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1.
BMC Res Notes ; 12(1): 596, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533815

RESUMO

OBJECTIVE: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. RESULTS: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg L-1), and were secreted at moderate to high percentages of the total extracellular protein (51-94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U L-1) that could allow their direct application.


Assuntos
Esterases/metabolismo , Ácido Glucurônico/metabolismo , Proteínas Recombinantes/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Esterases/genética , Espaço Extracelular/enzimologia , Fermentação , Metanol/química , Pichia/genética , Pichia/metabolismo
2.
Biosci Biotechnol Biochem ; 83(10): 1974-1984, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31216942

RESUMO

Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL-1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.


Assuntos
Burkholderia/genética , Regiões Promotoras Genéticas , Esterol Esterase/biossíntese , Espaço Extracelular/enzimologia , Genes Bacterianos , Proteínas Recombinantes/biossíntese , Análise de Sequência de RNA
3.
Ecotoxicol Environ Saf ; 181: 481-490, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31228824

RESUMO

Peroxidases and catalases are well-known antioxidant enzymes produced in almost all living organisms for the elimination of reactive oxygen species (ROS) and thus they prevent the occurrence of oxidative stress. In our study we focused on two soil fungi of the family Chaetomiaceae (mesophilic Chaetomium cochliodes and its thermophilic counterpart C. thermophilum var. dissitum) in order to explore the presence of peroxidase and catalase genes, formation of their native transcripts and protective effect of corresponding translation products in a case study. Predicted genes of our interest were confirmed by genomic PCR and their inducible transcripts by RT-PCR. We were able to quantify the expression levels of newly discovered fungal heme peroxidases and catalases with the reverse-transcription quantitative real-time PCR method. We compared obtained quantitative levels of mRNA production with the level of corresponding extracellular protein occurrence as detected with monitoring their specific peroxidase and catalase activities directly in the cultivation media at optimal growth temperatures. The presence of secretory Catalase 2 from C. thermophilum var. dissitum was detected and identified with mass spectrometry approach directly in the growth medium. This unique catalase is phylogenetically closely related with a previously described catalase-phenol oxidase thus representing an effective and versatile antioxidant in the environment of the fungal mycelia also involved in the catabolism of recalcitrant phenolic substances.


Assuntos
Ascomicetos/metabolismo , Catalase/metabolismo , Espaço Extracelular/enzimologia , Estresse Oxidativo , Peroxidases/metabolismo , Antioxidantes/metabolismo , Ascomicetos/classificação , Ascomicetos/enzimologia , Ascomicetos/genética , Catalase/genética , Meios de Cultura/metabolismo , Espaço Extracelular/metabolismo , Oxirredução , Peroxidases/genética , Filogenia , Temperatura
4.
Biomolecules ; 9(5)2019 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060228

RESUMO

Dietary and lifestyle changes are leading to an increased occurrence of non-alcoholic fatty liver disease (NAFLD). Using a hyperlipidemic murine model for non-alcoholic steatohepatitis (NASH), we have previously demonstrated that the lysosomal protease cathepsin D (CTSD) is involved with lipid dysregulation and inflammation. However, despite identifying CTSD as a major player in NAFLD pathogenesis, the specific role of extracellular CTSD in NAFLD has not yet been investigated. Given that inhibition of intracellular CTSD is highly unfavorable due to its fundamental physiological function, we here investigated the impact of a highly specific and potent small-molecule inhibitor of extracellular CTSD (CTD-002) in the context of NAFLD. Treatment of bone marrow-derived macrophages with CTD-002, and incubation of hepatic HepG2 cells with a conditioned medium derived from CTD-002-treated macrophages, resulted in reduced levels of inflammation and improved cholesterol metabolism. Treatment with CTD-002 improved hepatic steatosis in high fat diet-fed rats. Additionally, plasma levels of insulin and hepatic transaminases were significantly reduced upon CTD-002 administration. Collectively, our findings demonstrate for the first time that modulation of extracellular CTSD can serve as a novel therapeutic modality for NAFLD.


Assuntos
Catepsina D/antagonistas & inibidores , Espaço Extracelular/enzimologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Catepsina D/metabolismo , Células Cultivadas , Dieta Hiperlipídica , Células Hep G2 , Humanos , Inflamação/patologia , Lipoproteínas LDL , Fígado/efeitos dos fármacos , Fígado/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Inibidores de Proteases/farmacologia , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
5.
BMC Biotechnol ; 19(1): 29, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118018

RESUMO

BACKGROUND: Several types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatidyl groups into various phosphatidyl alcohols to produce modified phospholipids. Transphosphatidylation is a useful biocatalytic method for the synthesis of functional phospholipids from lecithin or phosphatidylcholine (PC), which are both easily accessible. Different PLD coding genes have been cloned from various sources from viral, prokaryotic, and eukaryotic organisms. Despite the catalytic potential of PLD, their low productivity has hampered their practical applications, probably because PLD, which is highly toxic to the host cells, when transformation of the PLD genes into the host cells, degrade PLs in the cell membrane. In this study, we designed a novel two-step expression system to produce and secrete recombinant PLD in extracellular medium, cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins. RESULTS: The engineered BL21 (DE3) host strain, which harbored the final expression vector pET28a-PLD-CBD-araC-ESN, was induced by IPTG and L-arabinose, the cell density decreased rapidly over a 2 h period and the enzymes released into the extracellular medium accounts owned 81.75% hydrolytic activity. Scanning electron microscopy results showed that there were obvious structural changes on the cell surface. The extracellularly secreted PLD-CBD powder was used to catalyze the transphosphatidylation reaction synthesis of phosphatidylserine, 2.3 U enzymes reacted for 12 h, during which the conversion rate reached 99% with very few by-products being produced. When the fused protein PLD-CBD immobilized on microcrystalline cellulose, the enzymes can be cycle used five times with 26% conversion rate was preserved. CONCLUSIONS: This study introduced an effective method for use in the expression of recombinant proteins and their extracellular secretion that simplifies the steps of sonication and purification and demonstrates great potential in the industrial application of enzymes. Cellulose as the most abundant renewable biomass resources in nature, and the cost is low, used for PLD immobilization make it more simple, effective and sustainable.


Assuntos
Celulose/metabolismo , Enzimas Imobilizadas/metabolismo , Espaço Extracelular/enzimologia , Fosfolipase D/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Autólise , Sítios de Ligação , Biocatálise , Enzimas Imobilizadas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Engenharia Genética/métodos , Cinética , Microscopia Eletrônica de Varredura , Fosfolipase D/genética , Fosfolipídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos Testes , Especificidade por Substrato
6.
Int J Biol Macromol ; 133: 987-997, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31029624

RESUMO

This study investigates the purification and biochemical characteristics of the protease secreted by Lactobacillus curvatus R5, which was isolated from Harbin dry sausages. The optimized fermentation conditions were fermentation time 36 h, initial pH 6 and fermentation temperature 37 °C. An extracellular protease was purified using ammonium sulfate precipitation, ion-exchange layer and gel filtration. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that molecular weight of the purified protease was 45.3 kDa. Protease produced by L. curvatus R5 reached a higher relative protease activity at pH 6, 40 °C, and the purified protease exhibited pH and thermal stability at pH 6 and 40 °C. The microbial protease activity can be inhibited by ethylene diamine tetraacetic acid disodium salt (EDTA). The Vmax and Km of the protease were 53 mg/min and 15.9 mg/mL, respectively. SDS-PAGE reflects the ability of the protease to hydrolyse myofibrillar protein and sarcoplasmic protein, especially on myosin heavy chain, actin, myosin light chain and phosphorylase. The 3D structure and the Ramachandran plot of L. curvatus R5 protease was obtained by homology modelling. The Ramachandran plot analysis revealed that the purified protease was composed of 366 amino acids, and its residues in favoured, allowed, generously allowed and disallowed regions were 84.6%, 11.3%, 3.2% and 0.9% residues, respectively. Molecular docking showed that the substrate actin bound to the protease active site by hydrogen bonding and hydrophobic interaction. This research provides a basis for understanding the enzymatic properties of L. curvatus R5 protease. In conclusion, L. curvatus R5 can be used as a starter culture or protease-producing strain to inoculate Harbin dry sausages.


Assuntos
Espaço Extracelular/enzimologia , Lactobacillus/citologia , Produtos da Carne/microbiologia , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Domínio Catalítico , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Hidrólise , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Simulação de Acoplamento Molecular , Miofibrilas/metabolismo , Peptídeo Hidrolases/química , Temperatura
7.
Int J Biol Macromol ; 132: 558-574, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928371

RESUMO

The present study investigated the purification, biochemical, and molecular characterization of a novel thermostable α-amylase (TfAmy48) from Tepidimonas fonticaldi strain HB23. MALDI-TOF/MS analysis indicated that the purified enzyme is a monomer with a molecular mass of 48,138.10 Da. The results from amino-acid sequence analysis revealed high homology between the 25 NH2-terminal residues of TfAmy48 and those of Gammaproteobacteria α-amylases. The optimum pH and temperature values for α-amylase activity were pH 8 and 80 °C, respectively. Thin-layer chromatography (TLC) analysis showed that the final hydrolyzed products of the enzyme from soluble potato starch were maltopentaose, maltose, and maltotriose, which indicate that TfAmy48 possessed an endo-acting pattern. Compared to Termamyl®300 L, TfAmy48 showed extreme stability and tolerance towards organic solvents and excellent compatibility with some commercial laundry detergents. These proprieties make TfAmy48 enzyme a potential candidate as a cleaning bioadditive in detergent composition. The Tfamy48 gene encoding TfAmy48 was cloned, sequenced, and heterologously-expressed in the extracellular fraction of Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rTfAmy48) were similar to those of native one. The highest sequence identity value (97%) was obtained with PsAmy1 α-amylase from Pseudomonas sp. strain KFCC10818, with only 16 amino-acid (aa) residues of difference.


Assuntos
Burkholderiales/enzimologia , Espaço Extracelular/enzimologia , Temperatura , alfa-Amilases/química , alfa-Amilases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Burkholderiales/genética , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Metais/farmacologia , Modelos Moleculares , Peso Molecular , Domínios Proteicos , Análise de Sequência de DNA , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/genética
8.
Biochimie ; 160: 200-209, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30898645

RESUMO

Here, for the first time, we report the presence of highly active extracellular carbonic anhydrase (CA) of α-class in cyanobacterial cells. The enzyme activity was confirmed both in vivo in intact cells and in vitro, using the recombinant protein. CA activity in intact cells of Cyanothece sp. ATCC 51142 reached ∼0.6 Wilbur-Anderson units (WAU) per 1 mg of total cell protein, and it was inhibited by a specific CAs inhibitor, ethoxyzolamide. The genes cce_4328 (ecaA) and cce_0871 (ecaB), encoding two potential extracellular CAs of Cyanothece have been cloned, and the corresponding proteins EcaA and EcaB, representing CAs of α- and ß-class, respectively, have been heterologously expressed in Escherichia coli. High specific activity (∼1.1 × 104 WAU per 1 mg of target protein) was detected for the recombinant EcaA only. The presence of EcaA in the outer cellular layers of Cyanothece was confirmed by immunological analysis with antibodies raised against the recombinant protein. The absence of redox regulation of EcaA activity indicates that this protein does not possess a disulfide bond essential for some α-class CAs. The content and activity of EcaA in a fraction of periplasmic proteins was higher in Cyanothece cells grown at ambient concentration of CO2 (0.04%) compared to those grown at an elevated CO2 concentration (1.7%). At the same time, the level of ecaA gene mRNA varied insignificantly in response to changes in CO2 supply. Our results indicate that EcaA is responsible for CA activity of intact Cyanothece cells and point to its possible physiological role under low-CO2 conditions.


Assuntos
Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Cyanothece/enzimologia , Espaço Extracelular/enzimologia , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Anidrases Carbônicas/genética , Anidrases Carbônicas/isolamento & purificação , Clonagem Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
9.
Int J Biol Macromol ; 130: 605-614, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30836186

RESUMO

Ruminococcus is one of the keystone bacteria of the human colonic microbiota and is highly specific for utilization of resistant starch via formation of amylosomes. Here, we present the characteristics of an extracellular amylase, Rbamy5, in Ruminococcus bromii. In an in silico study, it showed low homology with any other known amylases, but it was evolutionarily classified as a GH13_36 subfamily intermediary amylase. Recombinant Rbamy5 exhibited maximum activity toward amylose (384 ±â€¯26 U·mg-1) over soluble starch (254 ±â€¯3 U·mg-1), amylopectin (46.1 ±â€¯2.6 U·mg-1) and pullulan (72.5 ±â€¯0.41 U·mg-1) at 45 °C and pH 5.0. It was also able to degrade small substrates such as maltotriose (G3), maltotetraose (G4), and maltopentaose (G5) into maltose (G2). Despite lacking a specific N-terminal domain, Rbamy5 opened the cyclodextrin ring, which resembles those of neopullulanase. Moreover, it accumulated short α-(1 → 6)-branched maltooligosaccharides from soluble starch and maltosyl-ß-cyclodextrin (G2-ß-CD), while panose was solely derived from pullulan. These results suggest that Rbamy5 may have a role to provide branched maltooligosaccharides to stimulate the growth of beneficial microorganisms in the human intestine.


Assuntos
Glicosídeo Hidrolases/química , Ruminococcus/enzimologia , Ruminococcus/genética , alfa-Amilases/química , alfa-Amilases/genética , Sequência de Aminoácidos , Clonagem Molecular , Ativação Enzimática , Espaço Extracelular/enzimologia , Glicosídeo Hidrolases/metabolismo , Hidrólise , Filogenia , Análise de Sequência de DNA , Análise Espectral , Especificidade por Substrato , alfa-Amilases/isolamento & purificação , alfa-Amilases/metabolismo
10.
PLoS One ; 14(2): e0212120, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30763365

RESUMO

Deadwood is an important structural component in forest ecosystems and plays a significant role in global carbon and nutrient cycling. Relatively little is known about the formation and decomposition of CWD by microbial communities in situ and about the factors controlling the associated processes. In this study, we intensively analyzed the molecular fungal community composition and species richness in relation to extracellular enzyme activity and differences in decomposing sapwood and heartwood of 13 temperate tree species (four coniferous and nine deciduous species, log diameter 30-40 cm and 4 m long) in an artificial experiment involving placing the logs on the forest soil for six years. We observed strong differences in the molecular fungal community composition and richness among the 13 tree species, and specifically between deciduous and coniferous wood, but unexpectedly no difference was found between sapwood and heartwood. Fungal species richness correlated positively with wood extractives and negatively with fungal biomass. A distinct fungal community secreting lignocellulolytic key enzymes seemed to dominate the decomposition of the logs in this specific phase. In particular, the relative sequence abundance of basidiomycetous species of the Meruliaceae (e.g. Bjerkandera adusta) correlated with ligninolytic manganese peroxidase activity. Moreover, this study reveals abundant white-rot causing Basidiomycota and soft-rot causing Ascomycota during this phase of wood decomposition.


Assuntos
Fungos/isolamento & purificação , Fungos/metabolismo , Árvores/microbiologia , Biodiversidade , Biomassa , Fenômenos Químicos , Espaço Extracelular/enzimologia , Fungos/citologia , Microbiota , Árvores/química
11.
Microbes Environ ; 34(1): 83-88, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30799317

RESUMO

Marine microbes play a central role in driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEA) are the 'gatekeeper' of the marine carbon cycle, and these enzymes may be found attached to cells or dissolved (cell-free). Recent studies indicated that the proportion of dissolved enzymatic activity is generally similar to (if not higher than) cell-attached activity. Thus, it is critical to understand the sources and sinks of cell-free EEA in the ocean. We herein empirically tested whether bacterial stress and mortality (induced by mitomycin C) are a source of the cell-free EEA of alkaline phosphatase (APase), beta-glucosidase (BGase), and leucine aminopeptidase (LAPase). We found that bacterial stress and mortality caused relative increases in the proportion of dissolved relative to total EEA of up to 10.5% for APase, 13.5% for BGase, and 7.3% for LAPase. These relative increases in dissolved EEA corresponded to absolute increases in the cell-free pool of 4.8, 7.2, and 3.8% for APase, BGase and LAPase, respectively. Collectively, our results contribute relevant information on the origin of free dissolved extracellular enzymes in marine waters, indicating that bacterial stress and mortality are a source of cell-free enzymatic activity and suggesting a potential link between microbial interactions and the degradation of organic matter via the release of cell-free enzymes.


Assuntos
Fenômenos Fisiológicos Bacterianos , Espaço Extracelular/enzimologia , Água do Mar/microbiologia , Bactérias/enzimologia , Bactérias/crescimento & desenvolvimento , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Ecossistema , Viabilidade Microbiana/efeitos dos fármacos , Mitomicina/farmacologia , Água do Mar/química , Estresse Fisiológico/efeitos dos fármacos
12.
J Environ Sci (China) ; 76: 249-258, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30528015

RESUMO

A 120-day experiment was conducted to compare the removal of polycyclic aromatic hydrocarbons (PAHs) from agricultural soil after natural attenuation (NA), phytoremediation (P), mycoremediation (M), and plant-assisted mycoremediation (PAM) approaches in relation to the extracellular enzyme activities in soil. The NA treatment removed the total soil PAH content negligibly. The P treatment using maize (Zea mays) enhanced only the removal of low and medium molecular PAHs. The Pleurotus ostreatus cultivated on 30-50 mm wood chip substrate used in M treatment was the most successful in the removal of majority PAHs. Therefore, significantly (p < 0.05) highest total PAH removal by 541.4 µg/kg dw (dry weight) (36%) from all tested M treatments was observed. When using the same fungal substrate together with maize in PAM treatment, the total PAH removal was not statistically different from the previous M treatment. However, the maize-assisted mycoremediation treatment significantly boosted fungal biomass, microbial and manganese peroxidase activity in soil which strongly correlated with the removal of total PAHs. The higher PAH removal in that PAM treatment could be reflected in the following post-harvest time. Our suggested M and PAM approaches could be promising in situ bioremediation strategies for PAH-contaminated soils.


Assuntos
Espaço Extracelular/enzimologia , Peroxidases/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Poluentes do Solo/isolamento & purificação , Poluentes do Solo/metabolismo , Solo/química , Biodegradação Ambiental , Biomassa , Pleurotus/citologia , Pleurotus/metabolismo , Zea mays/citologia , Zea mays/metabolismo
13.
Appl Biochem Biotechnol ; 187(3): 894-912, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30099681

RESUMO

An extracellular laccase (Lacc10) was discovered in submerged cultures of Pleurotus ostreatus var. florida bleaching ß-carotene effectively without the addition of a mediator (650 mU/L, pH 4). Heterologous expression in P. pastoris confirmed the activity and structural analyses revealed a carotenoid-binding domain, which formed the substrate-binding pocket and is reported here for the first time. In order to increase activity, 106 basidiospore-derived monokaryons and crosses of compatible progenies were generated. These showed high intraspecific variability in growth rate and enzyme formation. Seventy-two homokaryons exhibited a higher activity-to-growth-rate-relation than the parental dikaryon, and one isolate produced a very high activity (1800 mU/L), while most of the dikaryotic hybrids showed lower activity. The analysis of the laccase gene of the monokaryons revealed two sequences differing in three amino acids, but the primary sequences gave no clue for the diversity of activity. The enzyme production in submerged cultures of monokaryons was stable over seven sub-cultivation cycles.


Assuntos
Técnicas de Cultura , Lacase/metabolismo , Pleurotus/enzimologia , Pleurotus/crescimento & desenvolvimento , beta Caroteno/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Espaço Extracelular/enzimologia , Lacase/química , Lacase/genética , Modelos Moleculares , Pleurotus/genética , Conformação Proteica
14.
Ultrason Sonochem ; 51: 315-324, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30322762

RESUMO

Herein the effect of low intensity ultrasound on the fermentation of skim milk medium by Lactobacillus paracasei were investigated to obtain optimum ultrasonic conditions for the highest yield of yoghurt peptides. The results showed that the fermented skim milk medium treated with ultrasound with its seed culture without ultrasonic treatment was an optimum scheme. In this scheme with the ultrasonic conditions of 28 kHz, ultrasonic pulsed model of on-time 100 s and off-time 10 s, 100 W/L for the treatment time of 30 min after the fermentation time of 9 h, the peptide content in the fermented skim milk media increased by 49.5% and the viable cells in the same media increased by 43.5% compared with those in the untreated samples. By response surface methodology (RSM) analysis and its verification experiments, a reasonably accurate empirical model was established for investigating and predicting the relationship between skim milk concentration, ultrasonic treatment time, power and the yield of yoghurt peptides. The former two parameters 12.6% w/v and 35 min were taken in the verification experiments in which the peptide content of the fermented media reached 5.9 mg/mL with an increase by 64.23% and the peptide yield was 14.2%, similar to its theoretical value of 14.6% according to the empirical model. The comparison of extracellular enzyme activities in the fermented skim milk media between with and without ultrasonic treatment under the conditions in the optimum scheme indicated that the mechanism of the ultrasound-activated peptide content increment might be the extracellular enzyme activities immediately activated by the ultrasound, effect of which would disappear in the progress of fermentation after the ultrasound was removed.


Assuntos
Fermentação , Lactobacillus paracasei/metabolismo , Leite/microbiologia , Peptídeos/metabolismo , Ondas Ultrassônicas , Iogurte/microbiologia , Animais , Biomassa , Espaço Extracelular/enzimologia , Concentração de Íons de Hidrogênio , Lactobacillus paracasei/citologia , Lactobacillus paracasei/crescimento & desenvolvimento
15.
Cell Mol Biol (Noisy-le-grand) ; 64(14): 53-60, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30511621

RESUMO

L-Asparaginase is an enzyme that hydrolyses the amino acid L-Asparagine into aspartic acid and ammonia. As a medication, L-Asparaginase is used in chemotherapy to treat acute lymphoblastic leukaemia by depleting circulating Asparagine and depriving tumor cells. Interest in Actinomycetes as potential producers of antibiotics and enzymes encouraged us to investigate an isolated strain (CA01) from soft wheat bran.The Actinomycete strain was characterized based on its morphological and biochemical characteristics and selected due to a proved promising ability to produce L-Asparaginase optimized in both solid and liquid media cultures.The conditions of enzyme production were standardized according to a one-factor-at-a-time (OFAT) experimental design.To obtain optimal medium combination, a Box-Behnken Response Surface Methodology (RSM) has been adopted by choosing the most influential factors. The optimal conditions for the enzyme production were (g/l): L-Asparagine 10.7; Glucose 2.7; starch 7, in based medium containing (g/l): K2HPO4 0.5; MgSO4, 7H2O 0.1, corresponding to an optimal enzymatic activity of 8.03 IU/ml at 27.83°C. The maximum production of enzyme was reached on the sixth day of experiment. The ANOVA test (P value ˂ 0.05) and adjusted R2 values close to the experimental R2 show that the obtained model of the active L-Asparaginase of CA01 strain production is significant with the following linear terms: temperature, substrate concentration, Glucose concentration and there squared.


Assuntos
Actinobacteria/enzimologia , Actinobacteria/isolamento & purificação , Asparaginase/biossíntese , Fibras na Dieta/microbiologia , Espaço Extracelular/enzimologia , Análise de Variância , Carbono/farmacologia , Cinética , Nitrogênio/farmacologia , Padrões de Referência , Fatores de Tempo
16.
Microb Cell Fact ; 17(1): 163, 2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30348150

RESUMO

BACKGROUND: Bacillus subtilis has been widely used as a host for heterologous protein expression in food industry. B. subtilis ATCC6051 is an alternative expression host for the production of industrial enzymes, and exhibits favorable growth properties compared to B. subtilis 168. Extracellular expression of pullulanase from recombinant B. subtilis is still limited due to the issues on promoters of B. subtilis expression system. This study was undertaken to develop a new, high-level expression system in B. subtilis ATCC6051. RESULTS: To further optimize B. subtilis ATCC6051 as a expression host, eight extracellular proteases (aprE, nprE, nprB, epr, mpr, bpr, vpr and wprA), the sigma factor F (spoIIAC) and a surfactin (srfAC) were deleted, yielding the mutant B. subtilis ATCC6051∆10. ATCC6051∆10 showed rapid growth and produced much more extracellular protein compared to the widetype strain ATCC6051, due to the inactivation of multiple proteases. Using this mutant as the host, eleven plasmids equipped with single promoters were constructed for recombinant expression of pullulanase (PUL) from Bacillus naganoensis. The plasmid containing the PspovG promoter produced the highest extracellular PUL activity, which achieved 412.9 U/mL. Subsequently, sixteen dual-promoter plasmids were constructed and evaluated using this same method. The plasmid containing the dual promoter PamyL-PspovG produced the maximum extracellular PUL activity (625.5 U/mL) and showed the highest expression level (the dry cell weight of 18.7 g/L). CONCLUSIONS: Taken together, we constructed an effective B. subtilis expression system by deleting multiple proteases and screening strong promoters. The dual-promoter PamyL-PspovG system was found to support superior expression of extracellular proteins in B. subtilis ATCC6051.


Assuntos
Bacillus subtilis/metabolismo , Biotecnologia/métodos , Espaço Extracelular/enzimologia , Expressão Gênica , Glicosídeo Hidrolases/biossíntese , Regiões Promotoras Genéticas
17.
Protein Pept Lett ; 25(10): 897-907, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30182832

RESUMO

BACKGROUND: Bacterial lipases find so many industrial applications. Therefore, new source of lipase suitable for industrial conditions is always required. Lipase zymography methods use costly chromogenic substrates and indicator dyes and are few in numbers. OBJECTIVE: The objectives of this work include lipase purification and its characterization from Acinetobacter radioresiens PR8 and development of new zymography method for lipase detection. METHOD: The lipase was purified using conventional method and cation exchange chromatography and it was characterized biochemically and analytically. Based on these characterization new in-gel lipase zymography method was developed. RESULTS: In this present work, an alkalophilic lipase producing bacterium was isolated from soil; screened for extracellular lipase activity and identified to be Acinetobacter radioresistens PR8 (Genbank accession ID: MF073322). Enzyme production kinetics showed maximum production (4.16 U/ml at pH 9) of enzyme after 72 h. The lipase activity was found to be highest in olive oil (1% v/v; 8.1 U/ml). Low molecular weight (27 kDa) alkaline (pH 9) cold active (20 °C) lipase was purified from Acinetobacter radioresistens PR8. Lipase was characterized using PMF, FT-IR and its high conformational stability (Transition temperature: 122.3 °C) was attributed from its DSC spectrum. The importance of magnesium and sodium ions for enhancing lipase activity was obtained from flux balance analysis. CONCLUSION: Based on the lipase activating role of Mn2+ and Na+ ions, optimum temperature, pH with no chromogenic substrates and indicator dyes, a new in gel zymography method for lipase detection was developed.


Assuntos
Acinetobacter/enzimologia , Temperatura Baixa , Eletroforese/métodos , Lipase/isolamento & purificação , Lipase/metabolismo , Acinetobacter/citologia , Estabilidade Enzimática , Espaço Extracelular/enzimologia , Lipase/química , Análise do Fluxo Metabólico
18.
J Theor Biol ; 456: 29-33, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063924

RESUMO

After gene duplication, paralogous genes evolve independently, and consequently, the new proteins encoded by these duplicated genes are exposed to changes in their subcellular location. Although there are increasing evidence that phylogenetically related proteins play different functions in different subcellular compartments, the number of evolutionary steps required for the emergence of a novel protein with a novel subcellular localization remains unclear. Regarding this intriguing topic, here we examine in depth our previous reports describing both intracellular and extracellular polyhydroxybutyrate polymerases (PhaC) in the Pseudomonadales group. The recapitulation of the intracellular-to-extracellular localization switch of PhaC in these strains shows a gradual evolution from a simple cytosolic PhaC form to a complex extracellular PhaC form specifically secreted via the type 1 secretion system. This gradual evolution includes several adaptive and pre-adaptive changes at the genomic, genetic and enzymatic levels, which are intimately related to the lifestyle of organisms during the evolution of protein localization. We conclude that the protein localization switch can be an extremely complex process in nature.


Assuntos
Aciltransferases/metabolismo , Citosol/enzimologia , Evolução Molecular , Espaço Extracelular/enzimologia , Pseudomonas/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Filogenia , Transporte Proteico/genética , Pseudomonas/genética , Alinhamento de Sequência
19.
Biosci Biotechnol Biochem ; 82(12): 2180-2190, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30122147

RESUMO

A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake (TmGLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0-6.0, and its activity is strongly inhibited by Ag+. It degrades α-1,4- and α-1,6-glycosidic linkages in various polysaccharides. Its gene (TmGlu1) was cloned using information from the enzyme's internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme.


Assuntos
Espaço Extracelular/enzimologia , Proteínas Fúngicas/química , Glucana 1,4-alfa-Glucosidase/química , Pichia/genética , Tricholoma/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
20.
Prep Biochem Biotechnol ; 48(6): 549-555, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29889602

RESUMO

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.


Assuntos
Amilases/biossíntese , Espaço Extracelular/enzimologia , Fermentação , Micélio/enzimologia , Saccharomycetales/enzimologia , Amilases/metabolismo , Biocatálise , Biomassa , Cátions , Meios de Cultura , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio
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