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1.
Biomacromolecules ; 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32559083

RESUMO

A family of poly(glycerol sebacate) analogs were synthesized by Candida antarctica lipase B (CALB) catalysis to tailor biomaterial properties. Different fractions of glycerol (G) units in PGS were replaced by 1,8-octanediol units (O) units. Poly(glycerol-1,8-octanediol-sebacate), PGOS, synthesized by CALB catalysis with a 1:3 molar ratio of G-to O- units has Mn and Mw values of 9,500 and 92,000, respectively. PGS undergoes fiber fusion during electrospinning and crosslinked PGS rapidly resorbs when implanted. By decreasing the molar ratio of glycerol-to-octanediol from 1:1 to 1:4, the peak melting temperature (Tm) increased from 27 to 47 oC. PGOS with 1:3 G- to O- units was electrospun into nanofibers without the need for a second component. The copolymer is semicrystalline and, when crosslinked, undergoes slow in vitro mass loss (3.5 ± 1.0% in 31 days) at pH 7.4 and 37 oC. Furthermore, PGOS crosslinked films have an Elastic modulus of 106.1 ± 18.6 MPa, which is more than 100-times that of crosslinked PGS. New PGOS polymers showed tunable molecular weights, better thermal properties, and excellent electrospinnability. This work expanded PGS analogs' function making these suitable biodegradable polymers for various biomedical applications.

2.
Biomacromolecules ; 21(2): 493-507, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31820938

RESUMO

Self-assembling peptide materials are promising next-generation materials with applications that include tissue engineering scaffolds, drug delivery, bionanomedicine, and enviro-responsive materials. Despite these advances, synthetic methods to form peptides and peptide-polymer conjugates still largely rely on solid-phase peptide synthesis (SPPS) and N-carboxyanhydride ring-opening polymerization (NCA-ROP), while green methods remain largely undeveloped. This work demonstrates a protease-catalyzed peptide synthesis (PCPS) capable of directly grafting leucine ethyl ester (Leu-OEt) from the C-terminus of a methoxy poly(ethylene glycol)-phenylalanine ethyl ester macroinitiator in a one-pot, aqueous reaction. By using the natural tendency of the growing hydrophobic peptide segment to self-assemble, a large narrowing of the (Leu)x distributions for both mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts, relative to oligo(Leu)x synthesized in the absence of a macroinitiator (mPEG45-Phe-OEt), was achieved. A mechanism is described where in situ ß-sheet coassembly of mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts during polymerization prevents peptide hydrolysis, providing a means to control the degree of polymerization (DP) and dispersity of diblock (Leu)x segments (matrix-assisted laser desorption time-of-flight (MALDI-TOF) x = 5.1, dispersity ≤ 1.02). The use of self-assembly to control the uniformity of peptides synthesized by PCPS paves the way for precise peptide block copolymer architectures with various polymer backbones and amino acid compositions synthesized by a green process.

3.
Biomacromolecules ; 21(2): 508-516, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31756098

RESUMO

This work demonstrates a general strategy for introducing remarkable changes in matrix organization and, consequently, functional properties of bacterial cellulose (BC). BC-producing cells were induced, using a well-defined atomized droplet nutrient delivery (ADND) system, to form pellicles with a regular layered morphology that persists throughout the mat depth. In contrast, the morphology of mats formed by conventional static medium nutrient delivery (SMND) is irregular with no distinguishable pattern. ADND also resulted in larger meso-scale average pore sizes but did not alter the fibril diameter (∼70 nm) and crystallinity index (92-95%). The specific modulus and specific tensile strength of ADND mats are higher than those of SMND mats. This is due to the regularity of dense layers that are present in ADND mats that are able to sustain tensile loads, when applied parallel to these layers. The density of BC films prepared by ADND is 1.63-fold lower than that of the SMND BC film. Consequently, the water contents (g/g) of ADND- and SMND-prepared BC mats are 263 ± 8.85 and 99.6 ± 2.04, respectively. A model that rationalizes differences in mat morphology resulting from these nutrient delivery methods based on nutrient and oxygen concentration gradients is proposed. This work raises questions as to the extent that ADND can be used to fine-tune the matrix morphology and how the resulting lower density mats will alter the diffusion of actives from the films to wound sites and increase the ability of cells to infiltrate the matrix during tissue engineering.

4.
Enzyme Microb Technol ; 133: 109467, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874689

RESUMO

The specific activity and enantioselectivity of immobilized cutinases from Aspergillus oryzae (AoC) and Humicola insolens (HiC) were compared with those of lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Lipase B from Candida antarctica (CALB) for menthol and its analogs that include isopulegol, trans-2-tert-butylcyclohexanol (2TBC), and dihydrocarveol (DHC). Common features of these alcohols are two bulky substituents: a cyclohexyl ring and an alkyl substituent. Dissimilarities are that the alkyl group reside at different positions or have dissimilar structures. The aim was to develop an understanding at a molecular level of similarities and differences in the catalytic behavior of the selected cutinases and lipases as a function of substrate structural elements. The experimental results reflect the (-)-enantioselectivity for AoC, HiC, TLL, and RML, while CALB is only active on DHC with (+)-enantioselectivity. In most cases, AoC has the highest activity while HiC is significantly more active than other enzymes on 2TBC. The E values of AoC, HiC, TLL, and RML for menthol are 27.8, 16.5, 155, and 125, respectively. HiC has a higher activity (>10-fold) on (-)-2TBC than AoC while they exhibit similar activities on menthol. Docking results reveal that the bulky group adjacent to the hydroxyl group determines the enantioselectivity of AoC, HiC, TLL, and RML. Amino acid residues that dominate the enantioselectivity of these enzymes are AoC's Phe195 aromatic ring; HiC's hydrophobic Leu 174 and Ile 169 groups; TLL's ring structures of Trp89, His258 and Tyr21; and Trp88 for RML. Results of this study highlight that cutinases can provide important advantages relative to lipases for enantioselective transformation, most notably with bulky and sterically hindered substrates.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Mentol/análogos & derivados , Catálise , Fungos/enzimologia , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Acoplamento Molecular
5.
Metab Eng ; 56: 39-49, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31449877

RESUMO

γ-Polyglutamic acid (γ-PGA) is a biodegradable polymer naturally produced by Bacillus spp. that has wide applications. Fermentation of γ-PGA using Bacillus species often requires the supplementation of L-glutamic acid, which greatly increases the overall cost. Here, we report a metabolically engineered Corynebacterium glutamicum capable of producing γ-PGA from glucose. The genes encoding γ-PGA synthase complex from B. subtilis (pgsB, C, and A) or B. licheniformis (capB, C, and A) were expressed under inducible promoter Ptac in a L-glutamic acid producer C. glutamicum ATCC 13032, which led to low levels of γ-PGA production. Subsequently, C. glutamicum F343 with a strong L-glutamic acid production capability was tested. C. glutamicum F343 carrying capBCA produced γ-PGA up to 11.4 g/L, showing a higher titer compared with C. glutamicum F343 expressing pgsBCA. By introducing B. subtilis glutamate racemase gene racE under Ptac promoter mutants with different expression strength, the percentage of L-glutamic acid units in γ-PGA could be adjusted from 97.1% to 36.9%, and stayed constant during the fermentation process, while the γ-PGA titer reached 21.3 g/L under optimal initial glucose concentrations. The molecular weight (Mw) of γ-PGA in the engineered strains ranged from 2000 to 4000 kDa. This work provides a foundation for the development of sustainable and cost-effective de novo production of γ-PGA from glucose with customized ratios of L-glutamic acid in C. glutamicum.


Assuntos
Corynebacterium glutamicum , Engenharia Metabólica , Ácido Poliglutâmico , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácido Poliglutâmico/biossíntese , Ácido Poliglutâmico/genética
6.
Front Chem ; 7: 159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30972325

RESUMO

One-hundred percent renewable triphenol-GTF-(glycerol trihydroferulate) and novel bisphenols-GDFx-(glycerol dihydroferulate) were prepared from lignocellulose-derived ferulic acid and vegetal oil components (fatty acids and glycerol) using highly selective lipase-catalyzed transesterifications. Estrogenic activity tests revealed no endocrine disruption for GDFx bisphenols. Triethyl-benzyl-ammonium chloride (TEBAC) mediated glycidylation of all bis/triphenols, afforded innocuous bio-based epoxy precursors GDFxEPO and GTF-EPO. GDFxEPO were then cured with conventional and renewable diamines, and some of them in presence of GTF-EPO. Thermo-mechanical analyses (TGA, DSC, and DMA) and degradation studies in acidic aqueous solutions of the resulting epoxy-amine resins showed excellent thermal stabilities (T d5% = 282-310°C), glass transition temperatures (T g ) ranging from 3 to 62°C, tunable tan α, and tunable degradability, respectively. It has been shown that the thermo-mechanical properties, wettability, and degradability of these epoxy-amine resins, can be finely tailored by judiciously selecting the diamine nature, the GTF-EPO content, and the fatty acid chain length.

7.
Biomacromolecules ; 19(12): 4576-4584, 2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30354071

RESUMO

A facile and effective method is described for the biosynthesis of ultrathin bacterial cellulose (BC) mats, which are green, inexpensive, lightweight, and flexible. Physical properties studied include thickness, morphology, reflectance, transmittance, and crystallinity index. BC mat thickness was varied by controlling the depth of the culture broth so that films with predictable thickness, between 113 and 1114 nm, were produced. These BC films have similar fiber morphology to corresponding mm thick BC films prepared under static culture conditions. To increase BC film hydrophobicity, surface trihexylsilylated BC (THSBC) mats with DSavg 0.015 were prepared. Both native and THSBC mats were investigated as antireflection coatings for silicon substrates. The 328 ± 42 nm thick BC mat demonstrated broadband, interference type antireflection over a spectral range of 500-1800 nm. Different reflection properties obtained as a function of BC film orientation reveals that engineered density gradients can be used to manipulate BC optical properties. Thus, optical quality and environmental friendly ultrathin BC films are promising biomaterials for next-generation optoelectronic devices.


Assuntos
Técnicas de Cultura de Células , Celulose/química , Gluconacetobacter xylinus/crescimento & desenvolvimento , Celulose/biossíntese , Gluconacetobacter xylinus/enzimologia , Interações Hidrofóbicas e Hidrofílicas , Silício/química , Propriedades de Superfície
8.
Org Biomol Chem ; 16(39): 7178-7190, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30252014

RESUMO

Structurally complex biosynthesized building blocks whose structures can be systematically varied are of great interest for the synthesis of manipulable self-organizing supramolecular systems. Sophorolipids (SLs) are an important class of glycolipid biosurfactants that consists of a sophorose (glucose disaccharide) polar head group that allows structural diversification by full or selective acetylation at the 6'- and 6''-positions. Porphyrins are a group of naturally-occurring heterocyclic macromolecular organic compounds that have efficient charge transfer properties. Herein we describe the synthesis of SL-porphyrin conjugates where the number of sophorolipid arms, availability of hydrogen bonding sophorose hydroxyl groups and rigidity of the lipid chain were systematically varied. SLs differing in 'sophorose acetylation' and 'lipid unsaturation' were conjugated to zinc-porphyrin dyes by copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) 'click' chemistry. Mono-, di-, and tetra-conjugation of SL-arms to the zinc-porphyrin core provided variation in SL-arm steric effects. UV-vis spectra in methanol/water reveal features indicative of supramolecular J-type aggregates. The synthesized compounds were designed to provide a library of unique bio-based molecules with built-in variation in non-covalent interactions, hydrogen-bonding, π-π stacking, metal-ligand coordination, dipole-dipole, van der Waals, and hydrophobic interactions for future interrogation of supramolecular self-assembly into functional materials for electro-optical applications.

9.
Biochemistry ; 57(7): 1190-1200, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29328676

RESUMO

Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature (Tg, i.e., 70 °C). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC's tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the production of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temperature for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temperature and concentration.


Assuntos
Actinomycetales/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Polietilenotereftalatos/metabolismo , Actinomycetales/química , Actinomycetales/metabolismo , Hidrolases de Éster Carboxílico/química , Estabilidade Enzimática , Glicosilação , Hidrólise , Modelos Moleculares , Agregados Proteicos , Termodinâmica
10.
Appl Microbiol Biotechnol ; 101(18): 6919-6928, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28761999

RESUMO

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.


Assuntos
Escherichia coli/enzimologia , Pichia/enzimologia , Sulfotransferases/metabolismo , Condroitina/metabolismo , Sulfatos de Condroitina/metabolismo , Escherichia coli/genética , Expressão Gênica , Glicosilação , Humanos , Cinética , Pichia/genética , Polissacarídeos/metabolismo , Sulfotransferases/genética , Transgenes
11.
mBio ; 8(3)2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28588129

RESUMO

Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies.IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts.


Assuntos
Antocianinas/biossíntese , Técnicas Bacteriológicas , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Trifosfato de Adenosina/metabolismo , Antocianinas/genética , Escherichia coli/genética , Fermentação , Flavonoides/biossíntese , Malonil Coenzima A/metabolismo , Engenharia Metabólica/economia , Redes e Vias Metabólicas
12.
Langmuir ; 33(23): 5760-5768, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28510440

RESUMO

Surfactants are ubiquitous constituents of commercial and biological systems that function based on complex structure-dependent interactions. Sophorolipid (SL) n-alkyl esters (SL-esters) comprise a group of modified naturally derived glycolipids from Candida bombicola. Herein, micellar self-assembly behavior as a function of SL-ester chain length was studied. Surface tensions as low as 31.2 mN/m and critical micelle concentrations (CMCs) as low as 1.1 µM were attained for diacetylated SL-decyl ester (dASL-DE) and SL-octyl ester, respectively. For deacetylated SL-esters, CMC values reach a lower limit at SL-ester chains above n-butyl (SL-BE, 1-3 µM). This behavior of SL-esters with increasing hydrophobic tail length is unlike other known surfactants. Diffusion-ordered spectroscopy (DOSY) and T1 relaxation NMR experiments indicate this behavior is due to a change in intramolecular interactions, which impedes the self-assembly of SL-esters with chain lengths above SL-BE. This hypothesis is supported by micellar thermodynamics where a disruption in trends occurs at n-alkyl ester chain lengths above those of SL-BE and SL-hexyl ester (SL-HE). Diacetylated (dA) SL-esters exhibit an even more unusual trend in that CMC increases from 1.75 to 815 µM for SL-ester chain lengths of dASL-BE and dASL-DE, respectively. Foaming studies, performed to reveal the macroscopic implications of SL-ester micellar behavior, show that the observed instability in foams formed using SL-esters are due to coalescence, which highlights the importance of understanding intermicellar interactions. This work reveals that SL-esters are an important new family of green high-performing surfactants with unique structure-property relationships that can be tuned to optimize micellar characteristics.


Assuntos
Ésteres/química , Glicolipídeos , Micelas , Tensão Superficial , Tensoativos
13.
Biotechnol J ; 12(8)2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28488758

RESUMO

Cutinases comprise a family of esterases with broad hydrolytic activity for chain and pendant ester groups. This work aimed to identify and improve an efficient cutinase for cellulose acetate (CA) deacetylation. The development of a mild method for CA fiber surface deacetylation will result in improved surface hydrophilicity and reactivity while, when combined with cellulases, a route to the full recycling of CA to acetate and glucose. In this study, the comparative CA deacetylation activity of four homologous wild-type (wt) fungal cutinases from Aspergillus oryzae (AoC), Thiellavia terrestris (TtC), Fusarium solani (FsC), and Humicola insolens (HiC) was determined by analysis of CA deacetylation kinetics. wt-HiC had the highest catalytic efficiency (≈32 [cm2 L-1 ]-1 h-1 ). Comparison of wt-cutinase catalytic constants revealed that differences in catalytic efficiency are primarily due to corresponding variations in corresponding substrate binding constants. Docking studies with model tetrameric substrates also revealed structural origins for differential substrate binding amongst these cutinases. Comparative docking studies of HiC point mutations led to the identification of two important rationales for engineering cutinases for CA deacetylation: (i) create a tight but not too closed binding groove, (ii) allow for hydrogen bonding in the extended region around the active site. Rationally designed HiC with amino acid substitutions I36S, predicted to hydrogen bond to CA, combined with F70A, predicted to remove steric constraints, showed a two-fold improvement in catalytic efficiency. Continued cutinase optimization guided by a detailed understanding of structure-activity relationships, as demonstrated here, will be an important tool to developing practical cutinases for commercial green chemistry technologies.


Assuntos
Hidrolases de Éster Carboxílico/genética , Celulose/análogos & derivados , Conformação Proteica , Sordariales/metabolismo , Acetilação , Sequência de Aminoácidos/genética , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Sítios de Ligação , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Catálise , Celulose/química , Celulose/genética , Celulose/metabolismo , Dicroísmo Circular , Fusarium/enzimologia , Fusarium/genética , Hidrólise , Cinética , Sordariales/enzimologia , Relação Estrutura-Atividade
14.
Chem Commun (Camb) ; 53(28): 4030-4033, 2017 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-28345083

RESUMO

This paper reports that the bulk polymerization of l-aspartic acid diethyl ester catalyzed by immobilized CAL-B at 80 °C for 24 h gives primarily (∼95%) α-linked poly(l-aspartate) in 70% yield with DPavg = 50 and regioselectivity (α/ß) = 94 : 6. Plots of log{[M]0/[M]t} vs. time and DPavgvs. conversion indicate that this polymerization proceeds in a controlled manner by a chain-growth mechanism up to 90% conversion. Thereafter, competition occurs between chain growth and step mechanisms.

15.
ACS Chem Neurosci ; 8(4): 752-758, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28140557

RESUMO

Peritoneal macrophages (PMACs) and spinal cord astrocytes were exposed to varying concentrations of soluble sophorolipid butyl ester diacetate (SLBEDA) in vitro. Macrophages and astrocytes demonstrated no decrease in viability in response to SLBEDA. Studying pro- and anti-inflammatory genes, PMACs did not show a shift toward a pro-inflammatory phenotype. However, at higher concentrations (3 and 30 µM), astrocytes showed an increase in their expression of glial acidic fibrillary protein. This novel category of compounds poses low risk to PMAC and astrocyte viability; however, the effect on PMAC polarization and astrocyte reactivity requires more elucidation.


Assuntos
Astrócitos/metabolismo , Polaridade Celular/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/biossíntese , Glicolipídeos/farmacologia , Macrófagos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL
16.
Appl Microbiol Biotechnol ; 101(7): 2843-2851, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27975137

RESUMO

Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.


Assuntos
Heparina/biossíntese , Pichia/enzimologia , Pichia/genética , Sulfotransferases/genética , Sulfotransferases/metabolismo , Endotoxinas , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Glicosilação , Heparina/química , Cinética , Pichia/metabolismo , Sulfotransferases/química
17.
Macromol Biosci ; 17(3)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27754612

RESUMO

Oligo(Glu70 -co-Leu30 ), a peptide synthesized by protease catalysis, is functionalized at the N-terminus with a 4-pentenoyl unit and grafted to polyLSL[6'Ac,6″Ac], a glycopolymer prepared by ring-opening metathesis polymerization of lactonic sophorolipid diacetate. First, polyLSL[6'Ac,6"Ac] fiber mats are fabricated by electrospinning. Oxidation of the fiber mats and subsequent reaction with cysteamine lead to thiol-functionalized fiber mats with no significant morphology changes. Grafting of the alkene-modified oligopeptide to thiol-functionalized polyLSL[6'Ac,6″Ac] fiber mats is achieved via "thiol-ene" click reaction. X-ray photoelectron spectroscopy analysis to characterize peptide grafting reveals that about 50 mol% of polyLSL[6'Ac,6''Ac] repeat units at fiber surfaces are decorated with a peptide moiety, out of which about 1/3 of the oligo(Glu70 -co-Leu30 ) units are physically adsorbed to polyLSL[6'Ac,6''Ac]. The results of this work pave the way to precise engineering of polyLSL fiber mats that can be decorated with a potentially wide range of molecules that tailor surface chemistry and biological properties.


Assuntos
Materiais Biocompatíveis/química , Biopolímeros/química , Glicoproteínas/química , Peptídeos/química , Materiais Biocompatíveis/síntese química , Glicoproteínas/síntese química , Peptídeos/síntese química , Espectroscopia Fotoeletrônica , Propriedades de Superfície
18.
Biotechnol Bioeng ; 114(1): 63-73, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27425027

RESUMO

Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.


Assuntos
Aspergillus oryzae/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/metabolismo , Sordariales/enzimologia , Aspergillus oryzae/genética , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Estabilidade Enzimática , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicosilação , Temperatura Alta , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sordariales/genética
19.
Genome Announc ; 4(6)2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27979935

RESUMO

We report here the 4.092-Mb high-quality draft genome assembly of a newly isolated poly-γ-glutamic acid-producing strain, Bacillus subtilis Ia1a. The genome sequence is considered a critical tool to facilitate the engineering of improved production strains. Exopolysaccharides and many industrially important enzymes can be produced by this new strain utilizing different carbon sources.

20.
Bioorg Med Chem Lett ; 26(13): 3089-3092, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27210435

RESUMO

A series of 5,7-dihydroxyflavanone derivatives were efficiently synthesized. Their antimicrobial efficacy on Gram-negative, Gram-positive bacteria and yeast were evaluated. Among these compounds, most of the halogenated derivatives exhibited the best antimicrobial activity against Gram-positive bacteria, the yeast Saccharomyces cerevisiae, and the Gram-negative bacterium Vibrio cholerae. The cytotoxicities of these compounds were low as evaluated on HepG2 cells using a cell viability assay. This study suggests that halogenated flavanones might represent promising pharmacological candidates for further drug development.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Flavanonas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Antifúngicos/síntese química , Antifúngicos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Flavanonas/síntese química , Flavanonas/química , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
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