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1.
Bioresour Bioprocess ; 11(1): 9, 2024 Jan 16.
Article in English | MEDLINE | ID: mdl-38647973

ABSTRACT

The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min-1 mg-1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg2+, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L-1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L-1 h-1 and a specific productivity of 357.6 mg mg-enzyme-1 h-1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L-1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L-1 h-1 and 583.4 mg mg-enzyme-1 h-1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde.

2.
Chembiochem ; 24(23): e202300556, 2023 12 01.
Article in English | MEDLINE | ID: mdl-37749055

ABSTRACT

A lipoxygenase from Pleurotus sajor-caju (PsLOX) was cloned, expressed in Escherichia coli, and purified as a soluble protein with a specific activity of 629 µmol/min/mg for arachidonic acid (AA). The native PsLOX exhibited a molecular mass of 146 kDa, including a 73-kDa homodimer, as estimated by gel-filtration chromatography. The major products converted from polyunsaturated fatty acids (PUFAs), including AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were identified as trioxilins (TrXs), namely 13,14,15-TrXB3 , 13,14,15-TrXB4 , and 15,16,17-TrXB5 , respectively, through high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The enzyme displayed its maximum activity at pH 8.0 and 20 °C. Under these conditions, the specific activity and catalytic efficiency of PsLOX for PUFAs exhibited the following order: AA>EPA>DHA. Based on HPLC analysis and substrate specificity, PsLOX was identified as an arachidonate 15-LOX. PsLOX efficiently converted 10 mM of AA, EPA, and DHA to 8.7 mM of 13,14,15-TrXB3 (conversion rate: 87 %), 7.9 mM of 13,14,15-TrXB4 (79 %), and 7.2 mM of 15,16,17-TrXB5 (72 %) in 15, 20, and 20 min, respectively, marking the highest conversion rates reported to date. Collectively, our results demonstrate that PsLOX is an efficient TrXs-producing enzyme.


Subject(s)
Lipoxygenase , Tandem Mass Spectrometry , Lipoxygenase/metabolism , Chromatography, Liquid , Fatty Acids, Unsaturated , Biotransformation , Docosahexaenoic Acids/metabolism
3.
J Agric Food Chem ; 71(10): 4328-4336, 2023 Mar 15.
Article in English | MEDLINE | ID: mdl-36856566

ABSTRACT

One-carbon chemicals (C 1s) are potential building blocks as they are cheap, sustainable, and abiotic components. Methanol-derived formaldehyde can be another versatile building block for the production of 2-keto-4-hydroxyacid derivatives that can be used for amino acids, hydroxy carboxylic acids, and chiral aldehydes. To produce 2-keto-4-hydroxybutyrate from C 1s in an environment-friendly way, we characterized an aldolase from Pseudomonas aeruginosa PAO1 (PaADL), which showed much higher catalytic activity in condensing formaldehyde and pyruvate than the reported aldolases. By applying a structure-based rational approach, we found a variant (PaADLV121A/L241A) that exhibited better catalytic activities than the wild-type enzyme. Next, we constructed a one-pot cascade biocatalyst system by combining PaADL and a methanol dehydrogenase (MDH) and, for the first time, effectively produced 2-keto-4-hydroxybutyrate as the main product from pyruvate and methanol via an enzymatic reaction. This simple process applied here will help design a green process for the production of 2-keto-4-hydroxyacid derivatives.


Subject(s)
Fructose-Bisphosphate Aldolase , Pyruvic Acid , Fructose-Bisphosphate Aldolase/metabolism , Pyruvic Acid/metabolism , Methanol/metabolism , Aldehyde-Lyases/chemistry , Formaldehyde
4.
Antioxidants (Basel) ; 11(6)2022 Jun 16.
Article in English | MEDLINE | ID: mdl-35740077

ABSTRACT

ß-Carotene 15,15'-oxygenase (BCO1) and ß-carotene 9',10'-oxygenase (BCO2) are potential producers of vitamin A derivatives, since they can catalyze the oxidative cleavage of dietary provitamin A carotenoids to retinoids and derivative such as apocarotenal. Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it, and are essential nutrients for humans and highly valuable in the food and cosmetics industries. ß-carotene oxygenases (BCOs) from various organisms have been overexpressed in heterogeneous bacteria, such as Escherichia coli, and their biochemical properties have been studied. For the industrial production of retinal, there is a need for increased production of a retinal producer and biosynthesis of retinal using biocatalyst systems improved by enzyme engineering. The current review aims to discuss BCOs from animal, plants, and bacteria, and to elaborate on the recent progress in our understanding of their functions, biochemical properties, substrate specificity, and enzyme activities with respect to the production of retinoids in whole-cell conditions. Moreover, we specifically propose ways to integrate BCOs into retinal biosynthetic bacterial systems to improve the performance of retinal production.

5.
Plants (Basel) ; 11(9)2022 May 05.
Article in English | MEDLINE | ID: mdl-35567244

ABSTRACT

Cytosolic lipid droplets (LDs) derived from the endoplasmic reticulum (ER) mainly contain neutral lipids, such as triacylglycerols (TAGs) and sterol esters, which are considered energy reserves. The metabolic pathways associated with LDs in eukaryotic species are involved in diverse cellular functions. TAG synthesis in plants is mediated by the sequential involvement of two subcellular organelles, i.e., plastids - plant-specific organelles, which serve as the site of lipid synthesis, and the ER. TAGs and sterol esters synthesized in the ER are sequestered to form LDs through the cooperative action of several proteins, such as SEIPINs, LD-associated proteins, LDAP-interacting proteins, and plant-specific proteins such as oleosins. The integrity and stability of LDs are highly dependent on oleosins, especially in the seeds, and oleosin degradation is critical for efficient mobilization of the TAGs of plant LDs. As the TAGs mobilize in LDs during germination and post-germinative growth, a plant-specific lipase-sugar-dependent 1 (SDP1)-plays a major role, through the inter-organellar communication between the ER and peroxisomes. In this review, we briefly recapitulate the different processes involved in the biogenesis and degradation of plant LDs, followed by a discussion of future perspectives in this field.

6.
Int J Mol Sci ; 23(7)2022 Apr 03.
Article in English | MEDLINE | ID: mdl-35409349

ABSTRACT

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDHLx) and DERATma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERATma and MDHLx.


Subject(s)
Escherichia coli , Methanol , Acetaldehyde , Escherichia coli/genetics , Ethanol , Formaldehyde , Methanol/chemistry
7.
J Agric Food Chem ; 70(4): 1203-1211, 2022 Feb 02.
Article in English | MEDLINE | ID: mdl-34994555

ABSTRACT

Enantiomerically pure d-amino acids are important intermediates as chiral building blocks for peptidomimetics and semisynthetic antibiotics. Here, a transcriptional factor-based screening strategy was used for the rapid screening of d-stereospecific amino acid amidase via an enzyme-specific amidophenol substrate. We used a d-threonine amidophenyl derivative to produce 2-aminophenol that serves as a putative enzyme indicator in the presence of d-threonine amidases. Comparative analyses of known bacterial species indicated that several Bacillus strains produce amidase and form putative indicators in culture media. The estimated amidase was cloned and subjected to rapid directed evolution through biosensor cells. Consequently, we characterized the F119A mutation that significantly improved the catalytic activity toward d-alanine, d-threonine, and d-glutamate. Its beneficial effects were confirmed by higher conversions and recurrent applications of the mutant enzyme, compared to the wild-type. This study showed that rapid directed evolution with biosensors coupled to designed substrates is useful to develop biocatalytic processes.


Subject(s)
Bacillus , Biosensing Techniques , Amidohydrolases/genetics , Amidohydrolases/metabolism , Amino Acids , Bacillus/genetics , Bacillus/metabolism , Mutation , Substrate Specificity
8.
Trends Biotechnol ; 40(3): 306-319, 2022 03.
Article in English | MEDLINE | ID: mdl-34462144

ABSTRACT

Aldol chemicals are synthesized by condensation reactions between the carbon units of ketones and aldehydes using aldolases. The efficient synthesis of diverse organic chemicals requires intrinsic modification of aldolases via engineering and design, as well as extrinsic modification through immobilization or combination with other catalysts. This review describes the development of aldolases, including their engineering and design, and the selection of desired aldolases using high-throughput screening, to enhance their catalytic properties and perform novel reactions. Aldolase-containing catalysts, which catalyze the aldol reaction combined with other enzymatic and/or chemical reactions, can efficiently synthesize diverse complex organic chemicals using inexpensive and simple materials as substrates. We also discuss the current challenges and emerging solutions for aldolase-based catalysts.


Subject(s)
Aldehyde-Lyases , Fructose-Bisphosphate Aldolase , Aldehyde-Lyases/chemistry , Catalysis , Substrate Specificity
9.
Trends Biotechnol ; 40(2): 166-179, 2022 02.
Article in English | MEDLINE | ID: mdl-34243985

ABSTRACT

Plastic contamination currently threatens a wide variety of ecosystems and presents damaging repercussions and negative consequences for many wildlife species. Sustainable plastic waste management is an important approach to environmental protection and a necessity in the current life cycle of plastics in nature. Plastic biodegradation by microorganisms is a notable possible solution. This opinion article includes a proposal to use hypothetical P450 enzymes with an engineered active site as potent trigger biocatalysts to biodegrade polyethylene (PE) via in-chain hydroxylation into smaller products of linear aliphatic alcohols and alkanoic acids based on cascade enzymatic reactions. Furthermore, we propose the adoption of P450 into plastic-eating synthetic bacteria for PE biodegradation. This strategy can be applicable to other dense plastics, such as polypropylene (PP) and polystyrene (PS).


Subject(s)
Ecosystem , Plastics , Bacteria/metabolism , Biodegradation, Environmental , Cytochrome P-450 Enzyme System/metabolism , Plastics/metabolism
10.
J Hazard Mater ; 416: 126239, 2021 08 15.
Article in English | MEDLINE | ID: mdl-34492990

ABSTRACT

Polystyrene (PS), a major plastic waste, is difficult to biodegrade due to its unique chemical structure that comprises phenyl moieties attached to long linear alkanes. In this study, we investigated the biodegradation of PS by mesophilic bacterial cultures obtained from various soils in common environments. Two new strains, Pseudomonas lini JNU01 and Acinetobacter johnsonii JNU01, were specifically enriched in non-carbonaceous nutrient medium, with PS as the only source of carbon. Their growth after culturing in basal media increased more than 3-fold in the presence of PS. Fourier transform infrared spectroscopy analysis, used to confirm the formation of hydroxyl groups and potentially additional chemical bond groups, showed an increase in the amount of oxidized PS samples. Moreover, field emission scanning electron microcopy analysis confirmed PS biodegradation by biofilms of the screened microbes. Water contact angle measurement additionally offered insights into the increased hydrophilic characteristics of PS films. Bioinformatics and transcriptional analysis of A. johnsonii JNU01 revealed alkane-1-monooxygenase (AlkB) to be involved in PS biodegradation, which was confirmed by the hydroxylation of PS using recombinant AlkB. These results provide significant insights into the discovery of novel functions of Pseudomonas sp. and Acinetobacter sp., as well as their potential as PS decomposers.


Subject(s)
Polystyrenes , Soil , Acinetobacter , Bacteria , Biodegradation, Environmental , Pseudomonas
11.
Front Bioeng Biotechnol ; 9: 681253, 2021.
Article in English | MEDLINE | ID: mdl-34336800

ABSTRACT

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

12.
J Agric Food Chem ; 69(30): 8492-8503, 2021 Aug 04.
Article in English | MEDLINE | ID: mdl-34282904

ABSTRACT

(-)-α-Bisabolol is a functional ingredient in various health and cosmetic products and has antibacterial, anti-inflammatory, and wound healing properties. (-)-α-Bisabolol is chemically synthesized and produced by steam distillation of essential oils extracted from Brazilian Candeia (Eremanthus erythropappus). To sustainably produce pure (-)-α-bisabolol, we previously engineered Escherichia coli to produce 9.1 g/L (-)-α-bisabolol via heterologous mevalonate pathways and (-)-α-bisabolol synthase (BOS) from German chamomile, Matricaria recutita (MrBOS). BOS has only been reported in MrBOS and Brazilian Candeia (EeBOS). The limited availability of BOS has made it difficult to achieve high titer and yield and large-scale (-)-α-bisabolol production. We identified a novel BOS in globe artichoke (CcBOS) and examined its functionality in vitro and in vivo. CcBOS showed higher catalytic efficiency and (-)-α-bisabolol production rates than those from MrBOS or EeBOS. In fed-batch fermentation, CcBOS generated the highest reported (-)-α-bisabolol titer to date (23.4 g/L). These results may facilitate economically viable industrial (-)-α-bisabolol production.


Subject(s)
Cynara scolymus , Cynara , Scolymus , Sesquiterpenes , Brazil , Cynara scolymus/genetics , Escherichia coli/genetics , Monocyclic Sesquiterpenes
13.
ChemSusChem ; 14(15): 3030, 2021 Aug 09.
Article in English | MEDLINE | ID: mdl-34272832

ABSTRACT

Invited for this month's cover is the joint research group of Prof. Chan Beum Park at the Korea Advanced Institute of Science and Technology (KAIST) and Prof. Chul-Ho Yun at the Chonnam National University (CNU). The image shows how the use of a natural photosensitizer, flavin mononucleotide, and visible light can lead to a cost-effective, green, and sustainable process for P450-catalyzed reactions in a whole-cell system. The Communication itself is available at 10.1002/cssc.202100944.


Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Flavin Mononucleotide/chemistry , Photosensitizing Agents/chemistry , Catalysis , Chlorzoxazone/chemistry , Escherichia coli/metabolism , Hydroxylation , Light , Nitrophenols/chemistry , Oxidation-Reduction , Photosynthesis , Solar Energy
14.
ChemSusChem ; 14(15): 3054-3058, 2021 Aug 09.
Article in English | MEDLINE | ID: mdl-34085413

ABSTRACT

Photobiocatalysis is a green platform for driving redox enzymatic reactions using solar energy, not needing high-cost cofactors and redox partners. Here, a visible light-driven whole-cell platform for human cytochrome P450 (CYP) photobiocatalysis was developed using natural flavins as a photosensitizer. Photoexcited flavins mediate NADPH/reductase-free, light-driven biocatalysis by human CYP2E1 both in vitro and in the whole-cell systems. In vitro tests demonstrated that the photobiocatalytic activity of CYP2E1 is dependent on the substrate type, the presence of catalase, and the acid type used as a sacificial electron donor. A protective effect of catalase was found against the inactivation of CYP2E1 heme by H2 O2 and the direct transfer of photo-induced electrons to the heme iron not by peroxide shunt. Furthermore, the P450 photobiocatalysis in whole cells containing human CYPs 1A1, 1A2, 1B1, and 3A4 demonstrated the general applicability of the solar-powered, flavin-mediated P450 photobiocatalytic system.

15.
Int J Mol Sci ; 22(3)2021 Feb 02.
Article in English | MEDLINE | ID: mdl-33540582

ABSTRACT

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.


Subject(s)
Alcohol Oxidoreductases/genetics , Bacillaceae/enzymology , Mutation , Alcohol Oxidoreductases/metabolism , Bacillaceae/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biosensing Techniques , Kinetics , Methanol/metabolism
16.
Front Bioeng Biotechnol ; 9: 787791, 2021.
Article in English | MEDLINE | ID: mdl-35004648

ABSTRACT

One-carbon (C1) chemicals are potential building blocks for cheap and sustainable re-sources such as methane, methanol, formaldehyde, formate, carbon monoxide, and more. These resources have the potential to be made into raw materials for various products used in our daily life or precursors for pharmaceuticals through biological and chemical processes. Among the soluble C1 substrates, methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Synthetic methylotrophy can be exploited to produce fuels and chemicals using methanol as a feedstock that integrates natural or artificial methanol assimilation pathways in platform microorganisms. In the methanol utilization in methylotrophy, methanol dehydrogenase (Mdh) is a primary enzyme that converts methanol to formaldehyde. The discovery of new Mdhs and engineering of present Mdhs have been attempted to develop synthetic methylotrophic bacteria. In this review, we describe Mdhs, including in terms of their enzyme properties and engineering for desired activity. In addition, we specifically focus on the application of various Mdhs for synthetic methylotrophy.

17.
Pharmaceuticals (Basel) ; 13(11)2020 Oct 22.
Article in English | MEDLINE | ID: mdl-33105851

ABSTRACT

Phloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min-1, respectively. However, CYP3A4 has a 3.3-fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h-1 productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6ß-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.

18.
J Biomed Nanotechnol ; 16(3): 304-314, 2020 Mar 01.
Article in English | MEDLINE | ID: mdl-32493541

ABSTRACT

We propose that nanogels (HLGs) prepared by simply blending an epidermal growth factor (EGF)-loaded hyaluronan (HA)-based nanoformulation and poloxamers can be efficient transdermal drug carriers. In particular, due to the thermogelling behavior of poloxamer, when the HLGs, which are liquid at room temperature, are applied to the skin's surface, they form a gel at skin temperature. First, lipid-based nanoformulations (EGF-LNs) were fabricated by the lipid thin film method and then chemically conjugated with HA on the surface of the films to prepare EGF-loaded HA-based nanoformulations (EGF-HLNs). Both EGF-LNs and EGF-HLNs exhibited a uniform size and spherical lamellar structure. The EGF-HLN was added to a poloxamer solution to form EGF-HLG, which is a liquid at room temperature and a gel at skin temperature. HLGs have been shown to be able to deliver and permeate EGF well into the skin using both in vitro and in vivo systems, thus serving as an effective transdermal delivery system. In addition, it has been confirmed that this system could be a possible implantable drug carrier. Therefore, HLGs, which are uncomplicated and easily prepared, are expected to be easily used not only in the pharmaceutical field but also in the cosmetic field.


Subject(s)
Nanogels , Wound Healing , Administration, Cutaneous , Drug Carriers , Epidermal Growth Factor , Skin
19.
J Microbiol ; 58(9): 725-733, 2020 Sep.
Article in English | MEDLINE | ID: mdl-32583284

ABSTRACT

Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.


Subject(s)
Aldose-Ketose Isomerases/metabolism , Bacteria/metabolism , Glucose/biosynthesis , Mannose-6-Phosphate Isomerase/metabolism , Ribose/biosynthesis , Hexoses/metabolism , Pentoses/metabolism , Substrate Specificity , Sweetening Agents/chemistry
20.
J Agric Food Chem ; 68(24): 6683-6691, 2020 Jun 17.
Article in English | MEDLINE | ID: mdl-32468814

ABSTRACT

In this study, we investigated an efficient enzymatic strategy for producing potentially valuable phloretin metabolites from phlorizin, a glucoside of phloretin that is rich in apple pomace. Almond ß-glucosidase efficiently removed phlorizin's glucose moiety to produce phloretin. CYP102A1 engineered by site-directed mutagenesis, domain swapping, and random mutagenesis catalyzed the highly regioselective C-hydroxylation of phloretin into 3-OH phloretin with high conversion yields. Under the optimal hydroxylation conditions of 15 g cells L-1 and a 20 mM substrate for whole-cell biocatalysis, phloretin was regioselectively hydroxylated into 3.1 mM 3-OH phloretin each hour. Furthermore, differentiation of 3T3-L1 preadipocytes into adipocytes and lipid accumulation were dramatically inhibited by 3-OH phloretin but promoted by phloretin. Consistent with these inhibitory effects, the expression of adipogenic regulator genes was downregulated by 3-OH phloretin. We propose a platform for the sustainable production and value creation of phloretin metabolites from apple pomace capable of inhibiting adipogenesis.


Subject(s)
Adipocytes/drug effects , Adipogenesis/drug effects , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/genetics , Phlorhizin/chemistry , Plant Extracts/chemistry , Adipocytes/cytology , Animals , Bacterial Proteins/metabolism , Biocatalysis , Cytochrome P-450 Enzyme System/metabolism , Fruit/chemistry , Growth Inhibitors/chemistry , Growth Inhibitors/pharmacology , Malus/chemistry , Mice , NADPH-Ferrihemoprotein Reductase/metabolism , Phloretin/chemistry , Phlorhizin/pharmacology , Plant Extracts/pharmacology , Protein Engineering
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