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1.
Sheng Wu Gong Cheng Xue Bao ; 38(1): 217-225, 2022 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-35142132

RESUMO

With the development of global economy, the dramatically increased production of polyethylene terephthalate (PET) plastics has led to a remarkably increased amount of plastic waste. PET waste can be treated by landfill, incineration, or biodegradation. While landfilling and incineration may cause secondary pollution, biodegradation has since received increased attentions due to its environmental friendliness. Recent studies have indicated that the carbohydrate binding module (CBM) can effectively enhance the binding of PET degrading enzymes to PET, and consequently increasing PET degradation rate. Here we constructed a fusion protein BaCBM2-Tfuc containing the BaCBM2 from Bacillus anthraci and the cutinase Tfuc from Thermobifida fusca, by megaprimer PCR of whole plasmids (MEGAWHOP). Notabaly, the PET film degradation efficiency (at 60 ℃) of BaCBM2-Tfuc was 2.8 times that of Tfuc. This study may provide technical support for constructing fusion proteins capable of efficiently degrading PET.


Assuntos
Hidrolases de Éster Carboxílico , Polietilenotereftalatos , Carboidratos , Thermobifida
2.
Sci Total Environ ; 808: 152107, 2022 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-34864034

RESUMO

Polyethylene terephthalate (PET) is a general plastic that produces a significant amount of waste due to its non-biodagradable properties. We obtained four bacteria (Stenotrophomonas pavanii JWG-G1, Comamonas thiooxydans CG-1, Comamonas koreensis CG-2 and Fulvimonas soli GM-1) that utilize PET as a sole carbon source through a novel stepwise screening and verification strategy. PET films pretreated with S. pavanii JWG-G1 exhibited weight loss of 91.4% following subsequent degradation by Thermobifida fusca cutinase (TfC). S. pavanii JWG-G1 was able to colonize the PET surface and maintain high cell viability (over 50%) in biofilm, accelerating PET degradation. Compared with PET films with no pretreatment, pretreatment with S. pavanii JWG-G1 caused the PET surface to be significantly rougher with greater hydrophilicity (contact angle of 86.3 ± 2° vs. 96.6 ± 2°), providing better opportunities for TfC to contact and act on PET. Our study indicates that S. pavanii JWG-G1 could be used as a novel pretreatment for efficiently accelerating PET biodegradation by TfC.


Assuntos
Hidrolases de Éster Carboxílico , Polietilenotereftalatos , Stenotrophomonas , Thermobifida
3.
Biotechnol Bioeng ; 119(2): 470-481, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34755331

RESUMO

Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.


Assuntos
Proteínas de Bactérias , Hidrolases de Éster Carboxílico , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Cinética , Simulação de Acoplamento Molecular , Polietilenotereftalatos/metabolismo , Especificidade por Substrato , Thermobifida/enzimologia
4.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445200

RESUMO

In the present work, different hydrolases were adsorbed onto polypropylene beads to investigate their activity both in short-esters and polyesters synthesis. The software MODDE® Pro 13 (Sartorius) was used to develop a full-factorial design of experiments (DoE) to analyse the thermostability and selectivity of the immobilized enzyme towards alcohols and acids with different chain lengths in short-esters synthesis reactions. The temperature optima of Candida antarctica lipase B (CaLB), Humicola insolens cutinase (HiC), and Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) were 85 °C, 70 °C, and 50 °C. CaLB and HiC preferred long-chain alcohols and acids as substrate in contrast to Thc_Cut1, which was more active on short-chain monomers. Polymerization of different esters as building blocks was carried out to confirm the applicability of the obtained model on larger macromolecules. The selectivity of both CaLB and HiC was investigated and best results were obtained for dimethyl sebacate (DMSe), leading to polyesters with a Mw of 18 kDa and 6 kDa. For the polymerization of dimethyl adipate (DMA) with BDO and ODO, higher molecular masses were obtained when using CaLB onto polypropylene beads (CaLB_PP) as compared with CaLB immobilized on macroporous acrylic resin beads (i.e., Novozym 435). Namely, for BDO the Mn were 7500 and 4300 Da and for ODO 8100 and 5000 Da for CaLB_PP and for the commercial enzymes, respectively. Thc_Cut1 led to polymers with lower molecular masses, with Mn < 1 kDa. This enzyme showed a temperature optimum of 50 °C with 63% of DMA and BDO when compared to 54% and 27%, at 70 °C and at 85 °C, respectively.


Assuntos
Ésteres/síntese química , Aromatizantes/síntese química , Poliésteres/síntese química , Biocatálise , Candida/enzimologia , Hidrolases de Éster Carboxílico/química , Enzimas Imobilizadas/química , Fungos do Gênero Humicola/enzimologia , Proteínas Fúngicas/química , Lipase/química , Polimerização , Thermobifida/enzimologia
5.
J Am Chem Soc ; 143(30): 11690-11702, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34283601

RESUMO

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.


Assuntos
Ácido Aspártico/análogos & derivados , Bacillales/genética , Peptídeos/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Bacillales/metabolismo , Peptídeos/química , Peptídeos/genética , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Thermobifida/genética , Thermobifida/metabolismo
6.
Appl Microbiol Biotechnol ; 105(11): 4551-4560, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34037842

RESUMO

Poly(ethylene terephthalate) (PET) is a major source of plastic pollution. Biodegradation technologies are of paramount interest in reducing or recycling PET waste. In particular, a synergistic microbe-enzyme treatment may prove to be a promising approach. In this study, a synergistic system composed of Microbacterium oleivorans JWG-G2 and Thermobifida fusca cutinase (referred to as TfC) was employed to degrade bis(hydroxyethyl) terephthalate (BHET) oligomers and a high crystalline PET film. A novel degradation product that was obtained by M. oleivorans JWG-G2 treatment alone was identified as ethylene glycol terephthalate (EGT). With the addition of TfC as a second biocatalyst, the highest synergy degrees for BHET oligomers and PET film degradation were 2.79 and 2.26, respectively. The largest amounts of terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) (47 nM and 330 nM, respectively) were detected after combined treatment of PET film with M. oleivorans JWG-G2 at 5 × 103 µL/cm2 and TfC at 120 µg/cm2, and the degree of PET film surface destruction was more significant than those produced by each treatment alone. The presence of extracellular PET hydrolases in M. oleivorans JWG-G2, including three carboxylesterases, an esterase and a lipase, was predicted by whole genome sequencing analysis, and a predicted PET degradation pathway was proposed for the synergistic microbe-enzyme treatment. The results indicated that synergistic microbe-enzyme treatment may serve as a potentially promising tool for the future development of effective PET degradation. KEY POINTS: • An ecofriendly synergistic microbe-enzyme PET degradation system operating at room temperature was first introduced for degrading PET. • A novel product (EGT) was first identified during PET degradation. • Potential PET hydrolases in M. oleivorans JWG-G2 were predicted by whole genome sequencing analysis.


Assuntos
Microbacterium , Polietilenotereftalatos , Hidrolases de Éster Carboxílico/genética , Etilenos , Hidrólise , Ácidos Ftálicos , Thermobifida
7.
Nat Commun ; 12(1): 2511, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947858

RESUMO

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.


Assuntos
Aminoácidos/química , Domínio Catalítico , Peptídeo Sintases/química , Peptídeos/química , Sideróforos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Coenzima A/química , Cristalografia por Raios X , Expressão Gênica , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sideróforos/biossíntese , Especificidade por Substrato , Thermobifida/química , Thermobifida/metabolismo
8.
J Am Chem Soc ; 143(15): 5917-5927, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33823110

RESUMO

Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation. Owing to a locked three-dimensional structure, lasso peptides can be unusually stable toward heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants to evaluate the substrate tolerance of the biosynthetic pathway. By evaluating more than 1000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Sistema Livre de Células , Ciclização , Família Multigênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Especificidade por Substrato , Thermobifida/metabolismo
9.
Sci Rep ; 11(1): 5290, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674702

RESUMO

Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 ß-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.


Assuntos
Sistemas de Secreção Bacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagênese , Proteínas Periplásmicas de Ligação/metabolismo , Via Secretória/genética , Bacillus subtilis/enzimologia , Proteínas de Escherichia coli/genética , Lipase/genética , Lipase/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microrganismos Geneticamente Modificados , Taxa de Mutação , Proteínas Periplásmicas de Ligação/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , Thermobifida/enzimologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
10.
Chem Commun (Camb) ; 57(4): 520-523, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33331834

RESUMO

Saturation mutagenesis at seven first-sphere residues of the cytochrome P450 monooxygenase 154E1 (CYP154E1) from Thermobifida fusca YX was applied to construct a variant with only three substitutions that enabled the effective two-step synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine. A recombinant E. coli whole-cell system was essential for GC/MS based medium-throughput screening and at the same time facilitated the oxidation of the substrate (R)-ketamine at a higher scale for product isolation and subsequent NMR analysis.


Assuntos
Antidepressivos/síntese química , Sistema Enzimático do Citocromo P-450/química , Ketamina/análogos & derivados , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular , Hidroxilação , Ketamina/síntese química , Ketamina/química , Ketamina/metabolismo , Simulação de Acoplamento Molecular , Mutação , Oxirredução , Ligação Proteica , Streptomyces coelicolor/enzimologia , Thermobifida/enzimologia
11.
Biochem Biophys Res Commun ; 532(3): 459-465, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32892948

RESUMO

cis-Prenyltransferases (cis-PTs) catalyze consecutive condensations of isopentenyl diphosphate to an allylic diphosphate acceptor to produce a linear polyprenyl diphosphate of designated length. Dimer formation is a prerequisite for cis-PTs to catalyze all cis-prenyl condensation reactions. The structure-function relationship of a conserved C-terminal RXG motif in cis-PTs that forms inter-subunit interactions and has a role in catalytic activity has attracted much attention. Here, we solved the crystal structure of a medium-chain cis-PT from Thermobifida fusca that produces dodecaprenyl diphosphate as a polyprenoid glycan carrier for cell wall synthesis. The structure revealed a characteristic dimeric architecture of cis-PTs in which a rigidified RXG motif of one monomer formed inter-subunit hydrogen bonds with the catalytic site of the other monomer, while the RXG motif of the latter remained flexible. Careful analyses suggested the existence of a possible long-range negative cooperativity between the two catalytic sites on the two monomeric subunits that allowed the binding of one subunit to stabilize the formation of the enzyme-substrate ternary complex and facilitated the release of Mg-PPi and subsequent intra-molecular translocation at the counter subunit so that the condensation reaction could occur in consecutive cycles. The current structure reveals the dynamic nature of the RXG motif and provides a rationale for pursuing further investigations to elucidate the inter-subunit cooperativity of cis-PTs.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transferases/química , Transferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Estabilidade Enzimática , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Thermobifida/enzimologia , Thermobifida/genética , Transferases/genética
12.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 8): 334-340, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32744244

RESUMO

The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Šresolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.


Assuntos
Proteínas de Bactérias/química , Coenzimas/química , Ferredoxinas/química , NAD/química , Oxirredutases/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Coenzimas/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , NAD/metabolismo , Oxirredutases/genética , Oxirredutases/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 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Thermobifida/química , Thermobifida/enzimologia
13.
J Biotechnol ; 323: 166-173, 2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-32841608

RESUMO

Cladribine (2-chloro-2'-deoxy-ß-d-adenosine) is a 2'-deoxyadenosine analogue, approved by the FDA for the treatment of hairy cell leukemia and more recently has been proved for therapeutic against many autoimmune diseases as multiple sclerosis. The biosynthesis of this compound using Thermomonospora alba CECT 3324 as biocatalyst is herein reported. This thermophilic microorganism was successfully entrapped in polyacrylamide gel supplemented with nanoclays such as bentonite. The immobilized biocatalyst (T. alba-Ac-Bent 1.00 %), was able to biosynthesize cladribine with a conversion of 89 % in 1 h of reaction and retains its activity for more than 270 reuses without significantly activity loss, showing better operational stability and mechanical properties than the natural matrix. A microscale assay using the developed system, could allow the production of at least 181 mg of cladribine in successive bioprocesses.


Assuntos
Biotransformação , Cladribina/metabolismo , Extremófilos/fisiologia , Resinas Acrílicas , Antineoplásicos/uso terapêutico , Vias Biossintéticas , Cladribina/uso terapêutico , Desoxiadenosinas , Geobacillus , Leucemia de Células Pilosas/tratamento farmacológico , Nanocompostos , Temperatura , Thermobifida/crescimento & desenvolvimento , Thermobifida/metabolismo
14.
Biochemistry (Mosc) ; 85(5): 575-582, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32571187

RESUMO

Phenylacetone monooxygenase (EC 1.14.13.92, PAMО) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. PAMО is a promising enzyme for biotechnological processes. In this work, we generated genetic constructs coding for PAMO from Thermobifida fusca, containing N- or C-terminal His6-tags (PAMO N and PAMO C, respectively), as well as PAMO L with the His6-tag attached to the enzyme C-terminus via a 19-a.a. spacer. All PAMO variants were expressed as catalytically active proteins in Escherichia coli BL21(DE3) cells; however, the expression level of PAMO N was 3 to 5 times higher than for the other two enzymes. The catalytic constants (kcat) of PAMO C and PAMO L were similar to that published for PAMO L produced in a different expression system; the catalytic constant for PAMO N was slightly lower (by 15%). The values of Michaelis constants with NADPH for all PAMО variants were in agreement within the published data for PAMO L (within the experimental error); however, the KM for benzylacetone was several times higher. Thermal inactivation studies and differential scanning calorimetry demonstrated that the thermal stability of PAMO N was 3 to 4 times higher compared to that of the enzymes with the C-terminal His6-tag.


Assuntos
Acetona/análogos & derivados , Histidina/química , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Oligopeptídeos/química , Proteínas Recombinantes/metabolismo , Acetona/metabolismo , Cinética , Relação Estrutura-Atividade , Especificidade por Substrato , Thermobifida/enzimologia
15.
J Biotechnol ; 314-315: 8-13, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32275938

RESUMO

Adipic acid is an important dicarboxylic acid, which is an essential building block to synthesize nylon 6-6 fiber. Adipic acid is primarily synthesized from chemical plant, however, this process is associated with a number of environmental concerns including heavy pollution, toxic catalyst and harsh reaction conditions. A decent amount of adipic acid was produced by reconstructing the reversed adipate-degradation pathway (RADP) from Thermobifida fusca in Escherichia coli. However, IPTG was used in the previous study, which was not feasible in the fermentation industry. In this study, strong promoter-5'-UTR complexes (PUTR) were chosen to construct a highly efficient induction-free system to produce adipic acid. First, comparisons of various exogenous 5'-UTR Complexes, as well as a series of E. coli host strains, demonstrated that those genes using E. coli K12 MG1655 as the host strain produced the highest titer of adipic acid. Subsequently, optimizations were applied to enhance the titer of adipate biosynthesizing strains. The highest titer of adipate of 57.6 g L-1 was achieved by fed-batch fermentation. This work offers a better way to enhance the industrial titer of adipate.


Assuntos
Adipatos/metabolismo , Escherichia coli/metabolismo , Regiões 5' não Traduzidas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Escherichia coli/genética , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermobifida/genética , Thermobifida/metabolismo
16.
Nature ; 580(7802): 216-219, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269349

RESUMO

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas , Reciclagem , Actinobacteria/enzimologia , Burkholderiales/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Ácidos Ftálicos/metabolismo , Polimerização , Thermobifida
17.
Viruses ; 11(11)2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717440

RESUMO

Thermobifida fusca is of biotechnological interest due to its ability to produce an array of plant cell wall hydrolytic enzymes. Nonetheless, only one T. fusca bacteriophage with genome information has been reported to date. This study was aimed at discovering more relevant bacteriophages to expand the existing knowledge of phage diversity for this host species. With this end in view, a thermostable T. fusca bacteriophage P318, which belongs to the Siphoviridae family, was isolated and characterized. P318 has a double-stranded DNA genome of 48,045 base pairs with 3'-extended COS ends, on which 52 putative ORFs are organized into clusters responsible for the order of genome replication, virion morphogenesis, and the regulation of the lytic/lysogenic cycle. In comparison with T. fusca and the previously discovered bacteriophage P1312, P318 has a much lower G+C content in its genome except at the region encompassing ORF42, which produced a protein with unknown function. P1312 and P318 share very few similarities in their genomes except for the regions encompassing ORF42 of P318 and ORF51 of P1312 that are homologous. Thus, acquisition of ORF42 by lateral gene transfer might be an important step in the evolution of P318.


Assuntos
Actinobacteria/virologia , Bacteriófagos/isolamento & purificação , Siphoviridae/isolamento & purificação , Bacteriófagos/genética , DNA Viral , Ontologia Genética , Transferência Genética Horizontal , Genoma Viral/fisiologia , Filogenia , Siphoviridae/genética , Thermobifida , Proteínas Virais/genética
18.
Methods Enzymol ; 627: 23-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31630742

RESUMO

The research on biocatalyzed polycondensation has delivered an array of polyesters having molecular weights below 20,000gmol-1 but characterized by controlled structures and desired functionalities. Their unique catalytic efficiency under mild conditions enables enzymes to catalyze the polycondensation of monomers bearing labile lateral moieties that can be easily accessed via post-polymerization modifications. Despite this great potential, nowadays biocatalysts are not employed for polycondensation on industrial scale due to some bottlenecks related to the formulation of biocatalysts and the process configuration, which make the enzymatic technology non-economic. Recycling the enzymatic catalysts is not only a matter of producing an active and robust formulation, but it also requires the optimal integration of such biocatalyst within a specific reactor and process configuration that must enable efficient mass-transfer while preserving the integrity of the enzymatic preparation. In this chapter, we describe examples of integrated experimental-computational approaches for the rational planning and implementation of enzymatic polycondensation using lipase B from Candida antarctica and cutinase 1 from Thermobifida cellulosilytica. They rely on molecular visualization, molecular modeling and chemometrics, which are methods requiring very modest computational power and approachable by operators who do not have specific computational background. The examples also address the sustainability issue, by describing solvent-free processes involving bio-based monomers and biocatalysts immobilized on renewable carriers.


Assuntos
Biocatálise , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Poliésteres/metabolismo , Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Candida/enzimologia , Biologia Computacional , Química Computacional , Modelos Moleculares , Poliésteres/síntese química , Thermobifida
19.
Sci Rep ; 9(1): 13526, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537863

RESUMO

A thermophilic Thermobifida fusca strain UPMC 901, harboring highly thermostable cellulolytic activity, was successfully isolated from oil palm empty fruit bunch compost. Its endoglucanase had the highest activity at 24 hours of incubation in carboxymethyl-cellulose (CMC) and filter paper. A maximum endoglucanase activity of 0.9 U/mL was achieved at pH 5 and 60 °C using CMC as a carbon source. The endoglucanase properties were further characterized using crude enzyme preparations from the culture supernatant. Thermal stability indicated that the endoglucanase activity was highly stable at 70 °C for 24 hours. Furthermore, the activity was found to be completely maintained without any loss at 50 °C and 60 °C for 144 hours, making it the most stable than other endoglucanases reported in the literature. The high stability of the endoglucanase at an elevated temperature for a prolonged period of time makes it a suitable candidate for the biorefinery application.


Assuntos
Actinobacteria/enzimologia , Celulase/isolamento & purificação , Celulase/metabolismo , Actinobacteria/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Carboximetilcelulose Sódica , Celulose , Estabilidade Enzimática/fisiologia , Concentração de Íons de Hidrogênio , Óleo de Palmeira , Temperatura , Thermobifida
20.
Biotechnol Bioeng ; 116(10): 2463-2472, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31184375

RESUMO

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well-studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/química , Celulase/química , Actinobacteria/genética , Aminoácidos Aromáticos , Proteínas de Bactérias/genética , Domínio Catalítico , Celulase/genética , Thermobifida
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