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1.
Arch Virol ; 165(5): 1057-1067, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144542

RESUMO

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.


Assuntos
Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Infecções por Vírus Respiratório Sincicial/patologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/isolamento & purificação , Células Th1/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas Virais de Fusão/genética , Viremia/imunologia
2.
Microb Cell Fact ; 19(1): 7, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931833

RESUMO

BACKGROUND: Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches. RESULTS: In this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry. CONCLUSION: This strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.


Assuntos
Glicoproteínas/biossíntese , Engenharia Metabólica/métodos , Pichia/metabolismo , Polissacarídeos/biossíntese , Produtos Biológicos , Biotecnologia , Glicoproteínas/química , Glicosilação , Pichia/genética , Polissacarídeos/química , Proteínas Recombinantes/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Enzyme Microb Technol ; 133: 109459, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874694

RESUMO

2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odor, which is widely used in cosmetics and food industries. Natural 2-PE is costly and cannot meet the market demand due to the relative low content of 2-PE in the plants. Thus, there is an increasing interest in the search for alternative routes for 2-PE production. Here we demonstrate the engineering of Pichia pastoris to produce 2-PE directly from simple sugars for the first time. We first demonstrated that improving downstream pathway from phenylpyruvate to 2-PE by overexpressing ARO10 and ADH6 could increase the biosynthesis of 2-PE. Then several genetic engineering strategies were developed to increase phenylpyruvate availability to improve 2-PE production. 1169 mg/L of 2-PE was accumulated in the final engineered strain. This study showed the potential of P. pastoris as a host strain to produce industrially interested 2-PE by metabolic engineering.


Assuntos
Engenharia Metabólica , Álcool Feniletílico/metabolismo , Pichia/genética , Pichia/metabolismo , Fermentação , Engenharia Genética , Glucose/metabolismo , Microbiologia Industrial
4.
Food Chem ; 305: 125447, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499289

RESUMO

A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2 U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9 kDa via SDS-PAGE. RmAmyA was optimally active at 75 °C and pH 6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16 h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.


Assuntos
Maltose/metabolismo , Rhizomucor/enzimologia , alfa-Amilases/metabolismo , Pão/análise , Indústria Alimentícia , Concentração de Íons de Hidrogênio , Hidrólise , Pichia/metabolismo , Amido/metabolismo , Especificidade por Substrato , Temperatura , alfa-Amilases/química , alfa-Amilases/genética
5.
PLoS One ; 14(12): e0227110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887188

RESUMO

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.


Assuntos
Clonagem Molecular/métodos , Vetores Genéticos , Proteínas Recombinantes/isolamento & purificação , Transfecção/métodos , Animais , Aurora Quinase A/genética , Aurora Quinase A/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Quinases Lim/genética , Quinases Lim/isolamento & purificação , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/isolamento & purificação , Quinases Associadas a rho/genética , Quinases Associadas a rho/isolamento & purificação
6.
Microb Cell Fact ; 18(1): 187, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675969

RESUMO

BACKGROUND: The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. RESULTS: The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. CONCLUSIONS: PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression-heterologous genes included-, thus directly influencing the production kinetics of recombinant protein.


Assuntos
Oxirredutases do Álcool/genética , Proteínas Fúngicas/metabolismo , Metanol/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Dosagem de Genes , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Pichia/genética , Regiões Promotoras Genéticas
7.
Sheng Wu Gong Cheng Xue Bao ; 35(10): 1986-2002, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31668043

RESUMO

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.


Assuntos
Adaptação Fisiológica , Meio Ambiente , Fermentação , Pichia/citologia , Pichia/metabolismo , Butanóis/metabolismo
8.
Parasitol Res ; 118(12): 3419-3427, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31724067

RESUMO

Opisthorchiasis affects millions of people in Southeast Asia and has been strongly associated with bile duct cancer. Current strategic control approaches such as chemotherapy and health education are not sustainable, and a prophylactic vaccine would be a major advance in the prevention of the disease. Tetraspanins are transmembrane proteins previously described as potential vaccine candidates for other helminth infections and are also found in the membranes of the tegument and extracellular vesicles of O. viverrini. Here, we investigated the potential of a recombinant protein encoding for the large extracellular loop of O. viverrini tetraspanin-2 (rOv-LEL-TSP-2) in a hamster vaccination model. Hamsters were vaccinated with 50 and 100 µg of rOv-LEL-TSP-2 produced from Pichia pastoris yeast combined with alum CpG adjuvant via the intraperitoneal route. The number of worms recovered from hamsters vaccinated with rOv-LEL-TSP-2 was significantly reduced compared to adjuvant control groups. Fecal egg output was also significantly reduced in vaccinated animals, and the average length of worms recovered from vaccinated animals was significantly shorter than that of the control group. Vaccinated animals showed significantly increased levels of anti-rOv-TSP-2 IgG in the sera after three immunizations, as well as increased levels of several T helper type 1 cytokines in the spleen including IFN-γ and IL-6 but not the Th2/regulatory cytokines IL-4 or IL-10. These results suggest that rOv-TSP-2 could be a potential vaccine against opisthorchiasis and warrants further exploration.


Assuntos
Opistorquíase/imunologia , Opistorquíase/prevenção & controle , Opisthorchis/imunologia , Vacinas Protozoárias/imunologia , Tetraspaninas/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Ductos Biliares Intra-Hepáticos/parasitologia , Cricetinae , Citocinas/sangue , Humanos , Pichia/metabolismo , Vacinas Protozoárias/administração & dosagem , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Vacinação
9.
Bioengineered ; 10(1): 689-696, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31739735

RESUMO

Epstein-Barr virus (EBV) associated with several diseases such as contagious mononucleosis chronic active EBV infection, and diverse sorts of malignant tumors. Therefore, using applicable vaccines could be advantageous for public health. Yet, the vaccine has been unavailable to protect from EBV so far. In the current study, to develop a multi-peptide vaccine for EBV and assess its expression in Pichia pastoris yeast system, three immunodominant sequences in glycoprotein (gp) 85, gp350 and latent membrane protein 1 (LMP1) were chosen. To construct fusion peptide, -GGGGS- liker was applied. After cloning the fusion peptide in the pPICZαA expression vector, this recombinant vector processed and transfected into Pichia pastoris host cells. The expression of high level of EBV fusion peptide was confirmed by dot blot and SDS-PAGE procedures. The Pichia pastoris is capable of supporting EBV fusion peptide expression. The application of this fusion peptide as a peptide vaccine to fight EBV is suggested.


Assuntos
Herpesvirus Humano 4/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genética , Vacinas Virais/biossíntese , Sequência de Aminoácidos , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/prevenção & controle , Linfoma de Burkitt/virologia , Clonagem Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Herpesvirus Humano 4/genética , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Mononucleose Infecciosa/imunologia , Mononucleose Infecciosa/prevenção & controle , Mononucleose Infecciosa/virologia , Glicoproteínas de Membrana/imunologia , Peptídeos/genética , Peptídeos/imunologia , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas de Subunidades , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologia
10.
Microb Cell Fact ; 18(1): 169, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601211

RESUMO

BACKGROUND: With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. RESULTS: Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. CONCLUSIONS: In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.


Assuntos
Dimetilaliltranstransferase , Pichia , Vitamina K 2/análogos & derivados , Vias Biossintéticas , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Regulação Fúngica da Expressão Gênica , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes , Vitamina K 2/metabolismo
11.
J Agric Food Chem ; 67(42): 11758-11768, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31577438

RESUMO

Patulin contamination not only is a menace to human health but also causes serious environmental problems worldwide due to the synthetic fungicides that are used to control it. This study focused on investigating the patulin degradation mechanism in Pichia caribbica at the molecular level. According to the results, P. caribbica (2 × 106 cells/mL) was able to degrade patulin from 20 µg/mL to an undetectable level in 72 h. The RNA-seq data showed patulin-induced oxidative stress and responses in P. caribbica. The deletion of PcCRG1 led to a significant decrease in patulin degradation by P. caribbica, whereas the overexpression of PcCRG1 accelerated the degradation of patulin. The study identified that PcCRG1 protein had the ability to degrade patulin in vitro. Overall, we demonstrated that the patulin degradation process in P. caribbica was more than one way; PcCRG1 was an S-adenosylmethionine-dependent methyltransferase and played an important role in the patulin degradation process in P. caribbica.


Assuntos
Proteínas Fúngicas/metabolismo , Fungicidas Industriais/metabolismo , Metiltransferases/metabolismo , Patulina/metabolismo , Pichia/metabolismo , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Metiltransferases/química , Metiltransferases/genética , Pichia/enzimologia , Pichia/genética , Alinhamento de Sequência
12.
Microb Cell Fact ; 18(1): 180, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31647018

RESUMO

BACKGROUND: Structurally stable α-galactosidases are of great interest for various biotechnological applications. More thermophilic α-galactosidases with high activity and structural stability have therefore to be mined and characterized. On the other hand, few studies have been performed to prominently enhance the AOX1 promoter activity in the commonly used Pichia pastoris system, in which production of some heterologous proteins are insufficient for further study. RESULTS: ReGal2 encoding a thermoactive α-galactosidase was identified from the thermophilic (hemi)cellulolytic fungus Rasamsonia emersonii. Significantly increased production of ReGal2 was achieved when ReGal2 was expressed in an engineered Pastoris pichia expression system with a modified AOX1 promoter and simultaneous fortified expression of Mxr1 that is involved in transcriptionally activating AOX1. Purified ReGal2 exists as an oligomer and has remarkable thermo-activity and thermo-tolerance, exhibiting maximum activity of 935 U/mg towards pNPGal at 80 °C and retaining full activity after incubation at 70 °C for 60 h. ReGal2 is insensitive to treatments by many metal ions and exhibits superior tolerance to protein denaturants. Moreover, ReGal2 efficiently hydrolyzed stachyose and raffinose in soybeans at 70 °C in 3 h and 24 h, respectively. CONCLUSION: A modified P. pichia expression system with significantly enhanced AOX1 promoter activity has been established, in which ReGal2 production is markedly elevated to facilitate downstream purification and characterization. Purified ReGal2 exhibited prominent features in thermostability, catalytic activity, and resistance to protein denaturants. ReGal2 thus holds great potential in relevant biotechnological applications.


Assuntos
Eurotiales/enzimologia , Pichia , Proteínas Recombinantes , alfa-Galactosidase , Clonagem Molecular , Proteínas Fúngicas/genética , Expressão Gênica , Cinética , Pichia/genética , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa-Galactosidase/química , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
13.
Enzyme Microb Technol ; 131: 109380, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615673

RESUMO

We previously described the fungus Penicillium chrysogenum 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the Pcfae1 gene from Penicillium chrysogenum and examined its synergistic effects on sugar beet pectin. The Pcfae1 gene was cloned and overexpressed in Pichia pastoris KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of Aspergillus oryzae, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0-7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3-8. PcFAE1 activity was only inhibited by Hg2+, and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl p-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified O-[6-O-feruloyl-ß-d-galactopyranosyl-(1→4)]-d-galactopyranose, O-[2-O-feruloyl-α-l-arabinofuranosyl-(1→5)]-l-arabinofuranose, and O-[5-O-feruloyl-α-l-arabinofuranosyl-(1→3)]-O-ß-d-xylopyranosyl-(1→4)-d-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both ß-d-galactopyranosidic and α-l-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-l-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates.


Assuntos
Arabinose/análogos & derivados , Hidrolases de Éster Carboxílico/metabolismo , Ácidos Cumáricos/metabolismo , Galactose/metabolismo , Pectinas/metabolismo , Penicillium chrysogenum/enzimologia , Arabinose/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Pichia/genética , Pichia/metabolismo , Especificidade por Substrato , Temperatura
14.
BMC Res Notes ; 12(1): 596, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533815

RESUMO

OBJECTIVE: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. RESULTS: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg L-1), and were secreted at moderate to high percentages of the total extracellular protein (51-94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U L-1) that could allow their direct application.


Assuntos
Esterases/metabolismo , Ácido Glucurônico/metabolismo , Proteínas Recombinantes/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Esterases/genética , Espaço Extracelular/enzimologia , Fermentação , Metanol/química , Pichia/genética , Pichia/metabolismo
15.
Enzyme Microb Technol ; 130: 109366, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421726

RESUMO

This study investigates how sorbitol/methanol mixed induction affects fermentation performance, dewatering characteristics of cells during harvesting and the profile of host cell proteins (HCP) in the process fluid when producing the target recombinant protein aprotinin. Compared to standard methanol induction, sorbitol/methanol (1:1, C-mol/C-mol) mixed induction improved cellular viability from 92.8 ±â€¯0.3% to 97.7 ±â€¯0.1% although resulted in a reduced product yield from 1.65 ±â€¯0.03 g L-1 to 1.12 ±â€¯0.07 g L-1. On the other hand, average oxygen consumption rate (OUR) dropped from 241.4 ±â€¯21.3 mmol L-1 h-1 to 145.5 ±â€¯6.7 mmol L-1 h-1. Cell diameter decreased over time in the mixed induction, resulting in a D50 value of 3.14 µm at harvest compared to 3.85 µm with methanol. The reduction in cell size enhanced the maximum dewatering efficiency from 78.1 ±â€¯3.9% to 84.5 ±â€¯3.3% as evaluated by using an established ultra scale-down methodology that models pilot and industrial scale disc stack centrifugation. Seventy host cell proteins (HCPs) were identified in clarified supernatant when using sorbitol/methanol mixed induction regimen. The total number of HCPs identified with standard methanol induction was nearly one hundred. The downstream process advantage of the mixed induction lies in improved product purity by reducing both cell mortality and level of released whole cell proteins. This needs to be balanced and optimised against the observed reduction in product yield during fermentation.


Assuntos
Centrifugação , Metanol/metabolismo , Pichia/metabolismo , Sorbitol/metabolismo , Biomassa , Sobrevivência Celular , Fermentação , Oxigênio/metabolismo , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes
16.
Food Microbiol ; 84: 103242, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421747

RESUMO

This paper describes a mixed fermentation model made by assembling block hosting models for the growth of lactic acid bacteria (Lactobacillus plantarum) and a yeast strain (Pichia kluyveri), metabolic production and the physical-chemical changes which occur during the fermentation of gowé. The growth model for P. kluyveri was developed on a synthetic medium following the gamma concept taking into account the effect of pH, temperature, concentrations in glucose, lactic acid and ethanol. Additional parameters for the previously defined L. plantarum growth model were also determined (glucose and ethanol concentrations). The model was validated in three different gowé processing conditions. Even if the model underestimates LAB growth, it explains what occurs in the product and enables in silico extrapolation to various fermentation conditions. The predicted hydrolysis rates of native and gelatinised starches showed that increasing malt content is not an efficient way to increase the sweetness of gowé in contrast to increasing the level of pre-cooking. The builing-block model developed in this study could be applied to many other fermented foods and particularly to non-alcoholic but acid and sweet cereal based beverages.


Assuntos
Fermentação , Lactobacillus plantarum/metabolismo , Modelos Biológicos , Pichia/metabolismo , Hidrólise , Lactobacillus plantarum/crescimento & desenvolvimento , Pichia/crescimento & desenvolvimento
17.
Environ Sci Pollut Res Int ; 26(28): 29366-29378, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31396876

RESUMO

The aim of this study was to improve the ethanol production from pomegranate peels (PPs). Therefore, the effect of enzymatic hydrolysis and different pretreatments on ethanol production by yeasts was examined. There were three different enzyme concentrations (3.6, 7.2, 14.4 FPU/g substrate) tested for enzymatic hydrolysis, and four different PP media, such as WSPP (whole slurry of PP), LFPP (liquid fraction of PP), WSFPP (washed solid fraction of PP) and N-WSFPP (non-washed solid fraction of PP), were prepared. Bioethanol production was monitored for 96 h. Maximum ethanol concentrations were obtained at WSPP medium as 12.69 g/L, 14.35 g/L and 4.23 g/L in Saccharomyces cerevisiae, Kluyveromyces marxianus and Pichia stipitis, respectively. On the other hand, the washing step of biomass increased the kinetic parameters dramatically and the highest theoretical ethanol yields and YP/S values were obtained from WSFPP medium in all tested yeasts. Theoretical ethanol yields were 97.8%, 98.7% and 35.5% for S. cerevisiae, K. marxianus and P. stipitis, respectively. Qp values were observed as 0.98 g/L h, 0.99 g/L h and 0.04 g/L h for the same yeasts. The highest YP/S values were detected as 0.50 g/g for S. cerevisiae, 0.50 g/g for K. marxianus and 0.30 g/g for P. stipitis in the washed pomegranate peel biomass.


Assuntos
Etanol/química , Lythraceae/química , Pichia/metabolismo , Saccharomyces cerevisiae/química , Biomassa , Etanol/metabolismo , Fermentação , Hidrólise/efeitos dos fármacos , Lythraceae/metabolismo , Pichia/química , /metabolismo
18.
J Biotechnol ; 304: 10-15, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400343

RESUMO

Huimcola insolens cutinase (HiC) was heterologously expressed in Pichia pastoris. To avoid a carbon starvation step, fermentation was conducted using combinations of sorbitol with glycerol and methanol in the cell growth and induction phases, respectively. The cutinase productivity (27.71 U mL-1 h-1) was 9.93 U mL-1 h-1 greater than that achieved using traditional two-phase methods, and a cutinase activity of 2660 U mL-1, using p-nitrophenyl butyrate as substrate, was achieved after only 96 h in a 3-L bioreactor. Subsequently, the combination of HiC with Thermobifida fusca cutinase (TfC) in cotton fabric bioscouring was evaluated by monitoring the wettability and dyeability of the fabric. Treatment with 20 U mL-1 of HiC at 80 °C for 5 min followed by 30 U mL-1 of TfC at 50 °C for 1 h gave the best results. The total treatment time was shorter and performance was better than those seen with the alkali method.


Assuntos
Hidrolases de Éster Carboxílico/genética , Pichia/crescimento & desenvolvimento , Saccharomycetales/enzimologia , Biodegradação Ambiental , Reatores Biológicos/microbiologia , Carbono/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Fibra de Algodão , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Têxteis
19.
Genes (Basel) ; 10(8)2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31409011

RESUMO

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.


Assuntos
Hirudinas/genética , Metanol/metabolismo , Pichia/genética , Transcriptoma , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hirudinas/metabolismo , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
J Agric Food Chem ; 67(37): 10505-10512, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31462045

RESUMO

An aspartic protease gene (Bsapa) was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris. The recombinant BsAPA showed maximal activity at pH 3.0 and 75 °C and remained stable at 70 °C and below, indicating the thermostable nature of BsAPA. However, heat inactivation still limits the application of BsAPA. To further improve its thermostability, an autocatalysis site (L205-F206) in BsAPA was identified and three mutants (F193W, K204P, and A371V) were generated based on the analysis of the structure neighboring the autocatalysis site. These mutants have improved thermostability, and their half-life at 75 °C increased by 0.5-, 0.2-, and 0.3-fold, respectively. A triple-site mutant (F193W/K204P/A371V) was generated, with 1.5-fold increased half-life at 80 and a 10.7 °C increased Tm, compared with those of the wild-type. These results indicate that autocatalysis of aspartic protease reduces enzyme thermostability. Furthermore, site-directed mutagenesis at regions near the autocatalysis site is an efficient approach to improve aspartic protease thermostability.


Assuntos
Ascomicetos/enzimologia , Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Ascomicetos/química , Ascomicetos/genética , Ácido Aspártico Proteases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Temperatura Alta , Cinética , Mutagênese Sítio-Dirigida , Mutação , Pichia/genética , Pichia/metabolismo
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