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1.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2936-2946, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472310

RESUMO

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 µmol/(min·mg), and 1 670 s⁻¹, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.


Assuntos
Estruturas Metalorgânicas , Amidoidrolases , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Glicina/análogos & derivados , Concentração de Íons de Hidrogênio , Temperatura
2.
Enzyme Microb Technol ; 150: 109865, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489024

RESUMO

In this study, we cross-linked aminated Thermothelomyces thermophilus laccase onto Immobead 150P epoxy carrier, and achieved an immobilization yield of 99.84 %. The optimum temperature and pH values for the oxidation of ABTS by laccase were determined to be 70 °C and pH 3.0. After 6 h at 50 °C, laccase activity was diminished by about 13 % in the free form and 28 %, in the immobilized form. Km values for both free and cross-linked laccase were 0.051 and 0.567 mM, whereas Vmax values were 2.027 and 0.854 µmol. min-1, respectively. The immobilized laccase was able to preserve its full activity for 6 weeks, retaining approximately 95 % and 78 % of its initial activity after 8 and 20 weeks, respectively. The contact angles were two-fold higher when the laccase enzyme was occupied in the biografting reaction, revealing that the hydrophobic compound bonded stably onto beechwood samples.


Assuntos
Enzimas Imobilizadas , Lacase , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lacase/metabolismo , Sordariales , Temperatura
3.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360752

RESUMO

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.


Assuntos
Quitosana , Enzimas Imobilizadas , Teste de Materiais , Nanopartículas/química , Fenilalanina Hidroxilase , Quitosana/química , Quitosana/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/farmacologia
4.
FASEB J ; 35(9): e21332, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34423867

RESUMO

Emerging research has highlighted the capacity of microRNA-23a-3p (miR-23a-3p) to alleviate inflammatory pain. However, the molecular mechanism by which miR-23a-3p attenuates inflammatory pain is yet to be fully understood. Hence, the current study aimed to elucidate the mechanism by which miR-23a-3p influences inflammatory pain. Bioinformatics was initially performed to predict the inflammatory pain related downstream targets of miR-23a-3p in macrophage-derived extracellular vesicles (EVs). An animal inflammatory pain model was established using Complete Freund's Adjuvant (CFA). The miR-23a-3p expression was downregulated in the microglia of CFA-induced mice, after which the inflammatory factors were determined by ELISA. FISH and immunofluorescence were performed to analyze the co-localization of miR-23a-3p and microglia. Interestingly, miR-23a-3p was transported to the microglia via M2 macrophage-EVs, which elevated the mechanical allodynia and the thermal hyperalgesia thresholds in mice model. The miR-23a-3p downstream target, USP5, was found to stabilize HDAC2 via deubiquitination to promote its expression while inhibiting the expression of NRF2. Taken together, the key findings of the current study demonstrate that macrophage-derived EVs containing miR-23a-3p regulates the HDAC2/NRF2 axis by decreasing USP5 expression to alleviate inflammatory pain, which may provide novel therapeutic targets for the treatment of inflammatory pain.


Assuntos
Vesículas Extracelulares/metabolismo , Histona Desacetilase 2/metabolismo , Inflamação/metabolismo , Macrófagos/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Dor/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Enzimas Desubiquitinantes/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Estabilidade Enzimática , Vesículas Extracelulares/genética , Inflamação/genética , Inflamação/terapia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Modelos Biológicos , Dor/genética , Manejo da Dor , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
5.
Bioresour Technol ; 339: 125599, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34303095

RESUMO

L-asparaginase shows great potential as a food enzyme to reduce acrylamide formation in fried and baked products. But for food applications, enzymes must be stable at high temperatures and have higher catalytic efficiency. These desirable characteristics are conferred by the immobilization of enzymes on a suitable matrix. The present study aimed to immobilize the L-asparaginase enzyme on magnetic nanoparticles to reduce acrylamide content in the food system. Immobilized preparations were characterized using SEM, TEM, FTIR, UV-spectrometry, and XRD diffraction analyses. These nanoparticles enhanced the thermal stability of the enzyme up to four-fold at 70 °C compared to the free enzyme. Kinetic parameters exhibited an increase in Vmax, Km, and catalytic efficiency by ~ 38% than the free counterpart. The immobilized preparations were reusable for up to five cycles. Moreover, their application in the pre-treatment coupled with blanching of potato chips led to a significant reduction (greater than 95%) of acrylamide formation.


Assuntos
Asparaginase , Nanopartículas de Magnetita , Acrilamida , Asparaginase/metabolismo , Catálise , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Cinética
6.
Enzyme Microb Technol ; 149: 109849, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311886

RESUMO

A lipase from Malassizia globose, named SMG1, is highly desirable for industrial application due to its substrate specificity towards mono- and diacylglycerol. To improve its thermostability, we constructed a mutant library using an error-prone polymerase chain reaction, which was screened for both initial and residual enzymatic activity. Selected mutants were further studied using purified proteins for their kinetic thermostability at 45 ℃, T50 (the temperature at which the enzyme loses half of its activity), and the optimal reaction temperature. Results showed that the majority of mutations with improved thermostability were on the protein surface. D245N and L270P showed the most significant thermostability enhancement with an approximately 3 ℃ increase in T50 compared to wild-type (WT). In addition, combining these two mutations resulted in an increase of T50 by 5 °C. Also, the optimal reaction temperatures of L270P and this double mutant are 10 ℃ higher than WT. The double mutant showed an approximately 100-fold increase in half-life at 45 ℃ and higher enzymatic activities at 30 ℃ and above compared to WT. High-temperature unfolding molecular dynamics simulation suggested that the double mutant stabilized a flexible loop in the catalytic pocket.


Assuntos
Basidiomycota/enzimologia , Lipase Lipoproteica , Estabilidade Enzimática , Cinética , Simulação de Dinâmica Molecular , Mutação , Temperatura
7.
Int J Biol Macromol ; 185: 966-982, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34237367

RESUMO

Herein, our suggestion is to immobilize enzymes in-situ on absorbable shape-memory stents instead of injecting therapeutic enzymes into the blood. Chitosan (CHI)-based stents were tailored as novel support and the enzyme-immobilizing ability was elucidated using L-asparaginase (L-ASNase). For developing shape-memory stents, CHI-glycerol (GLY) solution was prepared and further blended with different ratios of polyethylene glycol (PEG), and polyvinyl alcohol (PVA). Afterward, the blends were modified by ionic crosslinking with sodium tripolyphosphate to obtain a shape-memory character. L-ASNase was included in the blends by using in-situ method before ionic crosslinking. The prepared stents, with or without L-ASNase, were comprehensively characterized by using several techniques. Collectively, immobilized L-ASNase exhibited much better performance in immobilization parameters than free one, thanks to its improved stability and reusability. For instance, CHI/GLY/PEG-3@L-ASNase retained about 70% of the initial activity after storage at 30 °C for 2 weeks, whereas the free form lost half of its initial activity. Besides, it retained 73.4% residual activity after 15 consecutive cycles. Most importantly, stent formulations exhibited ~60% activity in the bioreactor system after 4 weeks of incubation. Given the above results, shape-memory stents can be a promising candidate as a new platform for immobilization, especially in the blood circulation system.


Assuntos
Asparaginase/farmacologia , Quitosana/química , Polietilenoglicóis/química , Álcool de Polivinil/química , Asparaginase/química , Estabilidade de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Polietilenoglicóis/farmacologia , Stents , Temperatura , Molhabilidade
8.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199271

RESUMO

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.


Assuntos
Amidoidrolases/genética , Técnicas Biossensoriais , Mutação/genética , Mononucleotídeo de Nicotinamida/metabolismo , Estabilidade Enzimática , Cinética , Mononucleotídeo de Nicotinamida/química , Multimerização Proteica , Espectrometria de Fluorescência , Temperatura , Triptofano/metabolismo
9.
Langmuir ; 37(28): 8474-8485, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34236863

RESUMO

Protein stability and performance in various natural and artificial systems incorporating many other macromolecules for therapeutic, diagnostic, sensor, and biotechnological applications attract increasing interest with the expansion of these technologies. Here we address the catalytic activity of lysozyme protein (LYZ) in the presence of a polyethylene glycol (PEG) crowder in a broad range of concentrations and temperatures in aqueous solutions of two different molecular mass PEG samples (Mw = 3350 and 10000 g/mol). The phase behavior of PEG-protein solutions is examined by using dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), while the enzyme denaturing is monitored by using an activity assay (AS) and circular dichroism (CD) spectroscopy. Molecular dynamic (MD) simulations are used to illustrate the effect of PEG concentration on protein stability at high temperatures. The results demonstrate that LYZ residual activity after 1 h incubation at 80 °C is improved from 15% up to 55% with the addition of PEG. The improvement is attributed to two underlying mechanisms. (i) Primarily, the stabilizing effect is due to the suppression of the enzyme aggregation because of the stronger PEG-protein interactions caused by the increased hydrophobicity of PEG and lysozyme at elevated temperatures. (ii) The MD simulations showed that the addition of PEG to some degree stabilizes the secondary structures of the enzyme by delaying unfolding at elevated temperatures. The more pronounced effect is observed with an increase in PEG concentration. This trend is consistent with CD and AS experimental results, where the thermal stability is strengthened with increasing of PEG concentration and molecular mass. The results show that the highest stabilizing effect is approached at the critical overlap concentration of PEG.


Assuntos
Polietilenoglicóis , Estabilidade Enzimática , Peso Molecular , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
10.
Enzyme Microb Technol ; 149: 109834, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311879

RESUMO

The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.


Assuntos
Clostridium cellulovorans , Xilanos , Clostridium cellulovorans/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Temperatura
11.
Enzyme Microb Technol ; 149: 109854, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311891

RESUMO

Xylanases are categorized into different family groups, two of which are glycoside hydrolases 10 (GH10) and 11 (GH11) families. These well-characterized xylanases demonstrate different modes of action in hydrolysis of xylans. Imitating certain types of microorganisms to produce bifunctional enzymes such as engineered xylanases has gained considerable attention among researchers. In this study, a recombinant chimeric enzyme (X11-10) was designed by fusing two thermostable xylanases through a peptide linker. The recombinant parental enzymes, xylanase 10 from fungus Bispora sp. MEY-1 (X10) and xylanase 11 from bacterium Thermobacillus xylanilyticus (X11), and their chimera were successfully expressed in Pichia pastoris (P. pastoris), purified, and characterized. Being active over a wide pH range, X11-10 chimera showed higher thermal stability, possessed a lower Km, and a higher catalytic efficiency (kcat/Km) in comparison to the parental enzymes. Also, molecular dynamics simulation (MDS) of X11-10 revealed that its active site residues were free to interact with substrate. This novel chimeric xylanase may have potential applications in different industrial processes since it can substitute two separate enzymes and therefore minimize the production costs.


Assuntos
Ascomicetos , Xilanos , Ascomicetos/metabolismo , Bacillales , Quimera/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Glicosídeo Hidrolases , Proteínas Recombinantes de Fusão/genética , Saccharomycetales , Especificidade por Substrato
12.
Appl Microbiol Biotechnol ; 105(13): 5281-5298, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34223948

RESUMO

The enzyme ß-galactosidase has great potential for application in the food and pharmaceutical industries due to its ability to perform the hydrolysis of lactose, a disaccharide present in milk and in dairy by-products. It can be used in free form, in batch processes, or in immobilized form, which allows continuous operation and provides greater enzymatic stability. The choice of method and support for enzyme immobilization is essential, as the performance of the biocatalyst is strongly influenced by the properties of the material used and by the interaction mechanisms between support and enzyme. Therefore, this review showed the main enzyme immobilization techniques, and the most used supports for the constitution of biocatalysts. Also, materials with the potential for immobilization of ß-galactosidases and the importance of their biotechnological application are presented. KEY POINTS: • The main methods of immobilization are physical adsorption, covalent bonding, and crosslinking. • The structural conditions of the supports are determining factors in the performance of the biocatalysts. • Enzymatic hydrolysis plays an important role in the biotechnology industry.


Assuntos
Enzimas Imobilizadas , Lactose , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , beta-Galactosidase/metabolismo
13.
Colloids Surf B Biointerfaces ; 205: 111917, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34120088

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Lactobacillus plantarum is a novel biocatalyst in the degradation of histamine, but its properties need enhancement before practical application. Herein, we used Fe3O4 magnetic nanoparticles (MNPs) as the carrier core to prepare immobilized GAPDH. GAPDH was cloned, expressed in E. coli and purified, followed by immobilization on Fe3O4 MNPs and characterization by TEM and FT-IR. Then, characteristic comparisons between immobilized enzyme and its free form showed that the optimal pH and temperature of the former shifted to 7.5 and 40 °C, respectively, and pH tolerance and thermostability were separately broadened to 4.5-8.5 and 50-60 °C. In a wine-making experiment, including grape and black raspberry wines, using the immobilized enzyme, the results showed that over 81 %, 75 % and 59 % of histamine was removed after each treatment. These findings demonstrate that immobilizing GAPDH onto Fe3O4 MNPs is facile and efficient for histamine removal in fermented beverages.


Assuntos
Nanopartículas de Magnetita , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Escherichia coli/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Histamina , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura
14.
Enzyme Microb Technol ; 148: 109807, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34116744

RESUMO

Oil palm leaves (OPL) silica (SiO2) can replace the energy-intensive, commercially produced SiO2. Moreover, the agronomically sourced biogenic SiO2 is more biocompatible and cost-effective enzyme support, which properties could be improved by the addition of magnetite (Fe3O4) and graphene oxide (GO) to yield better ternary support to immobilize enzymes, i.e., Candida rugosa lipase (CRL). This study aimed to optimize the Candida rugosa lipase (CRL immobilization onto the ternary OPL-silica-magnetite (Fe3O4)-GO (SiO2/Fe3O4/GO) support, for use as biocatalyst for ethyl valerate (EV) production. Notably, this is the first study detailing the CRL/SiO2/Fe3O4/GO biocatalyst preparation for rapid and high yield production of ethyl valerate (EV). AFM and FESEM micrographs revealed globules of CRL covalently bound to GL-A-SiO2/Fe3O4/GO; similar to Raman and UV-spectroscopy results. FTIR spectra revealed amide bonds at 3478 cm-1 and 1640 cm-1 from covalent interactions between CRL and GL-A-SiO2/Fe3O4/GO. Optimum immobilization conditions were 4% (v/v) glutaraldehyde, 8 mg/mL CRL, at 16 h stirring in 150 mM NaCl at 30 °C, offering 24.78 ± 0.26 mg/g protein (specific activity = 65.24 ± 0.88 U/g). The CRL/SiO2/Fe3O4/GO yielded 77.43 ± 1.04 % of EV compared to free CRL (48.75 ± 0.70 %), verifying the suitability of SiO2/Fe3O4/GO to hyperactivate and stabilize CRL for satisfactory EV production.


Assuntos
Óxido Ferroso-Férrico , Dióxido de Silício , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Esterificação , Grafite , Lipase/metabolismo , Saccharomycetales , Valeratos
15.
Enzyme Microb Technol ; 148: 109817, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34116748

RESUMO

Papain was immobilized onto Ti3C2 MXene nanosheets by physical adsorption and physical adsorption combined with covalent crosslinking with glutaraldehyde. Ti3C2 MXene nanosheets were prepared by hydrofluoric acid etching method. The resulting products were well characterized by SEM, BET, XRD, FTIR, XPS. The optimized immobilization conditions are pH 6.5, immobilization time of 20 h, immobilization temperature of 10℃, and 10 mL 2 mg mL-1 papain, the amount of papain immobilized was 156 mg g-1, the activity of the immobilized papain determined was 1701 U∙g-1. The immobilized papain exhibited enhanced pH and temperature endurances, immobilized papain also showed improved storage stability (39.25 % and 65.57 % after 20 days of storage at 4 °C). papain reusability was significantly improved after immobilization and it retained more than 50 % of its initial activity after 5 repeated cycles. Interestingly, the results of immobilized enzymes demonstrated that the immobilization of enzymes on Ti3C2 MXene is feasible. Such approach could be transferred to other support systems for anchoring enzyme.


Assuntos
Papaína , Titânio , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Glutaral , Concentração de Íons de Hidrogênio , Temperatura
16.
Enzyme Microb Technol ; 148: 109799, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34116753

RESUMO

Most of the presently known ß-glucosidases are sensitive to end-product inhibition by glucose, restricting their potential use in many industrial applications. Identification of novel glucose tolerant ß-glucosidase can prove a pivotal solution to eliminate end-product inhibition and enhance the overall lignocellulosic saccharification process. In this study, a novel gene encoding ß-glucosidase BglNB11 of 1405bp was identified in the genome of Saccharomonospora sp. NB11 and was successfully cloned and heterologously expressed in E. coli BL21 (DE3).The presence of conserved amino acids; NEPW and TENG indicated that BglNB11 belonged to GH1 ß-glucosidases. The recombinant enzyme was purified using a Ni-NTA column, with the molecular mass of 51 kDa, using SDS-PAGE analysis. BglNB11 showed optimum activity at 40 °C and pH 7 and did not require any tested co-factors for activation. The kinetic values, Km, Vmax, kcat, and kcat/Km of purified enzyme were 0.4037 mM, 5735.8 µmol/min/mg, 5042.16 s-1 and 12487.71 s-1 mM-1, respectively. The enzyme was not inhibited by glucose to a concentration of 4 M but was slightly stimulated in the presence of glucose. Molecular docking of BglNB11 with glucose suggested that the relative binding position of glucose in the active site channel might be responsible for modulating end product tolerance and stimulation. ß-glucosidase from BglNB11 is an excellent enzyme with high catalytic efficiency and enhanced glucose tolerance compared to many known glucose tolerant ß-glucosidases. These unique properties of BglNB11 make it a prime candidate to be utilized in many biotechnological applications.


Assuntos
Glucose , beta-Glucosidase , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Especificidade por Substrato , Temperatura , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
17.
Int J Biol Macromol ; 185: 287-296, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34153359

RESUMO

Cellulose microcrystalline (MCC) was widely used in pharmaceutical and chemical industries because of its low degree of polymerization and large specific surface area. As its modified form, dialdehyde cellulose (DAC) was used for cross-linking and immobilizing Rhizopus lipase together with magnetic nanoparticles (MNPs) due to its active aldehyde groups. In this study, in order to maintain the original enzyme activity as much as possible and improve the stability of lipase, the Rhizopus lipase was successfully immobilized on the magnetic dialdehyde cellulose nanoparticles (MDC). Specifically, the immobilization conditions including dosage of DAC, concentration of enzyme, immobilization time and temperature together with pH value of the reaction medium were optimized. Maximum immobilization yield (60.03 ± 0.49%) and recovery activity (88.88 ± 0.61%) can be obtained under the optimal process conditions. The changes in secondary structures of immobilized enzyme revealed the increment in conformational rigidity, which can be reflected in temperature and pH stability as well as tolerance of organic reagents. Additionally, the recovery activity of immobilized enzyme still reached 50.60 ± 0.59% after 30 d of storage and 52.10 ± 0.57% retained after 6 cycles. These results indicated the ideal application prospect of MDC in immobilized enzymes.


Assuntos
Celulose/análogos & derivados , Lipase/química , Nanopartículas de Magnetita/química , Rhizopus/enzimologia , Celulose/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Temperatura , Tempo
18.
Int J Biol Macromol ; 185: 1-19, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34146557

RESUMO

Nanostructured materials along with an added value of polymers-based support carriers have gained high interest and considered ideal for enzyme immobilization. The recently emerged nanoscience interface in the form of nanostructured materials combined with immobilized-enzyme-based bio-catalysis has now become research and development frontiers in advance and applied bio-catalysis engineering. With the involvement of nanoscience, various polymers have been thoroughly developed and exploited to nanostructured engineer constructs as ideal support carriers/matrices. Such nanotechnologically engineered support carriers/matrix possesses unique structural, physicochemical, and functional attributes which equilibrate principal factors and strengthen the biocatalysts efficacy for multipurpose applications. In addition, nano-supported catalysts are potential alternatives that can outstrip several limitations of conventional biocatalysts, such as reduced catalytic efficacy and turnover, low mass transfer efficiency, instability during the reaction, and most importantly, partial, or complete inhibition/deactivation. In this context, engineering robust and highly efficient biocatalysts is an industrially relevant prerequisite. This review comprehensively covered various biopolymers and nanostructured materials, including silica, hybrid nanoflower, nanotubes or nanofibers, nanomembranes, graphene oxide nanoparticles, metal-oxide frameworks, and magnetic nanoparticles as robust matrices for cellulase immobilization. The work is further enriched by spotlighting applied and industrially relevant considerations of nano-immobilized cellulases. For instance, owing to the cellulose-deconstruction features of nano-immobilized cellulases, the applications like lignocellulosic biomass conversion into industrially useful products or biofuels, improved paper sheet density and pulp beat in paper and pulp industry, fruit juice clarification in food industry are evident examples of cellulases, thereof are discussed in this work.


Assuntos
Biopolímeros/química , Celulases/metabolismo , Celulose/química , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Indústria Alimentícia , Nanoestruturas
19.
Arch Microbiol ; 203(7): 4433-4448, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34132850

RESUMO

Polysaccharides are biopolymers composed of simple sugars like glucose, galactose, mannose, fructose, etc. The major natural sources for the production of polysaccharides include plants and microorganisms. In the present work, four bacterial and two fungal polysaccharides (PS or EPS) were used for the modification and preservation of Pycnoporus sanguineus cellobiose dehydrogenase (CDH) activity. It was found that the presence of polysaccharide preparations clearly enhanced the stability of cellobiose dehydrogenase compared to the control value (4 °C). The highest stabilization effect was observed for CDH modified with Rh110EPS. Changes in the optimum pH in the samples of CDH incubated with the chosen polysaccharide modifiers were evidenced as well. The most significant effect was observed for Rh24EPS and Cu139PS (pH 3.5). Cyclic voltammetry used for the analysis of electrochemical parameters of modified CDH showed the highest peak values after 30 days of incubation with polysaccharides at 4 °C. In summary, natural polysaccharides seem to be an effective biotechnological tool for the modification of CDH activity to increase the possibilities of its practical applications in many fields of industry.


Assuntos
Desidrogenases de Carboidrato , Polyporaceae , Polissacarídeos , Bactérias/química , Desidrogenases de Carboidrato/metabolismo , Catálise/efeitos dos fármacos , Estabilidade Enzimática , Fungos/química , Polyporaceae/enzimologia , Polissacarídeos/metabolismo , Polissacarídeos/farmacologia
20.
Nat Commun ; 12(1): 3616, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127663

RESUMO

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.


Assuntos
Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Animais , Sítios de Ligação , Catálise , Estabilidade Enzimática , Cinética , Luciferases de Renilla/química , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Mamíferos , Camundongos , Mutagênese , Mutação , Células NIH 3T3 , Conformação Proteica , Temperatura
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