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1.
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
2.
Int J Biol Macromol ; 173: 1-12, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33476612

RESUMO

The catalytic mechanism of most lipases involves a step called "interfacial activation" which significantly increases lipases activity beyond the critical micellar concentration (CMC) of substrate. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of lipase interfacial activation beyond the CMC. Molecular dynamic (MD) simulations indicated the open- and closed-lid transitions and revealed that Phe113 was the critical site for RCL activation by its dynamic flipping. Such putative switch affecting interfacial activation has not been reported in lipase so far. The function of Phe113 was subsequently verified by mutation experiments. The F113W mutant increases the lipase catalytic efficiency (1.9 s-1·µM-1) to 280% at the optimum temperature (40 °C) and pH 8.5 with the addition of 0.12 µg protein in the 200 µL reaction system. MD simulations indicated that the fast flipping rate from the closed to the open state, the high open state proportion, and the exposure of the catalytic triad are the main reasons for the lipase activation. The mutual corroboration of simulations and site-directed mutagenesis results revealed the vital role of Phe113 in the lipase activation.


Assuntos
Butiratos/química , Proteínas Fúngicas/química , Lipase/química , Fenilalanina/química , Rhizopus/química , Sítios de Ligação , Biocatálise , Butiratos/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lipase/genética , Lipase/metabolismo , Simulação de Dinâmica Molecular , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhizopus/enzimologia , Especificidade por Substrato , Temperatura , Termodinâmica , Água/química
3.
Int J Biol Macromol ; 160: 1189-1200, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32485250

RESUMO

To improve the thermostability of r27RCL from Rhizopus chinensis and broaden its industrial applications, we used rational design (FoldX) according to ΔΔG calculation to predict mutations. Four thermostable variants S142A, D217V, Q239F, and S250Y were screened out and then combined together to generate a quadruple-mutation (S142A/D217V/Q239F/S250Y) variant, called m31. m31 exhibited enhanced thermostability with a 41.7-fold longer half-life at 60 °C, a 5 °C higher of topt, and 15.8 °C higher of T5030 compared to that of r27RCL expressed in Pichiapastoris. Molecular dynamics simulations were conducted to analyze the mechanism of the thermostable mutant. The results indicated that the rigidity of m31 was improved due to the decreased solvent accessible surface area, a newly formed salt bridge of Glu292:His171, and the increased ΔΔG of m31. According to the root-mean-square-fluctuation analysis, three positive mutations S142A, D217V, and Q239F located in the thermal weak regions and greatly decreased the distribution of thermal-fluctuated regions of m31, compared to that of r27RCL. These results suggested that to simultaneously implement MD simulations and ΔΔG-based rational approaches will be more accurate and efficient for the improvement of enzyme thermostability.


Assuntos
Proteínas Fúngicas/química , Temperatura Alta , Lipase/química , Simulação de Dinâmica Molecular , Desnaturação Proteica , Rhizopus/enzimologia , Substituição de Aminoácidos , Estabilidade Enzimática , Proteínas Fúngicas/genética , Lipase/genética
4.
Food Chem ; 309: 125709, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31708343

RESUMO

To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 ß-mannanase gene (mRmMan134A) from Rhizopus microsporus var. rhizopodiformis F518 was expressed in Pichia pastoris and a high expression level (3680 U mL-1) was obtained through high cell density fermentation. mRmMan134A exhibited maximum activity at pH 5.5 and 50 °C. It was then subjected to hydrolyze cassia gum with 70.6% of overall yield of manno-oligosaccharides. From the hydrolysate, seven components (F1-F7) were separated and identified as mannose, mannobiose, galactose, mannotriose, mannotetraose, 61-α-d-galactosyl-ß-d-mannobiose, and mannopentaose, respectively. According to in vitro fermentation, the manno-oligosaccharides were able to promote the growth of three Bifidobacterium strains and six Lactobaillus strains with 3.0-fold increment in culture absorbance, and these strains preferred manno-oligosaccharides with degree of polymerization (DP) 2-3 rather than those with DP 4-5. Novel manno-oligosaccharides from cassia gum with promising prebiotic activity were provided in the present study.


Assuntos
Cassia/metabolismo , Oligossacarídeos/metabolismo , Prebióticos , beta-Manosidase/metabolismo , Técnicas de Cultura Celular por Lotes , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Hidrólise , Manose/metabolismo , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/farmacologia , Rhizopus/enzimologia , Temperatura , beta-Manosidase/genética
5.
Int J Biol Macromol ; 154: 1314-1323, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733249

RESUMO

An in situ and real-time investigation was performed using an optical cell system and in-silico analysis to reveal the impacts of pressure and temperature on the conformational state and behaviours of Rhizopus chinensis lipase (RCL). The fluorescence intensity (FI) of RCL increased remarkably under high pressure, and part of this increase was recovered after depressurization. This result displayed the partially reversible conformational change of RCL, which may be associated with the local change of Trp224 near the catalytic centre. High temperature caused a significant loss of secondary structure, whereas the α-helical segments including the lid were preserved by high pressure even at temperatures over 60 °C. The parameters of enzymatic reaction monitored by UV showed that the hydrolysis rate was remarkably enhanced by the pressure of 200 MPa. In the pressure range of 0.1-200 MPa, the active volume measured by the in situ system decreased from -2.85 to -6.73 mL/mol with the temperature increasing from 20 °C to 40 °C. The high catalytic capacity of the lipase under high pressure and high temperature was primarily attributed to pressure protection on RCL.


Assuntos
Lipase/química , Lipase/metabolismo , Pressão , Rhizopus/enzimologia , Temperatura , Biocatálise , Simulação por Computador , Cinética , Modelos Moleculares , Conformação Proteica
6.
Prep Biochem Biotechnol ; 50(3): 226-233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31661372

RESUMO

Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by Rhizopus microsporus. The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 °C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 °C. Proteolytic activity was significantly reduced in the presence of Cu2+ and Al3+. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 ± 155 mM-1.s-1), Abz-KLRSSNQ-EDDnp (3,514 ± 130 mM-1.s-1), and Abz-KLRQSKQ-EDDnp (3,068 ± 386 mM-1.s-1). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca2+ and broad pH range of 4-7, in addition to its ability to efficiently clot milk.


Assuntos
Ácido Aspártico Proteases/química , Proteínas Fúngicas/química , Leite/química , Rhizopus/enzimologia , Animais , Bovinos , Concentração de Íons de Hidrogênio
7.
Molecules ; 24(23)2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31771200

RESUMO

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water-organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters.


Assuntos
Candida/enzimologia , Ésteres/química , Lipase/metabolismo , Mucor/enzimologia , Pseudomonas fluorescens/enzimologia , Rhizopus/enzimologia , Proteínas de Bactérias/metabolismo , Mimetismo Biológico , Catálise , Simulação por Computador , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura
8.
FEMS Yeast Res ; 19(6)2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505595

RESUMO

Microorganisms have evolved to produce specific end products for many reasons, including maintaining redox balance between NAD+ and NADH. The yeast Saccharomyces cerevisiae, for example, produces ethanol as a primary end product from glucose for the regeneration of NAD+. Engineered S. cerevisiae strains have been developed to ferment lignocellulosic sugars, such as xylose, to produce lactic acid by expression of a heterologous lactate dehydrogenase (ldhA from Rhizopus oryzae) without genetic perturbation to the native ethanol pathway. Surprisingly, the engineered yeast strains predominantly produce ethanol from glucose, but produce lactic acid as the major product from xylose. Here, we provide initial evidence that the shift in product formation from ethanol to lactic acid during xylose fermentation is at least partially dependent on the presence of functioning monocarboxylate transporter genes/proteins, including JEN1 and ADY2, which are downregulated and unstable in the presence of glucose, but upregulated/stable on xylose. Future yeast metabolic engineering studies may find the feedstock/carbon selection, such as xylose, an important step toward improving the yield of target end products.


Assuntos
L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Proteínas de Membrana Transportadoras/genética , Engenharia Metabólica , Rhizopus/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Regulação para Baixo , Etanol/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Glucose/metabolismo , L-Lactato Desidrogenase/genética , Transportadores de Ácidos Monocarboxílicos/genética , Rhizopus/genética , Saccharomyces cerevisiae/genética , Deleção de Sequência , Simportadores/genética , Transgenes
9.
Biochemistry ; 58(38): 3943-3954, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31436959

RESUMO

Members of an important group of industrial enzymes, Rhizopus lipases, exhibit valuable hydrolytic features that underlie their biological functions. Particularly important is their N-terminal polypeptide segment (NTPS), which is required for secretion and proper folding but is removed in the process of enzyme maturation. A second common feature of this class of lipases is the α-helical "lid", which regulates the accessibility of the substrate to the enzyme active site. Some Rhizopus lipases also exhibit "interfacial activation" by micelle and/or aggregate surfaces. While it has long been recognized that the NTPS is critical for function, its dynamic features have frustrated efforts to characterize its structure by X-ray crystallography. Here, we combine nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the structure and dynamics of Rhizopus chinensis lipase (RCL) with its 27-residue NTPS prosequence (r27RCL). Both r27RCL and the truncated mature form of RCL (mRCL) exhibit biphasic interfacial activation kinetics with p-nitrophenyl butyrate (pNPB). r27RCL exhibits a substrate binding affinity significantly lower than that of mRCL due to stabilization of the closed lid conformation by the NTPS. In contrast to previous predictions, the NTPS does not enhance lipase activity by increasing surface hydrophobicity but rather inhibits activity by forming conserved interactions with both the closed lid and the core protein structure. Single-site mutations and kinetic studies were used to confirm that the NTPS serves as internal competitive inhibitor and to develop a model of the associated process of interfacial activation. These structure-function studies provide the basis for engineering RCL lipases with enhanced catalytic activities.


Assuntos
Proteínas Fúngicas/química , Microbiologia Industrial , Lipase/química , Peptídeos/química , Rhizopus/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrólise , Cinética , Lipase/genética , Lipase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética , Peptídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
10.
J Oleo Sci ; 68(8): 781-792, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31366855

RESUMO

The possibility of improving brain function coupled with its preferential uptake in the brain has garnered attention for docosahexaenoic acid-bound lysophosphatidylcholine (DHA-LPC). However, studies focusing on the health benefits of dietary DHA-LPC are lacking. We prepared a dietary oil rich in DHA-LPC (DHA-LPC rich oil) via enzymatic modification of phospholipids (PL) extracted from squid (Todarodes pacificus) meal and purification of active carbon, ion exchange resin, and silica gel. We then examined the effects of dietary DHA-LPC rich oil on male Wistar rats by evaluating serum and liver lipid profiles, fatty acid (FA) metabolizing enzyme activity, and the FA composition of serum and brain. The rats were fed a basal diet containing either soybean oil alone (7%) or soybean oil (4.5%) with DHA-LPC rich oil (2.5%) for 28 days, and then evaluated. The rats fed the diet containing DHA-LPC rich oil showed reduced triacylglycerol concentration due, in part, to the enhancement of carnitine palmitoyltransferase 2 and acyl-CoA oxidase activities and suppression of acetyl-CoA carboxylase and glucose-6-phosphate dehydrogenase activities in the liver. Moreover, the dietary DHA-LPC rich oil moderately increased DHA in the FA composition of the rat hippocampus, which may be due to elevated DHA composition in serum LPC. These results suggest that DHA-LPC rich oil has hypolipidemic effect and moderate increase in hippocampal DHA amount in normal rats.


Assuntos
Encéfalo/metabolismo , Gorduras Insaturadas na Dieta/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Hipolipemiantes/farmacologia , Fígado/metabolismo , Lisofosfatidilcolinas/farmacologia , Administração Oral , Animais , Química Encefálica , Hidrolases de Éster Carboxílico/química , Decapodiformes/química , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Docosa-Hexaenoicos/administração & dosagem , Hipocampo/química , Hipocampo/metabolismo , Hipolipemiantes/administração & dosagem , Fígado/química , Lisofosfatidilcolinas/administração & dosagem , Masculino , Fosfolipídeos/química , Fosfolipídeos/isolamento & purificação , Ratos Wistar , Rhizopus/enzimologia
11.
Protein Expr Purif ; 163: 105443, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185288

RESUMO

The production of membrane-associated lipase from Rhizopus chinensis (RCL), which has a high ester synthesis activity and important potential applications, is difficult in heterologous expression system such as Escherichia coli and often leads to the formation of inclusion bodies. Here, we describe the soluble expression of mature RCL (mRCL) using maltose-binding protein (MBP) as a solubility-enhancing tag in the E. coli system. Although the MBP-mRCL fusion protein was soluble, mRCL was insoluble after removal of the MBP tag in E. coli BL21 (DE3). Using E. coli BL21 trxB (DE3) as an expression host, soluble mRCL was obtained and expression conditions were optimized. Furthermore, the ester synthesis activity of soluble mRCL was increased by detergent treatment and was found to be 3.5 and 1.5 times higher than those of the untreated enzyme and naturally occurring enzyme, respectively. Overall, this study provides a potential approach for producing active and soluble forms of eukaryotic lipases in a heterologous E. coli expression system.


Assuntos
Lipase/biossíntese , Rhizopus/enzimologia , Clonagem Molecular , Meios de Cultura , Escherichia coli , Ésteres/metabolismo , Lipase/genética , Lipase/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Proteínas Recombinantes de Fusão/metabolismo , Rhizopus/genética
12.
Prep Biochem Biotechnol ; 49(9): 858-867, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31219401

RESUMO

Cheese production is projected to reach 20 million metric tons by 2020, of which 33% is being produced using calf rennet (EC 3.4.23.4). There is shortage of calf rennet, and use of plant and microbial rennets, hydrolyze milk proteins non-specifically resulting in low curd yields. This study reports fungal enzymes obtained from cost effective medium, with minimal down streaming, whose activity is comparable with calf and Mucor rennet. Of the fifteen fungi that were screened, Mucor thermohyalospora (MTCC 1384) and Rhizopus azygosporus (MTCC 10195) exhibited the highest milk-clotting activity (MCA) of 18,383 ± 486 U/ml and 16,373 ± 558 U/ml, respectively. Optimization exhibited a 33% increase in enzyme production (30 g wheat bran containing 6% defatted soy meal at 30 °C, pH 7) for M. thermohyalospora. The enzyme was active from pH 5-10 and temperature 45-55 °C. Rhizopus azygosporus exhibited 31% increase in enzyme production (30 g wheat bran containing 4% defatted soy meal at 30 °C, pH 6) and the enzyme was active from pH 6-9 at 50 °C. Curd yields prepared from fungal enzyme extract decreased (5-9%), when compared with calf rennet and Mucor rennet. This study describes the potential of fungal enzymes, hitherto unreported, as a viable alternative to calf rennet.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Mucor/enzimologia , Rhizopus/enzimologia , Fermentação , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Mucor/metabolismo , Rhizopus/metabolismo , Especificidade por Substrato , Temperatura
13.
Carbohydr Polym ; 218: 324-332, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31221337

RESUMO

Dialdehyde starch (DAS) is a kind of modified starch which contains many active aldehyde groups and has good biocompatibility. In this study, magnetic dialdehyde starch nanoparticles were successfully used to immobilize lipase. The lipase was immobilized onto magnetic nanoparticles by using DAS instead of glutaraldehyde as a crosslinker. The parameters like DAS dosage, enzyme concentration and immobilization time were optimized. Enzymatic properties studies exhibited that after DAS cross-linking, the storage stability of the immobilized enzyme reached 82.5%, and the recycling rate reached 53.6%, whereas in case of glutaraldehyde cross linker, it was 79.4% and 46.8%, the former also exhibited better stability and durability. Compared with the free enzyme, the immobilized enzyme indicated higher acid-base tolerance and thermal stability, and had good enzymatic properties. Magnetic dialdehyde starch nanoparticles may have application prospects as an excellent enzyme carrier, which provides a reference for the preparation of other immobilized enzymes with excellent performance.


Assuntos
Hidrolases de Éster Carboxílico/química , Reagentes para Ligações Cruzadas/química , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Amido/análogos & derivados , Ensaios Enzimáticos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Oxirredução , Ácido Periódico/química , Rhizopus/enzimologia , Amido/química , Temperatura
14.
J Food Sci ; 84(6): 1382-1389, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31141617

RESUMO

Evolution of Rhizopus oryzae and Trichoderma reesei biomass in rice bran, their enzyme activity, and the profile of phenolic compounds released from the lignocellulosic matrices were determined and correlated by principal component analysis (PCA). PCA analysis confirms that cultivation of rice bran affected the release of methanol-soluble phenolic compounds (MSPC), ethanol-soluble phenolic compounds (ESPC), and bound phenolic compounds (BPC) positively, due to their enzymatic activity. The release of MSPC was influenced by the activity of cellulase and endoglucanase, which increased 110.6% and 136.3%, respectively, for Rhizopus oryzae and Trichoderma reesei. Gallic acid was the main component in the MSPC and ESPC compound fractions. Ferulic and syringic acids were found in its bound (BPC) form in the biomass. This study showed that bioactive compounds be released from lignocellulosic materials by fungus action and this process can be conducted to obtain specific phenolic compounds. PRACTICAL APPLICATION: Due the demand by natural compounds with biological activity, such as phenolic compounds, it is interesting to purpose alternatives to enhance their yield, like for instance, by fungal fermentation of lignocellulosic material. Therefore, understanding the relations among different phenolic compounds released and the production of fungal hydrolases during growth of Rhizopus oryzae and Trichoderma reesei in solid state cultivation using rice bran as a substrate is fundamental to control the process. This knowledge gets viable scale up to apply the phenolic compounds as preservative in food chain, because this becomes possible directing the process to obtain specific bioactive compounds in less time of cultivation and with low cost.


Assuntos
Celulase/metabolismo , Oryza/química , Fenóis/análise , Rhizopus/enzimologia , Trichoderma/enzimologia , Biomassa , Celulose/metabolismo , Ácidos Cumáricos/análise , Meios de Cultura , Fermentação , Ácido Gálico/análogos & derivados , Ácido Gálico/análise , Rhizopus/crescimento & desenvolvimento , Trichoderma/crescimento & desenvolvimento
15.
Int J Biol Macromol ; 130: 342-347, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30825565

RESUMO

Lipase r27RCL from Rhizopus chinensis was immobilized onto octyl-modified mesocellular foams (MCFs-C8) via two-step process of enzyme adsorption and cross-linking. Oxidized gum arabic was used as substitute for harmful glutaraldehyde to improve catalytic performance of immobilized enzyme for catalysis in non-aqueous phase. The parameters like aldehyde concentration, cross-linking time were optimized. Cross-linked enzyme aggregates (CLEAs) of lipase r27RCL prepared in MCFs-C8 by using oxidized gum arabic (GA-CLEAs@MCFs-C8) showed the highest esterification activity (145 µmol min-1 mg-1 protein) compared with lipase adsorbed onto MCFs-C8 (MCFs-C8-r27RCL) (98 µmol min-1 mg-1 protein), CLEAs of lipase in MCFs-C8 by glutaraldehyde (G-CLEAs@MCFs-C8) (88 µmol min-1 mg-1 protein) and immobilized lipase onto octyl/epoxy (1,1, v/v) modified MCFs (MCFs-octyl-epoxy-r27RCL) (35 µmol min-1 mg-1 protein). Moreover, GA-CLEAs@MCFs-C8 exhibited excellent thermal and mechanical stability, and could still maintain 69% of initial activity after 5 time cycles.


Assuntos
Enzimas Imobilizadas/química , Lipase/química , Agregados Proteicos , Rhizopus/enzimologia , Adsorção , Biocatálise , Enzimas Imobilizadas/metabolismo , Esterificação , Goma Arábica/química , Lipase/metabolismo , Fenômenos Mecânicos , Oxirredução , Temperatura
16.
Chem Commun (Camb) ; 55(27): 3951-3954, 2019 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-30874262

RESUMO

A far-red fluorescent probe has been developed for sensing fungal laccase. The probe was used to determine that Rhizopus oryzae had a high level endogenous laccase amongst 24 fungal strains. The Rhizopus oryzae was then used as a biocatalyst for the preparation of dicoumarin resulting in significant inhibition of Mycobacterium tuberculosis H37Ra.


Assuntos
Antituberculosos/farmacologia , Biocatálise , Dicumarol/farmacologia , Corantes Fluorescentes/química , Lacase/análise , Lacase/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Rhizopus/enzimologia , Antituberculosos/química , Antituberculosos/metabolismo , Dicumarol/química , Dicumarol/metabolismo , Testes de Sensibilidade Microbiana , Microscopia Confocal , Estrutura Molecular , Imagem Óptica
17.
Appl Biochem Biotechnol ; 188(3): 677-689, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30617446

RESUMO

Nano-sized Fe3O4 was synthesized by chemical co-precipitation and subsequently modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to introduce aldehyde group on its surface. With the help of "interface activation" by adding sucrose esters-11 as surfactant, lipase from Rhizopus oryzae was successfully immobilized onto the carrier with great enhancement of activity. The hydrolysis activity of immobilized enzyme were 9.16 times and 31.6 times of free enzyme when p-nitrophenol butyrate and p-nitrophenol palmitate were used as substrates. The thermo-stability of immobilized enzyme was also enhanced compared to free enzyme. The immobilized enzyme was successfully applied in synthesis of 1,3-diacyglycerols (1,3-DAG). The specific esterification activity of immobilized enzyme was about 1.5 times of the free enzyme. The immobilized enzyme showed good region-selectivity towards 1,3-diacyglycerols and retained nearly 80% of its activity after reused for 60 times, revealing a good industrial application prospect.


Assuntos
Diglicerídeos/síntese química , Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Rhizopus/enzimologia , Catálise , Esterificação , Cinética , Nanopartículas de Magnetita/química
18.
Int J Biol Macromol ; 127: 555-562, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30664969

RESUMO

The influence of polyols on Rhizopus chinensis lipase (RCL) was investigated under high pressure. The poor stability of RCL was observed at 500 MPa at 60 °C without polyols which protected RCL against the loss of activity. The lipase is more stable in phosphate buffer than in tris buffer despite the protection of polyols. The activity was maintained 63% by the sorbitol of 2 mol/L in Tris-HCl buffer but 73% in phosphate buffer after the treatment at 500 MPa and 60 °C for 25 min. The same protective effects could be observed at 1 mol/L of sorbitol, erythritol, xylitol, and mannitol. However, further increase of hydroxyl group number could not significantly improve the enzyme stability. The protection of polyols on RCL appears to depend on both of the polyol nature and the hydroxyl group number. Together with fluorescence spectra, circular dichroism spectra indicated that the chaotic conformation of RCL under high pressure became more ordered with 1 mol/L sorbitol. The results showed that sorbitol effectively stabilized the lipase conformation including the hydrophobic core under extreme conditions. It might be attributed to the interaction of polyols with RCL surface to modify intra-/intermolecular hydrogen bonds, maintaining the hydrophobic interactions within RCL.


Assuntos
Proteínas Fúngicas/química , Temperatura Alta , Lipase/química , Polímeros/química , Pressão , Rhizopus/enzimologia , Estabilidade Enzimática
19.
Appl Biochem Biotechnol ; 187(4): 1143-1157, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30175383

RESUMO

Xylitol is an important functional sugar alcohol which is widely used in industries. To efficiently produce xylitol from xylose by xylose reductase in a single step, the coupled system and the coexpression system using xylose reductase (XR) from Rhizopus oryzae and glucose dehydrogenase (GDH) from Exiguobacterium sibiricum were constructed, the latter of which was used for cofactor regeneration. One hundred fifty grams/liter of xylose could be fully converted to xylitol by employing the two-enzyme coupled system with cell-free extract, and xylitol productivity of 21.2 g/L/h was reached by biotransformation in vitro. The whole-cell biocatalyst coexpressing xylose reductase and glucose dehydrogenase was successfully used to synthesize xylitol via enzymatic reduction of xylose. After optimization of the induction condition, the enzyme activity reached 1533 U/L. Two hundred grams/liter of xylose could be completely converted to xylitol, and the highest xylitol productivity of 6.37 g/L/h was obtained under the optimal transformation conditions. Thus, recombinant Escherichia coli coexpressing xylose reductase and glucose dehydrogenase is a promising candidate for xylitol production from xylose.


Assuntos
Biotecnologia , D-Xilulose Redutase/genética , Escherichia coli/genética , Glucose 1-Desidrogenase/genética , Xilitol/biossíntese , Xilose/metabolismo , Bacillaceae/enzimologia , Bacillaceae/genética , Biocatálise , Biotransformação , D-Xilulose Redutase/metabolismo , Expressão Gênica , Glucose 1-Desidrogenase/metabolismo , Concentração de Íons de Hidrogênio , Rhizopus/enzimologia , Rhizopus/genética , Temperatura
20.
Am J Transplant ; 19(4): 1224-1228, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30282120

RESUMO

Transplant tourism, which is the practice of traveling to other countries for transplant, continues to be a major problem worldwide. We describe a patient who traveled to Pakistan and underwent commercial kidney transplant. He developed life-threatening infections from New Delhi metallo-ß-lactamase-1-producing Enterobacter cloacae and Rhizopus oryzae, resulting in a necrotizing kidney allograft infection and subsequent external iliac artery rupture. He survived after a prolonged course of nonstandardized antimicrobial therapy, including a combination of aztreonam and ceftazidime-avibactam, and aggressive surgical debridement with allograft nephrectomy. The early timing of infection with these unusual organisms localized to the allograft suggests contamination and substandard care at the time of transplant. This case highlights the challenges of caring for these infections and serves as a cautionary tale for the potential complications of commercial transplant tourism.


Assuntos
Infecções Bacterianas/complicações , Enterobacter cloacae/enzimologia , Transplante de Rim , Turismo Médico , Micoses/complicações , Rhizopus/enzimologia , beta-Lactamases/isolamento & purificação , Anti-Infecciosos/administração & dosagem , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Micoses/microbiologia
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