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1.
Food Chem ; 366: 130699, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34348221

RESUMO

In this work, magnetic chitosan (MCH) beads were synthesized by phase-inversion method, and grafted with polydopamine (PDA) and then used for direct immobilization of Candida rugosa lipase by Schiff base reaction. The amount of immobilized enzyme and the retained activity were found to be 47.3 mg/g and 72.8%, respectively, at pH 7.0, and at 25 °C. The apparent Km (9.7 mmol/L), and Vmax (384 U/mg) values of the immobilized lipase were significantly changed compared to the free lipase. The MCH@PDA-lipase was better thermal and storage stability at different temperatures than those of the free lipase. In hexane medium, the esterification reaction results showed that the maximum conversions of isoamylalcohol and isopentyl alcohol to isoamyl acetate and isopentyl acetate using the MCH@PDA-lipase were found to be 98.4 ± 1.3% and 73.7 ± 0.7%, respectively. These results showed that the MCH@PDA-lipase can be used as an operative immobilized enzyme system for many biotechnological applications.


Assuntos
Quitosana , Lipase , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Ésteres , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Fenômenos Magnéticos , Saccharomycetales
2.
Artigo em Inglês | MEDLINE | ID: mdl-34494946

RESUMO

Six yeast isolates were obtained from rotting wood samples in Brazil and frass of a cerambycid beetle larva in French Guiana. Sequence analysis of the ITS-5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the isolates represent a novel species of Cyberlindnera. This novel species is related to Cyberlindnera japonica, Cyberlindnera xylosilytica, Candida easanensis and Candida maesa. It is heterothallic and produces asci with two or four hat-shaped ascospores. The name Cyberlindnera dasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Cy. dasilvae is CBS 16129T and the designated paratype is CBS 16584. The MycoBank number is 838252. All isolates of Cy. dasilvae were able to convert xylose into xylitol with maximum xylitol production within 60 and 72 h. The isolates produced xylitol with values ranging from 12.61 to 31.79 g l-1 in yeast extract-peptone-xylose medium with 5% xylose. When the isolates were tested in sugarcane bagasse hydrolysate containing around 35-38 g l-1d-xylose, isolate UFMG-CM-Y519 showed maximum xylitol production.


Assuntos
Besouros/microbiologia , Filogenia , Saccharomycetales/classificação , Madeira , Xilitol , Animais , DNA Fúngico/genética , DNA Espaçador Ribossômico , Fezes/microbiologia , Larva/microbiologia , Técnicas de Tipagem Micológica , Saccharomycetales/isolamento & purificação , Análise de Sequência de DNA , Madeira/microbiologia , Xilitol/metabolismo
3.
Enzyme Microb Technol ; 150: 109859, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489049

RESUMO

Exploration of feruloyl esterase (FAE) with the resistance to heat and alkali conditions in biobleaching process to improve the separation efficiency of lignocellulose is the key to achieving green papermaking. Herein, we expressed FAEB of C. thermophilum and obtained a thermostable alkaline FAE that can effectively promote the removal of lignin from pulp. The faeB gene was successfully obtained through genomic Blast strategy and high-efficiency expressed under the control of strong alcohol oxidase promoter in Pichia pastoris. The recombinant CtFAEB has an optimal temperature of 65 °C and pH of 7.0. After treated at 65 °C for 1 h, CtFAEB can still retain 63.21 % of its maximum activity, showing a good thermal stability. In addition, the recombinant CtFAEB has broad pH stability and can retain about 56 % of the maximum activity even at pH 11.0. Compared with the effect of mesophilic FAE, pretreatment with thermostable CtFAEB can promote the delignification by laccase and alkaline hydrogen peroxide from the pulp at 70 °C and pH 9.0. Alignment of the protein sequences of CtFAEB and mesophilic FAE suggested that the percentage of amino acids that easily form alpha helix in CtFAEB increases, which enhances its structural rigidity and thereby improves its thermal stability and alkali tolerance. Our study provides an effective method to obtain thermostable and alkaline FAEs, which will promote its application in biobleaching and other biorefining industries.


Assuntos
Chaetomium , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Chaetomium/genética , Clonagem Molecular , Concentração de Íons de Hidrogênio , Saccharomycetales
4.
Food Sci Technol Int ; 27(6): 485-498, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34487460

RESUMO

The optimal conditions for elaborating oil/water nanoemulsions of lemongrass (LG), cinnamon bark (CB) and cinnamon leaves (CL) essential oils and their antimicrobial activity against Zygosaccharomyces bailii at pH 4.00 were studied. The effect of the emulsification methodology on the physical stability and antimicrobial activity of the nanoemulsions were also evaluated. Furthermore, the sensory impact of nanoemulsions added to an apple juice was tested. LG and CL nanoemulsions were elaborated by ultrasonication and CB nanoemulsion, by high-speed homogenization. They were stable for at least 120 days at 25 °C. They exhibited antimicrobial activity against Z. bailii being CB the most effective since it showed the smallest MIC value (156.3 mg/l), followed by LG (468.8 mg/l) and CL (1250.0 mg/l). A slight increase in growth rate was observed due to ultrasonication. An additive interaction in relation to the inhibitory effect between LG and CB nanoemulsions against Z. bailii was observed. While nanoemulsions obtained would be used as natural antimicrobial agents in food and beverage products, only LG nanoemulsion at MIC concentration diluted was acceptable in juice showing the sensory impact of essential oils on foods.


Assuntos
Cymbopogon , Óleos Voláteis , Zygosaccharomyces , Cinnamomum zeylanicum , Emulsões , Óleos Voláteis/farmacologia , Saccharomycetales
5.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34493582

RESUMO

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Engenharia de Proteínas/métodos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais , Sítios de Ligação , COVID-19/virologia , Vacinas contra COVID-19/economia , Humanos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Saccharomycetales/metabolismo , Vacinas de Subunidades
7.
J Agric Food Chem ; 69(38): 11502-11511, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34530618

RESUMO

Nature is harnessed since ancient times to fulfill human needs, and yeast culture has been mastered for bakery, brewery, or the preparation of beverages. In this context, the two recently discovered yeast species Starmerella reginensis and Starmerella kourouensis, belonging to a genus related to fermentative activities in the literature, were explored via untargeted metabolomics approaches. Ultrahigh-performance liquid chromatography hyphenated with tandem mass spectrometry and a deep investigation of molecular networks and spectral data allowed the annotation of, respectively, 439 and 513 metabolites for S. reginensis and S. kourouensis, with approximatively 30% compound annotations and 40% chemical class annotations for both yeast strains. These analyses and Fourier transform ion cyclotron resonance mass spectrometry accurate metabolic profiles unveiled a rich content of alkaloids, lipids, amino acids, and terpenoids for S. reginensis. S. kourouensis presents a similar profile with more sulfated compounds. In short, these results enrich the current knowledge about Starmerella yeast secondary metabolites and reveal their significant structural diversity of small molecules.


Assuntos
Metaboloma , Saccharomycetales , Humanos , Metabolômica , Filogenia , Saccharomycetales/genética , Espectrometria de Massas em Tandem
8.
G3 (Bethesda) ; 11(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34544138

RESUMO

Dissecting the genetic basis of complex trait remains a real challenge. The budding yeast Saccharomyces cerevisiae has become a model organism for studying quantitative traits, successfully increasing our knowledge in many aspects. However, the exploration of the genotype-phenotype relationship in non-model yeast species could provide a deeper insight into the genetic basis of complex traits. Here, we have studied this relationship in the Lachancea waltii species which diverged from the S. cerevisiae lineage prior to the whole-genome duplication. By performing linkage mapping analyses in this species, we identified 86 quantitative trait loci (QTL) impacting the growth in a large number of conditions. The distribution of these loci across the genome has revealed two major QTL hotspots. A first hotspot corresponds to a general growth QTL, impacting a wide range of conditions. By contrast, the second hotspot highlighted a trade-off with a disadvantageous allele for drug-free conditions which proved to be advantageous in the presence of several drugs. Finally, a comparison of the detected QTL in L. waltii with those which had been previously identified for the same trait in a closely related species, Lachancea kluyveri was performed. This analysis clearly showed the absence of shared QTL across these species. Altogether, our results represent a first step toward the exploration of the genetic architecture of quantitative trait across different yeast species.


Assuntos
Locos de Características Quantitativas , Saccharomycetales , Mapeamento Cromossômico , Dissecação , Ligação Genética , Genótipo , Fenótipo , Filogenia , Saccharomyces cerevisiae/genética , Saccharomycetales/genética
9.
Appl Microbiol Biotechnol ; 105(20): 7743-7755, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34545417

RESUMO

Fungal aryl-alcohol oxidases (AAOs) are attractive biocatalysts because they selectively oxidize a broad range of aromatic and aliphatic allylic primary alcohols while yielding hydrogen peroxide as the only by-product. However, their use is hampered by challenging and often unsuccessful heterologous expression. Production of PeAAO1 from Pleurotus eryngii ATCC 90787 in Pichia pastoris failed, while PeAAO2 from P. eryngii P34 with an amino acid identity of 99% was expressed at high yields. By successively introducing mutations in PeAAO1 to mimic the sequence of PeAAO2, the double mutant PeAAO1 ER with mutations K583E and Q584R was constructed, that was successfully expressed in P. pastoris. Functional expression was enhanced up to 155 U/l via further replacements D361N (variant NER) or V367A (variant AER). Fed-batch cultivation of recombinant P. pastoris yielded up to 116 mg/l of active variants. Glycosylated PeAAO1 variants demonstrated high stability and catalytic efficiencies similar to PeAAO2. Interestingly, P. pastoris expressing PeAAO1 variant ER contained roughly 13 gene copies but showed similar volumetric activity as NER and AER with one to two gene copies and four times lower mRNA levels. Additional H-bonds and salt bridges introduced by mutations K583E and Q584R might facilitate heterologous expression by enhanced protein folding.Key points• PeAAO1 not expressed in P. pastoris and PeAAO2 well-expressed in Pichia differ at 7 positions.• Expression of PeAAO1 in P. pastoris achieved through mutagenesis based on PeAAO2 sequence.• Combination of K583E and Q584R is essential for expression of PeAAO1 in P. pastoris.


Assuntos
Oxirredutases do Álcool/biossíntese , Pleurotus , Mutação , Pichia/genética , Pichia/metabolismo , Pleurotus/enzimologia , Pleurotus/genética , Proteínas Recombinantes/biossíntese , Saccharomycetales
10.
Bioresour Technol ; 340: 125700, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34426247

RESUMO

This study introduces the implication of immobilization technology in the fermentation process of sophorolipids (SLs) production by Candida bombicola. Firstly, an evaluation system was established for the performance of embedding immobilization and subsequently applied to guide the optimization of operating conditions for sodium alginate immobilization. Correspondingly, the SLs titer increased from 11.4 g/L to 14.6 g/L. Secondly, polyvinyl alcohol was introduced for composite embedding to improve the stability of immobilized beads. Then exogenous addition of 1.5% diatomite further enhanced the fermentation performance of immobilized cells, thereby increasing the SLs titer to 35.9 g/L, which was 2.1 times higher than the original immobilized cells method. Finally, the immobilized cells were tested for three repeated batches of SLs fermentation. Compared to the free cells fermentation, the SLs productivity and substrate conversion rate were increased by 35.5% and 9.1%, respectively. The obtained results showed high potential for application on an industrial scale.


Assuntos
Candida , Ácidos Oleicos , Fermentação , Saccharomycetales
11.
Bioresour Technol ; 340: 125677, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34358990

RESUMO

The xylitol production was performed with acidophilic Meyerozyma caribbica. The particle size of 0.02 ± 0.01 to 0.1 ± 0.05 mm was rich in glucose (12.0 ± 0.5 g/L), whereas 0.5 ± 0.25 to 2.0 ± 0.5 mm had a high content of xylose (8.0 ± 0.5 g/L). The xylitol production in the synthetic, non-detoxified and detoxified hydrolysate media was studied (50 ± 0.5 g/L) using 10% v/v non - induced cells of M. caribbica for 120 h. At the end of fermentation with the specific growth rate of 0.056 ± 0.01 (µ), xylitol yields of 45.00 ± 1.00%, 10.00 ± 1.00% and 54.00 ± 1.00% were obtained. The detoxification of the hydrolysate prepared using an identified corncob particle size of 0.5 ± 0.25 to 2.0 ± 0.5 mm could be used as the prospective pretreatment process for ecofriendly and industrial scale production of xylitol with M. caribbica.


Assuntos
Xilose , Zea mays , Fermentação , Hidrólise , Tamanho da Partícula , Polissacarídeos , Estudos Prospectivos , Saccharomycetales , Açúcares , Xilitol
12.
J Agric Food Chem ; 69(35): 10235-10245, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428899

RESUMO

Myoglobin (MG) is one of the eukaryotic heme-binding proteins that is closely associated with the real color and metallic taste of meat and can be used as a color additive in artificial meat alternatives. However, the traditional extraction methods are expensive and time-consuming and the heterologous biosynthesis of MG has never been reported. Herein, we achieved the secretory expression of porcine MG by engineered Komagataella phaffii using the suitable host (X33), signal peptide (α-factor signal peptide), and modified constitutive promoter (G1 promoter). In addition, the fermentation conditions for MG production were optimized at shaking-flask level (BMGY medium with 40 mg/L of hemin, 30 °C) and at fermenter level (30% DO, feeding 150 mg/L of hemin), resulting in the highest titer of 285.42 mg/L MG in fed-batch fermentations. Furthermore, a purification method for food-grade MG was developed, which can obtain 0.22 mol of heme/mol of MG with 88.0% purity and 66.1% recovery rate.


Assuntos
Mioglobina , Saccharomycetales , Animais , Reatores Biológicos , Fermentação , Mioglobina/genética , Suínos
13.
J Agric Food Chem ; 69(35): 10301-10310, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34449211

RESUMO

Fatty acids have great effects on the maintenance of the cell membrane structure, cell viability, and cell metabolisms. In this study, we sought to elucidate the effects of exogenous fatty acids on the salt tolerance of food yeast Zygosaccharomyces rouxii. Results showed that Z. rouxii can grow by using exogenous fatty acids (C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, and C18:2) as the sole carbon source. Four fatty acids (C12:0, C16:0, C16:1, and C18:1) can improve the salt tolerance of cells, enhance the formation of the cell biofilm, regulate the chemical compositions, restore growth in the presence of cerulenin, regulate the contents of membrane fatty acids, and control the expression of key genes in the fatty acid metabolism. Our results reveal that Z. rouxii can synthesize membrane fatty acids from exogenous fatty acids and the supplementation of these fatty acids can override the need for de novo fatty acid biosynthesis.


Assuntos
Zygosaccharomyces , Ácidos Graxos , Saccharomyces cerevisiae , Saccharomycetales , Tolerância ao Sal , Zygosaccharomyces/genética
14.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360963

RESUMO

The insect immune response is initiated by the recognition of invading microorganisms. Peptidoglycan recognition proteins (PGRPs) function primarily as pattern recognition receptors by specifically binding to peptidoglycans expressed on microbial surfaces. We cloned a full-length cDNA for a PGRP from the Asian corn borer Ostrinia furnacalis (Guenée) and designated it as PGRP1. PGRP1 mRNA was mainly detected in the fat bodies and hemocytes. Its transcript levels increased significantly upon bacterial and fungal challenges. Purified recombinant PGRP1 exhibited binding activity to the gram-positive Micrococcus luteus, gram-negative Escherichia coli, entomopathogenic fungi Beauveria bassiana, and yeast Pichia pastoris. The binding further induced their agglutination. Additionally, PGRP1 preferred to bind to Lys-type peptidoglycans rather than DAP-type peptidoglycans. The addition of recombinant PGRP1 to O. furnacalis plasma resulted in a significant increase in phenoloxidase activity. The injection of recombinant PGRP1 into larvae led to a significantly increased expression of several antimicrobial peptide genes. Taken together, our results suggest that O. furnacalis PGRP1 potentially recognizes the invading microbes and is involved in the immune response in O. furnacalis.


Assuntos
Imunidade Inata , Proteínas de Insetos/metabolismo , Lepidópteros/genética , Peptidoglicano/metabolismo , Animais , Beauveria/patogenicidade , Corpo Adiposo/metabolismo , Hemócitos/metabolismo , Proteínas de Insetos/genética , Lepidópteros/imunologia , Lepidópteros/microbiologia , Micrococcus luteus/patogenicidade , Monofenol Mono-Oxigenase/metabolismo , Peptidoglicano/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Saccharomycetales/patogenicidade
15.
Appl Microbiol Biotechnol ; 105(16-17): 6345-6354, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34410438

RESUMO

VP28 is an envelope protein of White Spot Syndrome Virus (WSSV), which has been shown in previous studies to induce a high immune response in shrimp. VP28 has been produced in some host systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris as free protein. Here we showed a new strategy of anchoring VP28 on the Saccharomyces cerevisiae yeast surface and using the yeast cell extract combined with probiotic as an oral vaccine for shrimp farming. We have successfully constructed a recombinant yeast cell capable of expressing VP28 on the cell surface. The feeding diet combined with VP28 anchored yeast cell extract provided significant assurance to Litopenaeus vannamei, challenged by WSSV, resulting in a relative percent survival (RPS) of 87.10 ± 2.15%. Interestingly, the utilization of VP28 anchored yeast cell extract could enhance the efficiency of probiotic strains like Lactobacillus and Bacillus on shrimp farming. The results in both laboratory scales and field trials using extract of VP28 displaying Saccharomyces showed a growth-promoting effect in shrimp, assessed through average shrimp weight. Taken together, our results in this study demonstrated a new successful strategy of using yeast cell surface as a tool to produce VP28-based oral vaccine for shrimp aquaculture. KEY POINTS: • A new strategy of using VP28 antigen as anchored protein on S. cerevisiae yeast cell surface (S. cerevisiae::VP28) • The utilization of VP28 antigen and yeast as S. cerevisiae::VP28 extract enhanced potential protection of Litopenaeus vannamei against White Spot Syndrome Virus (RPS 87.10%) • The use of S. cerevisiae::VP28 extract increased efficiency of probiotic on shrimp growth-promoting effect either lab-scale or field trial.


Assuntos
Penaeidae , Saccharomyces cerevisiae , Agricultura , Animais , Antígenos de Superfície , Saccharomyces cerevisiae/genética , Saccharomycetales , Proteínas do Envelope Viral
16.
Methods Mol Biol ; 2351: 289-303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382196

RESUMO

Interactions between regulatory proteins and specific genomic regions are critical for all chromatin-based processes, including transcription, DNA replication, and DNA repair. Genome-wide mapping of such interactions is most commonly performed with chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), but a number of orthogonal methods employing targeted enzymatic activity have also been introduced. We previously described a genome-wide implementation of chromatin endogenous cleavage (ChEC-Seq), wherein a protein of interest is fused to micrococcal nuclease (MNase) to enable targeted, calcium-dependent genomic cleavage. Here, we describe the ChEC-Seq protocol for use in budding yeast though it can be used in other organisms in conjunction with appropriate methods for introduction of an MNase fusion protein.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estudo de Associação Genômica Ampla , Estudo de Associação Genômica Ampla/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
17.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361722

RESUMO

Off-flavors produced by undesirable microbial spoilage are a major concern in wineries, as they affect wine quality. This situation is worse in warm areas affected by global warming because of the resulting higher pHs in wines. Natural biotechnologies can aid in effectively controlling these processes, while reducing the use of chemical preservatives such as SO2. Bioacidification reduces the development of spoilage yeasts and bacteria, but also increases the amount of molecular SO2, which allows for lower total levels. The use of non-Saccharomyces yeasts, such as Lachancea thermotolerans, results in effective acidification through the production of lactic acid from sugars. Furthermore, high lactic acid contents (>4 g/L) inhibit lactic acid bacteria and have some effect on Brettanomyces. Additionally, the use of yeasts with hydroxycinnamate decarboxylase (HCDC) activity can be useful to promote the fermentative formation of stable vinylphenolic pyranoanthocyanins, reducing the amount of ethylphenol precursors. This biotechnology increases the amount of stable pigments and simultaneously prevents the formation of high contents of ethylphenols, even when the wine is contaminated by Brettanomyces.


Assuntos
Brettanomyces/metabolismo , Aromatizantes/metabolismo , Tecnologia de Alimentos/métodos , Odorantes/análise , Saccharomycetales/metabolismo , Vinho/análise , Antocianinas/metabolismo , Carboxiliases/metabolismo , Fermentação , Proteínas Fúngicas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Dióxido de Enxofre/farmacologia , Vitis/metabolismo , Vitis/microbiologia , Vinho/microbiologia
18.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360993

RESUMO

The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant glycoforms HexNAc2Hex8 and HexNAc2Hex11 are found at Asn119, Asn378, and Asn743, three of the canonical four N-glycosylation sites of human CP. The availability of high-quality recombinant human CP represents a significant advancement in the field of CP biology. However, productivity needs to be increased and further careful glycoengineering of the SM5 strain is mandatory in order to evaluate the possible therapeutic use of the recombinant protein for enzyme replacement therapy of aceruloplasminemia patients.


Assuntos
Ceruloplasmina/genética , Microbiologia Industrial/métodos , Engenharia de Proteínas/métodos , Saccharomycetales/genética , Ceruloplasmina/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
19.
Molecules ; 26(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34443298

RESUMO

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.


Assuntos
Antracenos/farmacologia , Corantes/farmacologia , Perileno/análogos & derivados , Fármacos Fotossensibilizantes/farmacologia , Plâncton/crescimento & desenvolvimento , Quinonas/farmacologia , Saccharomycetales/crescimento & desenvolvimento , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fluorescência , Morfogênese/efeitos dos fármacos , Perileno/farmacologia , Plâncton/efeitos dos fármacos , Saccharomycetales/citologia , Saccharomycetales/efeitos dos fármacos
20.
J Agric Food Chem ; 69(33): 9608-9615, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34387482

RESUMO

The development of new preservatives is an ongoing investigation in the food industry, especially those which are safe and environmentally friendly. In this study, biosurfactant sophorolipids (SLs) functionalized with amino acids were developed as efficient preservative agents. SLs were first isolated from fermentation broth by Candida bombicola ATCC 22214, hydrolyzed, and purified by extraction. The typical recovery is around 70%, while the extracted material consists of over 90% deacetylated acidic SLs (SL-COOH). Four types of SL derivatives were then synthesized via dicyclohexylcarbodiimide amidation reactions from prepared SL-COOH. Among the derivatives produced, the arginine SL conjugates (SL-d-Arg) displayed the highest activity against Gram-positive bacteria and fungi and even inhibited the cell growth of Gram-negative bacteria and mildew. Furthermore, the arginine conjugates performed the broadest antimicrobial activity among the derivatives evaluated. The sterilization dosage of the arginine conjugates against the food-spoilage pathogen Bacillus spp. was 63-125 mg/L, in contrast to 250 mg/L for the enterotoxin producer Staphylococcus aureus and 500 mg/L for fungi. More importantly, SL-d-Arg displayed excellent biocompatibility, with a therapeutic index of over 7.94. SL-d-Arg has excellent potential as an alternative to traditional chemical preservatives.


Assuntos
Aminoácidos , Glicolipídeos , Ácidos Oleicos , Saccharomycetales
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