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1.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1590-1606, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559742

RESUMO

Panax ginseng is a traditional Chinese medicine with significant pharmaceutical effects and wide application. Through orientational modification and transformation of ginsenoside glycosyl, rare ginsenosides with high antitumor activities can be generated. Traditional chemical methods cannot be applied in clinic. because of extremely complex preparation technologies and very high cost Transformations using microorganisms and their enzymatic systems provide the most feasible methods for solving the main problems. At present, the key problems in enzymatic synthesis of ginsenosides include low specific enzyme activities, identity of enzymes involved in the enzymatic synthesis, and their catalytic mechanisms, as well as nonsystematic studies on structural bioinformatics; specificity of enzymatic hydrolysis for saponin glycosyl has been rarely studied. Many reviews have been reported on glycosidase molecular recognition, immobilization, and biotransformation in ionic liquids (ILs), whereas ginsenoside transformation and application have not been systematically studied. To evaluate theoretical and applied studies on ginsenoside-oriented biotransformation, by reviewing the latest developments in related fields and evaluating the widely applied biocatalytic strategy, this review aims to evaluate the ginsenoside-oriented transformation method with improved product specificity, increased biocatalytic efficiency, and industrial application prospect based on the designed transformations of enzyme and solvent engineering of ILs. Therefore, useful theoretical and experimental evidence can be obtained for the development of ginsenoside anticancer drugs, large-scale preparation, and clinical applications in cancer therapy.


Assuntos
Biocatálise , Panax , Ginsenosídeos , Glicosídeo Hidrolases , Saponinas
2.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1787-1796, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559759

RESUMO

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.


Assuntos
Biocatálise , Quitina/análogos & derivados , Quitinases , Concentração de Íons de Hidrogênio , Reação em Cadeia da Polimerase
3.
Microb Cell Fact ; 18(1): 159, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542050

RESUMO

BACKGROUND: Xylanases randomly cleave the internal ß-1,4-glycosidic bonds in the xylan backbone and are grouped into different families in the carbohydrate-active enzyme (CAZy) database. Although multiple xylanases are detected in single strains of many filamentous fungi, no study has been reported on the composition, synergistic effect, and mode of action in a complete set of xylanases secreted by the same microorganism. RESULTS: All three xylanases secreted by Penicillium chrysogenum P33 were expressed and characterized. The enzymes Xyl1 and Xyl3 belong to the GH10 family and Xyl3 contains a CBM1 domain at its C-terminal, whereas Xyl2 belongs to the GH11 family. The optimal temperature/pH values were 35 °C/6.0, 50 °C/5.0 and 55 °C/6.0 for Xyl1, Xyl2, and Xyl3, respectively. The three xylanases exhibited synergistic effects, with the maximum synergy observed between Xyl3 and Xyl2, which are from different families. The synergy between xylanases could also improve the hydrolysis of cellulase (C), with the maximum amount of reducing sugars (5.68 mg/mL) observed using the combination of C + Xyl2 + Xyl3. Although the enzymatic activity of Xyl1 toward xylan was low, it was shown to be capable of hydrolyzing xylooligosaccharides into xylose. Xyl2 was shown to hydrolyze xylan to long-chain xylooligosaccharides, whereas Xyl3 hydrolyzed xylan to xylooligosaccharides with a lower degree of polymerization. CONCLUSIONS: Synergistic effect exists among different xylanases, and it was higher between xylanases from different families. The cooperation of hydrolysis modes comprised the primary mechanism for the observed synergy between different xylanases. This study demonstrated, for the first time, that the hydrolysates of GH11 xylanases can be further hydrolyzed by GH10 xylanases, but not vice versa.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimologia , Polissacarídeos/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucuronatos/metabolismo , Temperatura Alta , Hidrólise , Família Multigênica , Oligossacarídeos/metabolismo , Penicillium chrysogenum/química , Penicillium chrysogenum/genética , Domínios Proteicos , Xilanos/metabolismo
4.
Phys Chem Chem Phys ; 21(32): 17821-17835, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31373340

RESUMO

The rise of New Delhi metallo-beta-lactamase-1 (NDM-1) producers is a major public health concern due to carbapenem resistance. Infections caused by carbapenem-resistant enterobacteria (CRE) are classified as a serious problem. To understand the structure and function of NDM-1, an amino acid replacement approach is considered as one of the methods to get structural insight. Therefore, we have generated novel mutations (N193A, S217A, G219A and T262A) near active sites and an omega-like loop to study the role of conserved residues of NDM-1. The minimum inhibitory concentrations (MICs) of ampicillin, imipenem, meropenem, cefotaxime, cefoxitin and ceftazidime for all mutants were found to be reduced 2 to 6 fold, compared to a wild type NDM-1 producing strain. The Km values increased while Kcat and Kcat/Km values were decreased compared to wild type. The affinity as well as the catalysis properties of these mutants were reduced considerably for imipenem, meropenem, cefotaxime, cefoxitin, and ceftazidimem compared to wild type, hence the catalytic efficiencies (Kcat/Km) of all mutant enzymes were reduced owing to the poor affinity of the enzyme. The IC50 values of these mutants with respect to each drug were reduced compared to wild type NDM-1. MD simulations and docking results from the mutant protein models, along with the wild type example, showed stable and consistent RMSD, RMSF and Rg behavior. The α-helix content values of all mutant proteins were reduced by 13%, 6%, 14% and 9% compared to NDM-1. Hence, this study revealed the impact role of active sites near residues on the enzyme catalytic activity of NDM-1.


Assuntos
Antibacterianos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , beta-Lactamases/química , Antibacterianos/farmacologia , Biocatálise , Domínio Catalítico , Farmacorresistência Bacteriana , Cinética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica , beta-Lactamases/genética , beta-Lactamases/metabolismo
5.
J Agric Food Chem ; 67(37): 10392-10400, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31461615

RESUMO

The specificity of fructooligosaccharides as prebiotics depends on their size and structure, which in turn depend on their origin or the synthesis procedure. In this work we describe the application of an inulosucrase (IslA) from Leuconostoc citreum CW28 to produce high molecular weight inulin from sucrose alongside a commercial endoinulinase (Novozym 960) produced by Aspergillus niger for a simultaneous or sequential reaction to synthesize fructooligosaccharides (FOS). The simultaneous reaction resulted in a higher substrate conversion and a wide diversity of FOS when compared to the sequential reaction. A shotgun MS analysis of the commercial endoinulinase preparation surprisingly revealed an additional enzymatic activity: a fructosyltransferase, responsible for the synthesis of FOS from sucrose. Consequentially, the range of FOS obtained in reactions combining inulosucrase from Ln. citreum with the fructosyltransferase and endoinulinase from A. niger with sucrose as substrate may be extended and regulated.


Assuntos
Proteínas de Bactérias/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Hexosiltransferases/química , Inulina/química , Leuconostoc/enzimologia , Oligossacarídeos/química , Aspergillus niger/enzimologia , Biocatálise , Sacarose/química
6.
J Agric Food Chem ; 67(38): 10604-10613, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31466448

RESUMO

The aim of this study was to investigate the dipeptidyl peptidase-IV (DPP-IV) inhibition and metabolic stability of a casein-derived peptide Val-Pro-Tyr-Pro-Gln (VPYPQ) and its fragments as well as their release from casein following hydrolysis. Results showed that VPYPQ was the most potent DPP-IV inhibitory peptide among them with an IC50 value of 41.45 µM. This might be due to its two internal Pro residues at positions 2 and 4. Moreover, VPYPQ was resistant to hydrolysis by gastrointestinal enzymes and was relatively more stable to hydrolysis by DPP-IV and peptidases in plasma compared with its fragments. Additionally, oral administration of VPYPQ at a dose of 90 µmol/kg body weight could reduce the postprandial blood glucose levels in mice. More importantly, VPYPQ could be released efficiently from casein following hydrolysis by a combination of papain and in vitro digestion, reaching up to 3211.15 µg/g. Therefore, VPYPQ was a promising casein-derived DPP-IV inhibitor.


Assuntos
Caseínas/química , Preparações de Ação Retardada/química , Inibidores da Dipeptidil Peptidase IV/química , Peptídeos/química , Animais , Biocatálise , Glicemia/metabolismo , Preparações de Ação Retardada/administração & dosagem , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Teste de Tolerância a Glucose , Humanos , Hidrólise , Camundongos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Peptídeos/administração & dosagem , Ratos , Ratos Sprague-Dawley
7.
J Enzyme Inhib Med Chem ; 34(1): 1506-1510, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31431090

RESUMO

Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton. In humans there are 15 isoforms among which only 12 are catalytically active. Since active human (h) CAs show different efficiency, the understanding of the molecular determinants affecting it is a matter of debate. Here we investigated, by a site-specific mutagenesis approach, residues modulating the catalytic features of one of the least investigated cytosolic isoform, i.e. hCA XIII. Results showed that residues assisting the formation of an ordered solvent network within the catalytic site as well as those forming a histidine cluster on the protein surface are important to guarantee an efficient proton transfer.


Assuntos
Biocatálise , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Mutagênese Sítio-Dirigida , Anidrases Carbônicas/química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
8.
J Phys Chem A ; 123(36): 7710-7719, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31433182

RESUMO

Maleamate amidohydrolase (NicF) is a key enzyme in vitamin B3 metabolism that catalyzes the hydrolysis of maleamate to produce maleic acid and ammonia. Unlike most members from the amidohydrolase superfamily it does not require a metal ion. Here, we use multiscale computational enzymology to investigate the catalytic mechanism, substrate binding, oxyanion hole, and roles of key active site residues of NicF from Bordetella bronchiseptica. In particular, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) and QTAIM methods have been applied. The mechanism of the NicF-catalyzed reaction proceeds by a nucleophilic addition-elimination sequence involving the formation of a thioester enzyme intermediate (IC2 in stage 1) followed by hydrolysis of the thioester bond to form the products (stage 2). Consequently, the formation of IC2 in stage 1 is the rate-limiting step with a barrier of 88.8 kJ·mol-1 relative to the reactant complex, RC. Comparisons with related metal-dependent enzymes, particularly the zinc-dependent nicotinamidase from Streptococcus pneumonia (SpNic), have also been made to further illustrate unique features of the present mechanism. Along with -NH- donor groups of the oxyanion hole (i.e., HN-Thr146, HN-Cys150), the active site ß-hydroxyl of threonine (HO-ßThr146) is concluded to play a role in stabilizing the carbonyl oxygen of maleamate during the mechanism.


Assuntos
Amidoidrolases/química , Amidoidrolases/metabolismo , Biocatálise , Maleatos/metabolismo , Simulação de Dinâmica Molecular , Teoria Quântica , Bordetella bronchiseptica/enzimologia , Hidrólise , Maleatos/química , Estrutura Molecular
9.
J Agric Food Chem ; 67(32): 8967-8976, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31334650

RESUMO

Protein oxidation in milk products may entail flavor changes through reactions at methionine residues. However, little is known about the extent of methionine oxidation in milk and milk products. In the present study, a method for quantitation of methionine, methionine sulfoxide, and methionine sulfone by a stable isotope dilution assay using HILIC-ESI-MS/MS was established. For the quantitation of protein-bound analytes, anaerobic enzymatic hydrolysis was optimized to suppress artificial methionine oxidation. Moreover, the method allowed for monitoring of artificial oxidation by coincubation of the labeled probe [2H8]methionine. The percentage of oxidized methionine was low in UHT milk (up to 1.6%) and evaporated milk (up to 8.8%), but higher in beverages such as cocoa milk drinks (up to 19.0%) and coffee milk drinks (up to 32.8%), resulting in methionine sulfoxide concentrations of up to 6.7 g/kg protein in the latter. These products are important dietary sources of methionine sulfoxide. Model studies revealed that methionine residues can be oxidized strongly in the presence of phenolic compounds such as catechin, caffeic acid, and gallic acid, which are present in cocoa and coffee and may account for the high extent of oxidation in commercial samples.


Assuntos
Bebidas/análise , Metionina/análogos & derivados , Leite/química , Espectrometria de Massas em Tandem/métodos , Anaerobiose , Animais , Biocatálise , Dipeptidases/química , Hidrólise , Leucil Aminopeptidase/química , Metionina/análise , Oxirredução
10.
Bioresour Technol ; 291: 121802, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31352164

RESUMO

In this study, the potential of the pyrolysis method to overcome the negative effects of Azolla-filiculoides in infected areas was thoroughly investigated. Non-catalytic pyrolysis experiments were conducted at a temperature range of 400-700 °C. The highest possible bio-oil yield (35 wt%) was attained at 500 °C. To achieve the best chemical composition of bio-oil and higher amount of synthesis gas the catalytic pyrolysis were conducted in a dual-bed quartz reactor at the optimum temperature (500 °C). Although, all three catalysts (pyro-char, modified pyro-char (MPC), and Mg-Ni-Mo/MPC) showed almost an impressive performance in promotion of the common reactions, Mg-Ni-Mo/MPC catalyst have illustrated the stunning results by increasing the percentage of furan compounds from 5.25% to 33.07%, and decreasing the acid compounds from 25.56% to 9.09%. Using GC-MS and GC-FID liquid and gaseous products were fully analyzed. The carbon-based catalysts were also evaluated via FTIR, FESEM, EDX, and BET analyses.


Assuntos
Polifenóis/biossíntese , Traqueófitas/metabolismo , Biocatálise , Cromatografia Gasosa-Espectrometria de Massas , Temperatura Alta , Óleos Vegetais , Pirólise
11.
Inorg Chem ; 58(15): 9557-9561, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31313577

RESUMO

An iron(III) methoxide complex reacts with para-substituted triarylmethyl radicals to give iron(II) and methoxyether products. Second-order rate constants for the radical derivatives were obtained. Hammett and Marcus plots suggest the radical transfer reactions proceed via a concerted process. Calculations support the concerted nature of these reactions involving a single transition state with no initial charge transfer. These findings have implications for the radical "rebound" step invoked in nonheme iron oxygenases, halogenases, and related synthetic catalysts.


Assuntos
Compostos Férricos/metabolismo , Compostos Ferrosos/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Oxigenases/metabolismo , Biocatálise , Compostos Férricos/química , Compostos Ferrosos/química , Estrutura Molecular , Oxirredutases/química , Oxigênio/química , Oxigenases/química
12.
J Agric Food Chem ; 67(31): 8548-8558, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31266305

RESUMO

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.


Assuntos
Proteínas de Bactérias/química , Esterases/química , Ésteres/química , Gordonia (Bactéria)/enzimologia , Ácidos Ftálicos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Biodegradação Ambiental , Dietilexilftalato/química , Dietilexilftalato/metabolismo , Esterases/genética , Esterases/metabolismo , Ésteres/metabolismo , Gordonia (Bactéria)/genética , Gordonia (Bactéria)/isolamento & purificação , Gordonia (Bactéria)/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Alinhamento de Sequência , Microbiologia do Solo
13.
J Agric Food Chem ; 67(31): 8573-8580, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31293156

RESUMO

Glycosylation endows both natural and synthetic small molecules with modulated physicochemical and biological properties. Plant and bacterial glycosyltransferases capable of decorating various privileged scaffolds have been extensively studied, but those from kingdom Fungi still remain underexploited. Here, we use a combination of genome mining and heterologous expression techniques to identify four novel glycosyltransferase-methyltransferase (GT-MT) functional modules from Hypocreales fungi. These GT-MT modules display decent substrate promiscuity and regiospecificity, methylglucosylating a panel of natural products such as flavonoids, stilbenoids, anthraquinones, and benzenediol lactones. Native GT-MT modules can be split up and regrouped into hybrid modules with similar or even improved efficacy as compared with native pairs. Methylglucosylation of kaempferol considerably improves its insecticidal activity against the larvae of oriental armyworm Mythimna separata (Walker). Our work provides a set of efficient biocatalysts for the combinatorial biosynthesis of small molecule glycosides that may have significant importance to the pharmaceutical, agricultural, and food industries.


Assuntos
Proteínas Fúngicas/química , Glicosiltransferases/química , Hypocreales/enzimologia , Metiltransferases/química , Fenóis/química , Animais , Biocatálise , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hypocreales/genética , Inseticidas/química , Inseticidas/farmacologia , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mariposas/efeitos dos fármacos , Fenóis/farmacologia , Especificidade por Substrato
14.
Bioresour Technol ; 289: 121673, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31260936

RESUMO

d-Allulose is a promising low-calorie sweetener especially for diabetes and obesity patients. The functionalized polyhydroxyalkanoate (PHA) nano-beads decorated with d-tagatose 3-epimerase (DTE) was produced in recombinant endotoxin-free ClearColi, whereby the expression, purification, and immobilization of the active DTE were efficiently combined into one step. The immobilized DTE exhibited remarkable enzyme activity of 649.3 U/g beads and extremely high stability at a harsh working condition (pH 7.0-8.0, 65 °C). When DTE-PHA beads were subjected to enzymatic synthesis of d-allulose, a maximum conversion rate of 33% can be achieved at pH 7.0 and 65 °C for 3 h, and DTE-PHA beads retained about 80% of its initial activity after 8 continuous cycles. Moreover, the d-allulose/d-fructose binary mixture can be simply separated by a single cation exchange resin-equipped chromatography. Taken together, DTE-PHA beads are promising and robust nano-biocatalysts that will remarkably simplify the production procedures of d-allulose, contributing to its cost-effective production.


Assuntos
Frutose/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Biocatálise , Análise Custo-Benefício , Concentração de Íons de Hidrogênio , Nanoestruturas , Racemases e Epimerases/metabolismo
15.
Chem Commun (Camb) ; 55(62): 9148-9151, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31304493

RESUMO

Using superoxide reductase as a model system, a computational approach reveals how histidine tautomerism tunes the redox properties of metalloenzymes to enable their catalytic function. Inspired by these experimentally inaccessible insights, non-canonical histidine congeners are introduced as new versatile tools for the rational engineering of biological transition metal sites.


Assuntos
Oxirredutases/química , Oxirredutases/metabolismo , Elementos de Transição/metabolismo , Biocatálise , Modelos Moleculares , Estrutura Molecular , Oxirredução , Elementos de Transição/química
16.
J Microbiol Biotechnol ; 29(7): 1043-1052, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31353877

RESUMO

Active lipase-producing bacterium Burkholderia gladioli Bps-1 was rapidly isolated using a modified trypan blue and tetracycline, ampicillin (TB-TA) plate. The electro-phoretically pure enzyme was obtained by purification using ethanol precipitation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight was 34.6 kDa and the specific activity was determined to be 443.9 U/mg. The purified lipase showed the highest activity after hydrolysis with p-NPC16 at a pH of 8.5 and 50°C, and the Km, kcat, and kcat/Km values were 1.05, 292.95 s-1 and 279 s-1mM-1, respectively. The lipase was highly stable at 7.5 ≤ pH ≤ 10.0. K+ and Na+ exerted activation effects on the lipase which had favorable tolerance to short-chain alcohols with its residual enzyme activity being 110% after being maintained in 30% ethanol for 1 h. The results demonstrated that the lipase produced by the strain B. gladioli Bps-1 has high enzyme activity and is an alkaline lipase. The lipase has promising chemical properties for a range of applications in the food-processing and detergent industries, and has particularly high potential for use in the manufacture of biodiesel.


Assuntos
Burkholderia gladioli/enzimologia , Burkholderia gladioli/isolamento & purificação , Lipase/metabolismo , Biocatálise , Biocombustíveis , Burkholderia gladioli/crescimento & desenvolvimento , Burkholderia gladioli/metabolismo , Meios de Cultura , Detergentes , Estabilidade Enzimática , Etanol/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Lipase/química , Lipase/isolamento & purificação , Peso Molecular , Especificidade por Substrato , Temperatura Ambiente
17.
J Agric Food Chem ; 67(33): 9314-9324, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352776

RESUMO

Trehalose, a stable nonreducing disaccharide, protects biomolecules against environmental stress. However, trehalose production using secretory trehalose synthase (TreS) by Bacillus subtilis has not been well studied. In this study, a mutant TreS was successfully secreted and expressed in B. subtilis WB800N. The extracellular enzyme activity of TreS regulated by the P43 promoter and SPPhoD signal peptide in recombinant B. subtilis WB800N reached 23080.6 ± 1119.4 U/L in a 5-L fermenter after optimizing the culture medium, while xpF, skfA, lytC, and sdpC were knocked out. To reduce maltose consumption, malP and amyE corresponding to maltose transporters were further deleted. To simplify the trehalose production process, we invented a fermentation-coupling biocatalysis process involving recombinant bacteria fermentation to secrete TreS and simultaneous conversion of maltose to trehalose by TreS and found that the conversion rate of maltose to trehalose reached 75.5%, suggesting that this is an efficient strategy for large-scale trehalose production using recombinant B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Trealose/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biocatálise , Fermentação , Maltose/metabolismo , Engenharia Metabólica
18.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
19.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1348-1358, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328491

RESUMO

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.


Assuntos
Streptomyces coelicolor , Biocatálise , Clonagem Molecular , Escherichia coli , Glucosiltransferases , Trealose
20.
J Agric Food Chem ; 67(29): 8212-8226, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31309827

RESUMO

The factors that determine the digestion rate of starches were revealed using different forms of starches and a mixture of α-amylase and amyloglucosidase. Gelatinized starch samples with a degree of gelatinization (DG) from 12.2 to 100% for potato starch and from 7.1 to 100% for lotus seed starch were obtained. With an increasing DG, the short- and long-range molecular orders of both starches were disrupted progressively. The first-order digestion rate constant (k) of both starches increased with an increasing DG, although the positive linear relationships between DG and k differed (R2 = 0.87 for potato starch, and R2 = 0.74 for lotus seed starch). The mean fluorescence intensity showed a positive linear correlation with DG, which was strong for potato starch (R2 = 0.99) and relatively weaker for lotus seed starch (R2 = 0.54). These results indicated that DG is a major determinant for the digestion rate of potato starch and lotus seed starch and that the access/binding of enzymes to starch was the main rate-limiting factor for digestion of starches.


Assuntos
Glucana 1,4-alfa-Glucosidase/química , Lotus/química , Extratos Vegetais/química , Solanum tuberosum/química , Amido/química , alfa-Amilases/química , Biocatálise , Digestão , Hidrólise , Cinética , Sementes/química
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