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1.
Polymers (Basel) ; 16(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38337299

RESUMO

The total rate of plastic production is anticipated to surpass 1.1 billion tons per year by 2050. Plastic waste is non-biodegradable and accumulates in natural ecosystems. In 2020, the total amount of plastic waste was estimated to be 367 million metric tons, leading to unmanageable waste disposal and environmental pollution issues. Plastics are produced from petroleum and natural gases. Given the limited fossil fuel reserves and the need to circumvent pollution problems, the focus has shifted to biodegradable biopolymers, such as polyhydroxyalkanoates (PHAs), polylactic acid, and polycaprolactone. PHAs are gaining importance because diverse bacteria can produce them as intracellular inclusion bodies using biowastes as feed. A critical component in PHA production is the downstream processing procedures of recovery and purification. In this review, different bioengineering approaches targeted at modifying the cell morphology and synchronizing cell lysis with the biosynthetic cycle are presented for product separation and extraction. Complementing genetic engineering strategies with conventional downstream processes, these approaches are expected to produce PHA sustainably.

2.
Int J Biol Macromol ; 151: 609-617, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32061700

RESUMO

ß-Glucosidases (BGL) are key members of the cellulase enzyme complex that determine efficiency of lignocellulosic biomass degradation, which have shown great functional importance to many biotechnological systems. A previous reported BGL from Neosartorya fischeri (NfBGL) showed much higher activity than other BGLs. Screening the important residues based on sequence alignment, analyzing a homology model, and subsequent alteration of individually screened residues by site-directed mutagenesis were carried out to investigate the molecular determinants of the enzyme's high catalytic efficiency. Tyr320, located in the wild-type NfBGL substrate-binding pocket was identified as crucial to the catalytic function of NfBGL. The replacement of Tyr320 with aromatic amino acids did not significantly alter the catalytic efficiency towards p-nitrophenyl ß-d-glucopyranoside (pNPG). However, mutants with charged and hydrophilic amino acids showed almost no activity towards pNPG. Computational studies suggested that an aromatic acid is required at position 320 in NfBGL to stabilize the enzyme-substrate complex formation. This knowledge on the mechanism of action of the molecular determinants can also help rational protein engineering of BGLs.


Assuntos
Aspergillus/enzimologia , Tirosina/química , beta-Glucosidase/química , Sequência de Aminoácidos , Aspergillus/genética , Catálise , Domínio Catalítico , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificação
3.
Appl Microbiol Biotechnol ; 97(23): 10003-11, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23604557

RESUMO

An extracellular ß-glucosidase (BGL) from Fusarium oxysporum was purified to homogeneity by a single chromatography step on a gel filtration column. The optimum activity of BGL on cellobiose was observed at pH 5.0 and 60 °C. Under the same conditions, the K(m) and V(max) values for p-nitrophenyl ß-D-glucopyranoside and cellobiose were 2.53 mM, 268 U mg protein(-1) and 20.3 mM, 193 U mg protein(-1), respectively. The F. oxysporum BGL enzyme was highly stable at acidic pH (t 1/2 = 470 min at pH 3). A commercial BGL Novo188 (Novozymes) and F. oxysporum BGL were compared in their ability to supplement Celluclast 1.5 L (Novozymes). In comparison with the commercial Novo188 (267 mg g substrate(-1)), F. oxysporum BGL supplementation released more reducing sugars (330 mg g substrate(-1)) from cellulose under simulated gastric conditions. These properties make F. oxysporum BGL a good candidate as a new commercial BGL to improve the nutrient bioavailability of animal feed.


Assuntos
Ração Animal/análise , Aditivos Alimentares/química , Fusarium/enzimologia , beta-Glucosidase/química , Estabilidade Enzimática , Aditivos Alimentares/isolamento & purificação , Fusarium/química , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Especificidade por Substrato , Temperatura , beta-Glucosidase/isolamento & purificação , beta-Glucosidase/metabolismo
4.
J Microbiol Biotechnol ; 23(3): 351-6, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23462008

RESUMO

A high beta-glucosidase (BGL)-producing strain, Stereum hirsutum, was identified and isolated and showed a maximum BGL activity (10.4 U/ml) when cultured with Avicel and tryptone as the carbon and nitrogen sources, respectively. In comparison with other BGLs, BGL obtained from S. hirsutum showed a higher level of activity to cellobiose (V(max) = 172 U/mg, and k(cat) = 281/s). Under the optimum conditions (600 rpm, 30°C, and pH 6.0), the maximum BGL activity of 10.4 U/ml with the overall productivity of 74.5 U/l/h was observed. BGL production was scaled up from a laboratory scale (7-L fermenter) to a pilot scale (70-L fermenter). When S. hirsutum was cultured in fed-batch culture with rice straw as the carbon source in a 70-L fermenter, a comparable productivity of 78.6 U/l/h was obtained. Furthermore, S. hirsutum showed high levels of activity of other lignocellulases (cellobiohydrolase, endoglucanase, xylanase, and laccase) that are involved in the saccharification of biomasses. Application of S. hirsutum lignocellulases in the hydrolysis of Pinus densiflora and Catalpa ovata showed saccharification yields of 49.7% and 43.0%, respectively, which were higher than the yield obtained using commercial enzymes.


Assuntos
Basidiomycota/enzimologia , Basidiomycota/crescimento & desenvolvimento , Biotecnologia/métodos , Metabolismo dos Carboidratos , beta-Glucosidase/metabolismo , Bignoniaceae/metabolismo , Biomassa , Carbono/metabolismo , Celulose/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Hidrólise , Lignina/metabolismo , Nitrogênio/metabolismo , Oryza/metabolismo , Peptonas/metabolismo , Pinus/metabolismo , Caules de Planta/metabolismo , Temperatura
5.
Mol Biosyst ; 8(12): 3103-6, 2012 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-23059854

RESUMO

A novel type III polyketide synthase (RePKS) from Rhizobium etli produced a heptaketide pyrone using acetyl-CoA and six molecules of malonyl-CoA. Its catalytic efficiency (k(cat)/K(m) = 5230 mM(-1) min(-1)) for malonyl CoA was found to be the highest ever reported. Molecular dynamics studies revealed the unique features of RePKS.


Assuntos
Malonil Coenzima A/metabolismo , Policetídeo Sintases/metabolismo , Pironas/metabolismo , Rhizobium etli/enzimologia , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Malonil Coenzima A/química , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Pironas/química , Rhizobium etli/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
6.
Mol Biosyst ; 8(11): 2864-7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22945364

RESUMO

BPKS from Botrytis cinerea is a novel type III polyketide synthase that accepts C(4)-C(18) aliphatic acyl-CoAs and benzoyl-CoA as the starters to form pyrones, resorcylic acids and resorcinols through sequential condensation with malonyl-CoA. The catalytic efficiency (k(cat)/K(m)) of BPKS was 2.8 × 10(5) s(-1) M(-1) for palmitoyl-CoA, the highest ever reported. Substrate docking analyses addressed the unique features of BPKS such as its high activity and high specificity toward long chain acyl-CoAs.


Assuntos
Acil Coenzima A/metabolismo , Botrytis/enzimologia , Proteínas Fúngicas/metabolismo , Policetídeo Sintases/metabolismo , Catálise , Especificidade por Substrato
7.
Bioresour Technol ; 120: 264-72, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22831905

RESUMO

A basidiomycetous fungus, identified as Pholiota adiposa SKU0714 on the basis of morphological and phylogenetic analyses, was found to secrete efficient lignocellulose degrading enzymes. The strain showed maximum endoglucanase, cellobiohydrolase and ß-glucosidase activities of 26, 32 and 39 U/mL, respectively and also secreted xylanase, laccase, mannanase, and lignin peroxidase with activities of 1680, 0.12, 65 and 0.41 U/mL, respectively when grown with rice straw as a carbon source. Among the various plant biomasses tested for saccharification, poplar biomass produced the maximum amount of reducing sugar. Response surface methodology was used to optimize hydrolysis parameters. A maximum saccharification yield of 83.4% (667 mg/g-substrate), the highest yield from any plant biomass, was obtained with Populus biomass after 24h of hydrolysis. P. adiposa was proven to be a good choice for the production of reducing sugars from cellulosic biomass.


Assuntos
Biomassa , Metabolismo dos Carboidratos , Celulase/metabolismo , Lignina/metabolismo , Pholiota/enzimologia , Populus/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Carbono/farmacologia , Celulose 1,4-beta-Celobiosidase/metabolismo , Eletroforese em Gel de Poliacrilamida , Hidrólise/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Nitrogênio/farmacologia , Pholiota/efeitos dos fármacos , Pholiota/isolamento & purificação , Filogenia , Populus/efeitos dos fármacos , Análise de Regressão , Reprodutibilidade dos Testes , Tensoativos/farmacologia , beta-Glucosidase/metabolismo
8.
Bioresour Technol ; 117: 310-6, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22617039

RESUMO

Enzymatic saccharification of woody biomasses was performed using glycoside hydrolases from Stereum hirsutum, a newly isolated fungal strain found to secrete efficient glycoside hydrolases. The strain showed the highest ß-glucosidase, cellobiohydrolase, endoglucanase, endoxylanase, laccase, and filter paper activity of 10.3, 1.7, 10.3, 29.9, 0.12, and 0.58 U/ml, respectively. Among the various biomasses tested for saccharification, pine biomass produced maximum reducing sugar. Response surface methodology was used to optimize the hydrolysis of pine biomass to achieve the highest level of sugars. The parameters including enzyme, substrate concentration, temperature and pH were found to be critical for the conversion of pine biomass into sugars. Maximum saccharification of 49.7% (435 mg/g-substrate) was obtained after 96 h of hydrolysis. A close agreement between the experimental results and the model predictions was achieved. S. hirsutum could be a good choice for the production of reducing sugars from cellulosic biomasses.


Assuntos
Basidiomycota/enzimologia , Biomassa , Metabolismo dos Carboidratos , Glicosídeo Hidrolases/metabolismo , Madeira/metabolismo , Basidiomycota/efeitos dos fármacos , Basidiomycota/isolamento & purificação , Metabolismo dos Carboidratos/efeitos dos fármacos , Celulase/metabolismo , Celulose 1,4-beta-Celobiosidase/metabolismo , Eletroforese em Gel de Poliacrilamida , Hidrólise/efeitos dos fármacos , Lacase/biossíntese , Modelos Biológicos , Nitrogênio/farmacologia , Filogenia , Pinus/efeitos dos fármacos , Pinus/metabolismo , Análise de Regressão , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos dos fármacos , Tensoativos/farmacologia , Fatores de Tempo , Madeira/efeitos dos fármacos
9.
J Biol Chem ; 287(23): 19429-39, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22500022

RESUMO

The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theory-based screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (K(d)) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of the MDR superfamily enzymes.


Assuntos
Álcool Desidrogenase/química , Proteínas Fúngicas/química , Glicina/química , Neurospora crassa/enzimologia , Zinco/química , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Motivos de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicina/genética , Glicina/metabolismo , Mutagênese Sítio-Dirigida , Neurospora crassa/genética , Estrutura Terciária de Proteína , Zinco/metabolismo
10.
Biotechnol Lett ; 34(7): 1307-13, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22421976

RESUMO

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.


Assuntos
Enzimas Imobilizadas/metabolismo , Nanopartículas , Oligossacarídeos/metabolismo , Pholiota/enzimologia , Dióxido de Silício/metabolismo , Xilosidases/metabolismo , Ligação Proteica
11.
Enzyme Microb Technol ; 50(4-5): 255-62, 2012 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-22418266

RESUMO

NADH oxidase (Nox) catalyzes the conversion of NADH to NAD(+). A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 453 amino acid residues. The molecular mass of the purified LrNox enzyme was estimated to be ~50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 100 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had optimal activity at pH 5.6 and temperature 65 °C, and k(cat)/K(m) of 3.77×10(7) s(-1) M(-1), the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of LrNox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H(2)O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications such as NAD(+) regeneration.


Assuntos
Clonagem Molecular , Escherichia coli/enzimologia , Lacticaseibacillus rhamnosus/enzimologia , Complexos Multienzimáticos , NADH NADPH Oxirredutases , Sequência de Aminoácidos , Biotecnologia/métodos , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/genética , Temperatura Alta , Cinética , Lacticaseibacillus rhamnosus/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Água/metabolismo
12.
Appl Microbiol Biotechnol ; 94(2): 413-23, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22042231

RESUMO

An isolated gene from Neosartorya fischeri NRRL181 encoding a ß-glucosidase (BGL) was cloned, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,467 bp, capable of encoding a polypeptide of 488 amino acid residues. The gene was over-expressed in Escherichia coli, and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified recombinant BGL showed a high level of catalytic activity, with V (max) of 886 µmol min(-1) mg-protein(-1) and a K (m) of 68 mM for p-nitrophenyl-ß-D: -glucopyranoside (pNPG). The optimal temperature for enzyme activity was about 40°C, and the optimal pH was about 6.0. A homology model of N. fischeri BGL1 was constructed based on the X-ray crystal structure of Phanerochaete chrysosporium BGLA. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose shed light on the unique substrate specificity of N. fischeri BGL1 only towards pNPG.


Assuntos
Neosartorya/enzimologia , Neosartorya/genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Temperatura , beta-Glucosidase/química
13.
J Microbiol Biotechnol ; 21(7): 711-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21791957

RESUMO

A highly efficient cellobiohydrolase (CBH)-secreting basidiomycetous fungus, Agaricus arvensis KMJ623, was isolated and identified based on its morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular CBH was purified to homogeneity from A. arvencis culture supernatant using sequential chromatography. The relative molecular mass of A. arvencis CBH was determined to be 65 kDa by SDSPAGE and 130 kDa by size-exclusion chromatography, indicating that the enzyme is a dimer. A. arvencis CBH showed a catalytic efficiency (kcat/Km) of 31.8 mM⁻¹ s⁻¹ for p-nitrophenyl-beta-D-cellobioside, the highest level seen for CBH-producing microorganisms. Its internal amino acid sequences showed significant homology with CBHs from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, A. arvencis CBH is distinguished from other CBHs by its high catalytic efficiency.


Assuntos
Agaricus/classificação , Agaricus/enzimologia , Celulose 1,4-beta-Celobiosidase/metabolismo , Agaricus/genética , Agaricus/isolamento & purificação , Sequência de Aminoácidos , Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/isolamento & purificação , Cromatografia em Gel , Cromatografia Líquida , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Glucosídeos/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Multimerização Proteica , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
14.
Curr Pharm Biotechnol ; 12(8): 1194-204, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21470154

RESUMO

Glycopeptide antibiotics, vancomycin and teicoplanin, inhibit cell wall synthesis in Gram-positive bacteria by interacting with peptidoglycan D-Ala-D-Ala peptide stem termini of the pentapeptide side chains of the peptidoglycan precursors. In glycopeptide-resistant bacteria, multiresistance poses major therapeutic problems. New potent antibacterial agents are needed to combat these resistance problems, resulting in the explosion of novel glycopeptides in recent years. The glycosylation patterns of glycopeptides and the chemical modifications of the glycosyl moieties greatly influence their antibiotic activity, and certain combinations have resulted in highly active new compounds. Considerable efforts have been made to produce semisynthetic glycopeptides with improved pharmacokinetic and pharmacodynamic properties and activity towards resistant strains. This review provides an overview of the chemistry, the antimicrobial activity, the pharmacokinetics and the toxicology of teicoplanin and other glycopeptide antibiotic derivatives.


Assuntos
Antibacterianos/farmacologia , Glicopeptídeos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Farmacorresistência Bacteriana , Glicopeptídeos/síntese química , Glicopeptídeos/química , Relação Estrutura-Atividade
15.
Appl Microbiol Biotechnol ; 90(2): 499-507, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21246353

RESUMO

Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t(1/2) of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K(m) of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into L-xylulose. A∼71% conversion and a productivity of 10.7 g h(-1)l(-1) were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to L-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production.


Assuntos
D-Xilulose Redutase/química , D-Xilulose Redutase/metabolismo , Enzimas Imobilizadas , Nanopartículas/química , Rhizobium etli/enzimologia , Dióxido de Silício/química , Biotransformação , Catálise , Escherichia coli/metabolismo , Xilulose/biossíntese , Xilulose/metabolismo
16.
Appl Microbiol Biotechnol ; 89(3): 635-44, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20852996

RESUMO

Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an L-rhamnose isomerase (L-RhI). The identified L-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k(cat) of 8,971 min⁻¹ and a k(cat)/K(m) of 17 min⁻¹mM⁻¹ for L-rhamnose. Although L-RhIs have been characterized from several other sources, B. halodurans L-RhI is distinguished from other L-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans L-RhI a good choice for industrial applications. This work describes one of the most thermostable L-RhI characterized thus far.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Bacillus/enzimologia , Ramnose/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Bacillus/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Fases de Leitura Aberta , Multimerização Proteica , Análise de Sequência de DNA , Temperatura
17.
Appl Microbiol Biotechnol ; 89(2): 337-44, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20811797

RESUMO

An efficient ß-1,4-glucosidase (BGL) secreting strain, Agaricus arvensis, was isolated and identified. The relative molecular weight of the purified A. arvensis BGL was 98 kDa, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. Using a crude enzyme preparation, A. arvensis BGL was covalently immobilized onto functionalized silicon oxide nanoparticles with an immobilization efficiency of 158%. The apparent V (max) (k (cat)) values of free and immobilized BGL under standard assay conditions were 3,028 U mg protein(-1) (4,945 s(-1)) and 3,347 U mg protein(-1) (5,466 s(-1)), respectively. The immobilized BGL showed a higher optimum temperature and improved thermostability as compared to the free enzyme. The half-life at 65 °C showed a 288-fold improvement over the free BGL. After 25 cycles, the immobilized enzyme still retained 95% of the original activity, thus demonstrating its prospects for commercial applications. High specific activity, high immobilization efficiency, improved stability, and reusability of A. arvensis BGL make this enzyme of potential interest in a number of industrial applications.


Assuntos
Agaricus/enzimologia , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Nanopartículas/química , beta-Glucosidase/química , Agaricus/química , Agaricus/genética , Agaricus/isolamento & purificação , Estabilidade Enzimática , Enzimas Imobilizadas/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Cinética , Peso Molecular , Dióxido de Silício/química , Microbiologia do Solo , beta-Glucosidase/isolamento & purificação
18.
Appl Biochem Biotechnol ; 163(1): 25-39, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20582640

RESUMO

A high cellobiohydrolase (CBH)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 according to the morphology and comparison of internal transcribed spacer rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, a maximum CBH activity of 2.6 U mg-protein(-1), one of the highest among CBH-producing microorganisms, was obtained. The optimum temperature and pH for CBH production were 30 °C and 4.0, respectively. The increased production of CBH in P. purpurogenum culture at 30 °C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. The internal amino acid sequences of P. purpurogenum CBH showed a significant homology with hydrolases from glycoside hydrolase family 7. The extracellular CBH was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast-protein liquid chromatography. The purified CBH was a monomeric protein with a molecular weight of 60 kDa and showed broad substrate specificity with maximum activity towards p-nitrophenyl ß-D: -cellobiopyranoside. P. purpurogenum CBH showed t (1/2) value of 4 h at 60 °C and V (max) value of 11.9 µmol min(-1) mg-protein(-1) for p-nitrophenyl-D: -cellobiopyranoside. Although CBHs have been reported, the high specific activity distinguishes P. purpurogenum CBH.


Assuntos
Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Penicillium/enzimologia , Penicillium/isolamento & purificação , Sequência de Aminoácidos , Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/isolamento & purificação , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Dados de Sequência Molecular , Penicillium/classificação , Penicillium/genética , Filogenia , Alinhamento de Sequência , Microbiologia do Solo
19.
Appl Microbiol Biotechnol ; 89(5): 1435-42, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21038097

RESUMO

Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t1/2) increased from 2 to 275 h) at 50 °C following immobilization.


Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Bacillus/enzimologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Dióxido de Silício/química , Aldose-Cetose Isomerases/isolamento & purificação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Temperatura
20.
J Microbiol Biotechnol ; 20(10): 1415-23, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21030826

RESUMO

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular weight of F. pinicola xylanase was determined to be 58 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of 70 degreesC. The enzyme showed t(1/2) value of 33 h at 70 degrees C and catalytic efficiency (k(cat) = 77.4 s⁻¹, k(cat)/K(m) = 22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.


Assuntos
Agaricales/enzimologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Agaricales/química , Agaricales/genética , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Temperatura Alta , Hidrólise , Cinética , Peso Molecular , Especificidade por Substrato
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