.5 g of product per day. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Open AccessArticle
Simultaneous Optimal Production of Flavonol Aglycones and Degalloylated Catechins from Green Tea Using a Multi-Function Food-Grade Enzyme
Catalysts 2019, 9(10), 861; https://doi.org/10.3390/catal9100861 - 16 Oct 2019
Cited by 2 | Viewed by 897
Abstract
(1) Background: Green tea (GT) contains well-known phytochemical compounds; namely, it is rich in flavan-3-ols (catechins) and flavonols comprising all glycoside forms. These compounds in GT might show better biological activities after a feasible enzymatic process, and the process on an industrial scale [...] Read more.
(1) Background: Green tea (GT) contains well-known phytochemical compounds; namely, it is rich in flavan-3-ols (catechins) and flavonols comprising all glycoside forms. These compounds in GT might show better biological activities after a feasible enzymatic process, and the process on an industrial scale should consider enzyme specificity and cost-effectiveness. (2) Methods: In this study, we evaluated the most effective method for the enzymatic conversion of flavonoids from GT extract. One enzyme derived from Aspergillus niger (molecular weight 80–90 kDa) was ultimately selected, showing two distinct but simultaneous activities: intense glycoside hydrolase activity via deglycosylation and weak tannin acyl hydrolase activity via degalloylation. (3) Results: The optimum conditions for producing flavonol aglycones were pH 4.0 and 50 °C. Myricetin glycosides were cleaved 3.7–7.0 times faster than kaempferol glycosides. Flavonol aglycones were produced effectively by both enzymatic and hydrochloride treatment in a time-course reaction. Enzymatic treatment retained 80% (w/w) catechins, whereas 70% (w/w) of catechins disappeared by hydrochloride treatment. (4) Conclusions: This enzymatic process offers an effective method of conditionally producing flavonol aglycones and de-galloylated catechins from conversion of food-grade enzyme. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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Review

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Open AccessReview
Kramers’ Theory and the Dependence of Enzyme Dynamics on Trehalose-Mediated Viscosity
Catalysts 2020, 10(6), 659; https://doi.org/10.3390/catal10060659 - 11 Jun 2020
Cited by 1 | Viewed by 687
Abstract
The disaccharide trehalose is accumulated in the cytoplasm of some organisms in response to harsh environmental conditions. Trehalose biosynthesis and accumulation are important for the survival of such organisms by protecting the structure and function of proteins and membranes. Trehalose affects the dynamics [...] Read more.
The disaccharide trehalose is accumulated in the cytoplasm of some organisms in response to harsh environmental conditions. Trehalose biosynthesis and accumulation are important for the survival of such organisms by protecting the structure and function of proteins and membranes. Trehalose affects the dynamics of proteins and water molecules in the bulk and the protein hydration shell. Enzyme catalysis and other processes dependent on protein dynamics are affected by the viscosity generated by trehalose, as described by the Kramers’ theory of rate reactions. Enzyme/protein stabilization by trehalose against thermal inactivation/unfolding is also explained by the viscosity mediated hindering of the thermally generated structural dynamics, as described by Kramers’ theory. The analysis of the relationship of viscosity–protein dynamics, and its effects on enzyme/protein function and other processes (thermal inactivation and unfolding/folding), is the focus of the present work regarding the disaccharide trehalose as the viscosity generating solute. Finally, trehalose is widely used (alone or in combination with other compounds) in the stabilization of enzymes in the laboratory and in biotechnological applications; hence, considering the effect of viscosity on catalysis and stability of enzymes may help to improve the results of trehalose in its diverse uses/applications. Full article
(This article belongs to the Special Issue Biocatalytic Process Optimization)
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