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1.
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
2.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 816-826, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223000

RESUMO

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.


Assuntos
Hidrolases de Éster Carboxílico , Engenharia de Proteínas , Hidrolases de Éster Carboxílico/metabolismo , Catálise , Peso Molecular , Pichia/genética , Pichia/metabolismo , Polimerização , Especificidade por Substrato
3.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
4.
World J Microbiol Biotechnol ; 35(6): 84, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134444

RESUMO

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.


Assuntos
Dosagem de Genes , Pectinas/metabolismo , Pichia/genética , Poligalacturonase/biossíntese , Poligalacturonase/genética , Talaromyces/enzimologia , Talaromyces/genética , Reatores Biológicos , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Hidrólise , Pichia/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/genética , Temperatura Ambiente , Fatores de Tempo
5.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067833

RESUMO

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-ß-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters Km and Vmax were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0-8.0) and was highly thermostable at 55 °C for 72 h. Low Cu2+ concentrations (0.1-1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse "in natura", sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.


Assuntos
Aspergillus fumigatus/enzimologia , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Lignina/metabolismo , Pichia/metabolismo , Aspergillus fumigatus/genética , Biomassa , Celulase/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Hidrólise , Pichia/genética , Pichia/crescimento & desenvolvimento , Termotolerância
6.
Vet Microbiol ; 232: 79-83, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030849

RESUMO

Classical swine fever virus (CSFV) envelope glycoprotein Erns has been shown to bind to cell surface sulphated-heparin-like glycosaminoglycans (GAGs), which participate in cell attachment of the virus. In this study, the CSFV Erns gene was codon optimized for expression in the yeast Pichia pastoris. A C-terminally truncated Erns recombinant protein lacking the previously identified heparin-binding domain (HBD) bound to heparin column, suggesting the presence of another HBD in CSFV Erns. Sequence analyses of the CSFV Erns coding region revealed a common potential N-terminal HBD at residues 301-311. Site-directed mutagenesis of the basic amino acids at K303 and K306 significantly reduced the heparin-binding affinity of the protein. Further mutations of both T310 and H311 had little effect. Thus, a novel potential heparin-binding site near the N-terminus of CSFV strain TD96 Erns has been detected, and the two basic amino acids K303 and K306 are crucial for binding activity to heparin matrix and cell-surface GAGs.


Assuntos
Vírus da Febre Suína Clássica/química , Glicoproteínas/química , Heparina/metabolismo , Proteínas do Envelope Viral/química , Aminoácidos/metabolismo , Sítios de Ligação , Vírus da Febre Suína Clássica/genética , Glicoproteínas/genética , Mutagênese Sítio-Dirigida , Mutação , Fases de Leitura Aberta , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas do Envelope Viral/genética
7.
Prep Biochem Biotechnol ; 49(6): 606-615, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929565

RESUMO

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (kLa) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by MutS P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2 kW m-3. Specific growth rate, and then dextranase production, increased as kLa and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2 L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut+ strain. After a scale-up process, at constant kLa, the specific growth rate was kept at 30 L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30 L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.


Assuntos
Dextranase/biossíntese , Pichia/genética , Proteínas Recombinantes/biossíntese , Oxirredutases do Álcool/genética , Biomassa , Reatores Biológicos , Dextranase/genética , Fermentação , Proteínas Fúngicas/análise , Glicerol/análise , Glicerol/metabolismo , Engenharia Metabólica/métodos , Metanol/análise , Metanol/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Talaromyces/enzimologia , Talaromyces/genética
8.
Molecules ; 24(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991754

RESUMO

Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules.


Assuntos
Proteínas Fúngicas , Expressão Gênica , Pichia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
9.
Prep Biochem Biotechnol ; 49(7): 679-685, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30990115

RESUMO

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.


Assuntos
Asparaginase/genética , Clonagem Molecular/métodos , Pectobacterium chrysanthemi/enzimologia , Pectobacterium chrysanthemi/genética , Asparaginase/química , Expressão Gênica , Genes Bacterianos , Glicosilação , Modelos Moleculares , Pectobacterium chrysanthemi/química , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
10.
Enzyme Microb Technol ; 125: 13-20, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885320

RESUMO

The cell wall integrity and stress response component (WSC) domain was first described in the Wsc-family protein of the yeast Saccharomyces cerevisiae, and later found in diverse eukaryotic organisms. Due solely to their presence in the Wsc-family proteins working as a plasma membrane sensor for surface stress and in a fungal ß-1,3-exoglucanse, WSC domains have been presumed to possess carbohydrate-binding property without any experimental evidence. Aiming at elucidation of function(s) of WSC domains, we characterized a WSC domain-containing alcohol oxidase from the rice blast pathogen Pyricularia oryzae (PoAlcOX). Recombinant PoAlcOX produced with Pichia pastoris showed alcohol oxidase activity toward a wide range of substrates including two aliphatic alcohols, a branched-chain alcohol, a diol, and a polyol. Deletion of the WSC domain virtually unaffected oxidation of these substrates by PoAlcOX, indicating that the domain makes no contribution to the catalytic activity. In analogy to some carbohydrate-binding modules, we inferred that the WSC domain plays a role in protein anchoring, and evaluated binding capability of PoAlcOX to a set of polysaccharide components of fungal and plant cell walls. This revealed that PoAlcOX binds to xylans and fungal chitin/ß-1,3-glucan in the WSC domain-dependent manner, demonstrating for the first time the carbohydrate-binding property of the domain. Additionally, we provide evidence that PoAlcOX immobilized on birch wood xylan retains the catalytic activity. Overall, the data we collected suggest that the role of the WSC domain of PoAlcOX is not recognition of substrates but attaching the enzyme to plant and/or fungal cell wall.


Assuntos
Oxirredutases do Álcool/química , Ascomicetos/enzimologia , Parede Celular/metabolismo , Proteínas Fúngicas/química , Polissacarídeos/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Oryza , Pichia/genética , Pichia/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Xilanos/metabolismo
11.
Enzyme Microb Technol ; 125: 53-62, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885325

RESUMO

The thermostable ß-fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short-chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60-70 °C). In this research, FOS produced by BfrA were characterized by HPAE-PAD analysis as a mixture of 1-kestotriose, 6G-kestotriose (neokestose), and to a major extent 6-kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non-saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized 6-kestotriose as the major component of a FOS mixture that includes minor amounts of tetra- and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3-fold higher than that of the wild-type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg-1, represents an attractive candidate for the continuous production of FOS-containing invert syrup at pasteurization temperatures.


Assuntos
Proteínas de Bactérias/metabolismo , Oligossacarídeos/biossíntese , Thermotoga maritima/enzimologia , beta-Frutofuranosidase/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Oligossacarídeos/química , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sacarose/metabolismo , Temperatura Ambiente , Thermotoga maritima/genética , beta-Frutofuranosidase/genética
12.
Int J Mol Sci ; 20(5)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841519

RESUMO

Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.


Assuntos
Aspergillus niger/enzimologia , Biocatálise , Dissacarídeos/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Quercetina/metabolismo , Aspergillus niger/genética , Dissacarídeos/química , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Microbiologia Industrial/métodos , Pichia/genética , Pichia/metabolismo , Quercetina/química , Rutina/química , Rutina/metabolismo
13.
Microb Cell Fact ; 18(1): 45, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845994

RESUMO

BACKGROUND: Pectinolytic enzymes, which are used in several industries, especially in the clarification process during wine and fruit juice production, represent approximately 10% of the global enzyme market. To prevent the proliferation of undesired microorganisms, to retain labile and volatile flavor compounds, and to save energy, the current trend is to perform this process at low temperatures. However, the commercially available pectinases are highly active at temperatures approximately 50 °C and poorly active at temperatures below 35 °C, which is the reason why there is a constant search for cold-active pectinases. In preliminary studies, pectinolytic activity was detected in cold-adapted yeasts and yeast-like microorganisms isolated from Antarctica. The aim of the present work was to characterize pectinases secreted by these microorganisms and to express the best candidate in Pichia pastoris. RESULTS: Degradation of pectin by extracellular protein extracellular extracts obtained from 12 yeast cultures were assayed in plates at 4 °C to 37 °C and pH from 5.4 to 7.0, obtaining positive results in samples obtained from Dioszegia sp., Phenoliferia glacialis and Tetracladium sp. An enzyme was purified from Tetracladium sp., analyzed by peptide mass fingerprinting and compared to genome and transcriptome data from the same microorganism. Thus, the encoding gene was identified corresponding to a polygalacturonase-encoding gene. The enzyme was expressed in Pichia pastoris, and the recombinant polygalacturonase displayed higher activity at 15 °C than a mesophilic counterpart. CONCLUSIONS: Extracellular pectinase activity was found in three yeast and yeast-like microorganisms from which the highest activity was displayed by Tetracladium sp., and the enzyme was identified as a polygalacturonase. The recombinant polygalacturonase produced in P. pastoris showed high activity at 15 °C, representing an attractive candidate to be applied in clarification processes in the production of fermented beverages and fruit juices.


Assuntos
Ascomicetos/enzimologia , Temperatura Baixa , Poligalacturonase/biossíntese , Regiões Antárticas , Ascomicetos/genética , Basidiomycota/enzimologia , Basidiomycota/genética , Fermentação , Pichia/genética , Pichia/metabolismo , Poligalacturonase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
14.
Biomed Res Int ; 2019: 8010635, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30915359

RESUMO

ß-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The ß-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a ß-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0-8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of ß-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.


Assuntos
Proteínas de Bactérias , Bifidobacterium animalis/enzimologia , Bifidobacterium animalis/genética , Pichia , Proteínas Recombinantes de Fusão , beta-Galactosidase , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , beta-Galactosidase/biossíntese , beta-Galactosidase/genética , beta-Galactosidase/isolamento & purificação
15.
Yeast ; 36(5): 329-339, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30903803

RESUMO

Production of fuel ethanol is one of the possible ways to utilize crude glycerol, substantial amounts of which are produced by biodiesel industry. Earlier, we have described construction of the recombinant strains of methylotrophic thermotolerant yeast Ogataea polymorpha with simultaneous overexpression of the genes PDC1 and ADH1, which produced increased amounts of ethanol from glycerol. In this work, we have further improved these strains by overexpression of genes involved either in oxidative (through dihydroxyacetone) or phosphorylative (through glycerol-3-phosphate) pathway of glycerol catabolism, as well as heterologous gene coding for glycerol transporter FPS1 from Komagataella phaffii (formerly, Pichia pastoris). Obtained recombinant strains produced up to 10.7 g/L of ethanol (with ethanol productivity 30 mg/g of biomass/hr and yield 132 mg/g of consumed glycerol) from pure glycerol and up to 3.55 g/L of ethanol (with ethanol productivity 11.6 mg/g of biomass/hr and yield 72.3 mg/g of consumed glycerol) from crude glycerol as a carbon source, which is approximately 15 times more relative to that of the O. polymorpha wild-type strain and 2.2 more relative to the earlier constructed strain.


Assuntos
Etanol/metabolismo , Glicerol/metabolismo , Redes e Vias Metabólicas/genética , Saccharomycetales/genética , Termotolerância , Biomassa , Metabolismo dos Carboidratos , Fermentação , Fosforilação Oxidativa , Pichia/genética , Pichia/metabolismo , Saccharomycetales/metabolismo
16.
Yeast ; 36(5): 285-296, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30912856

RESUMO

Pichia pastoris is a very popular yeast for recombinant protein production, mainly due to the strong, methanol-inducible PAOX1 promoter. Methanol induction however poses several drawbacks. One approach to improve processes is to use MutS strains with reduced methanol catabolic ability. Various reports claim that MutS allows higher recombinant protein production levels than Mut+ but scarcely elaborate on reasons for differences. In this study, enhanced green fluorescent protein was used as a PAOX1 -driven reporter for the investigation of expression differences between Mut+ and MutS strains. Mut+ exhibited higher responses to methanol, with faster growth (0.07 vs. 0.01 hr-1 ) and higher consumption of methanol (2.25 vs. 1.81 mmol/gDCW .hr) and oxygen (2.2 vs. 0.66 mmol/gDCW .hr) than MutS. Mut+ yielded more biomass than MutS (2.3 vs. 1.3 gDCW /L), and carbon dioxide analysis of bioreactor off-gas suggested that considerable amounts of methanol were consumed by Mut+ via the dissimilatory pathway. In contrast, it was demonstrated that the MutS population switched to an induced state more rapidly than Mut+. In addition, MutS exhibited 3.4-fold higher fluorescence levels per cell (77,509 vs. 23,783 SFU) indicative of higher recombinant protein production. The findings were verified by similar results obtained during the expression of a lipase. Based on the differences in response to methanol versus recombinant protein production, it was proposed that higher energy availability occurs in MutS for recombinant protein synthesis, contrary to Mut+ that uses the energy to maintain high levels of methanol catabolic pathways and biomass production.


Assuntos
Proteínas de Fluorescência Verde/genética , Redes e Vias Metabólicas/genética , Metanol/metabolismo , Pichia/genética , Proteínas Recombinantes/biossíntese , Biomassa , Reatores Biológicos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Fenótipo , Pichia/metabolismo , Proteínas Recombinantes/genética
17.
J Sci Food Agric ; 99(10): 4518-4523, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30868593

RESUMO

BACKGROUND: Transglutaminase (TGase) catalyzes post-translational modification of proteins by γ-glutamyl-ϵ-lysine chain links, covalent conjugation of polyamines, and deamidation. Zea mays TGase (TGZ) is a plant TGase with potential application prospects in the food industry. In this study, two promoter types, PFLD1 and PTEF1 , were compared to improve the expression of TGZ, and the cross-linking effect of recombinant TGZ on the properties of acid-induced milk protein concentrate (MPC) gel was assessed. RESULTS: A higher expression of TGZ was obtained under the induction of PFLD1 with a production of 635 U L-1 . After purification using chromatography, TGZ activity was 0.4 U mg-1 . The results indicated that TGZ treatment has effectively improved the textural properties of MPC gel at strength level and water-holding capacity. Optimal texture of MPC gel was achieved after TGZ treatment using 2 U g-1 TGZ for 2 h at 35 °C and pH 7. CONCLUSION: Comparative analysis of the promoters has greatly contributed to the production of TGZ in the industrial field. Furthermore, the modification of MPC gel texture by TGZ indicated that this recombinant enzyme has a practical value in dairy product, especially in yoghurt industry. © 2019 Society of Chemical Industry.


Assuntos
Expressão Gênica , Proteínas do Leite/química , Pichia/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Transglutaminases/genética , Zea mays/enzimologia , Animais , Bovinos , Proteínas do Leite/metabolismo , Pichia/metabolismo , Proteínas de Plantas/metabolismo , Transglutaminases/metabolismo , Zea mays/genética
18.
Res Vet Sci ; 124: 200-211, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30921567

RESUMO

Myostatin (MSTN) was identified as a negative regulator of skeletal muscle growth. MSTN inhibition by myostatin propeptide (MSPP) increased skeletal muscle mass, myofiber growth and muscle force. Thus, this study was designed to produce wild-type porcine MSPP (WT-MSPP) and its mutated form (D75A-MSPP) in yeast Pichia pastoris and to investigate its potential enhancement of myoblast growth and differentiation. In an in vitro study, C2C12 myoblasts were treated with the purified WT-MSPP or D75A-MSPP (10 µg/mL) in either a regular culture medium or in a differentiation medium for 72 h. In an animal trial, post-weaning C57BL/6 mice fed with a high-fat diet (HFD) were administered WT-MSPP or D75A-MSPP for 6 weeks. The results showed that C2C12 myoblasts treated with the purified WT-MSPP or D75A-MSPP could dramatically promote cell proliferation. Both myoD and myogenin were significantly increased (p < .05) after WT-MSPP or D75A-MSPP treatment. D75A-MSPP was particularly more effective than WT-MSPP in promoting myotube formation (p < .05). The post-weaning mice treated with D75A-MSPP significantly increased both body and muscle weights compared with the mock and WT-MSPP groups (p < .05). Furthermore, the mice treatment with D75A-MSPP could prevent increased glucose injection from inducing glucose elevation. Our data indicated that a mutant-type MSPP (D75A-MSPP) was superior to WT-MSPP in effectively enhancing myofiber growth due to the highly resistant to proteolytic cleavage by the bone morphogenetic protein-1/tolloid (BMP-1/TLD) and thus has potential applications for clinical muscle wasting diseases or for increasing muscle mass in meat-producing animals.


Assuntos
Intolerância à Glucose/veterinária , Mioblastos/fisiologia , Miostatina/metabolismo , Pichia/genética , Sus scrofa/fisiologia , Doenças dos Suínos/tratamento farmacológico , Animais , Dieta Hiperlipídica/efeitos adversos , Intolerância à Glucose/tratamento farmacológico , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Suínos
19.
N Biotechnol ; 51: 67-79, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30822538

RESUMO

AID/APOBEC3 enzymes are cytidine deaminases that mutate antibody and retroviral genes and also mediate extensive tumor genome mutagenesis. The study of purified AID/APOBEC3 proteins is challenged by difficulties with their expression and purification arising from genotoxicity in expression hosts, extensive non-specific protein-protein/DNA/RNA interactions and haphazard oligomerization. To date, expression hosts for purification of AID/APOBEC3 enzymes include bacteria, insect and mammalian cells. Here the establishment and optimization of a yeast expression/secretion system for AID/APOBEC3s are reported, followed by comparison with the same enzymes expressed in bacterial and mammalian hosts. AID and APOBEC3G were expressed successfully in Pichia pastoris, each either with an N-terminal GST tag, C-terminal V5-His tag or as untagged native form. It was verified that the yeast-expressed enzymes exhibit identical biochemical properties to those reported using bacterial and mammalian expression, indicating high fidelity of protein folding. It was demonstrated that the system can be adapted for secretion of the enzymes into the media which was used directly in various enzyme assays. The system is also amenable to elimination of bulky fusion tags, providing native untagged enzymes. Thus, P. pastoris is an advantageous expression factory for AID/APOBEC3 enzymes, considering the cost, time, efficiency and quality of the obtained enzymes. The first report is also provided here of a functionally active, untagged, secreted AID, which may become a useful research reagent. A comprehensive comparison is made of the effect of fusion tags and expression hosts on the biochemical actions of AID and APOBEC3G.


Assuntos
Desaminases APOBEC/biossíntese , Desaminases APOBEC/genética , Citidina Desaminase/biossíntese , Citidina Desaminase/genética , Imunidade , Neoplasias/enzimologia , Pichia/genética , Desaminases APOBEC/isolamento & purificação , Citidina Desaminase/isolamento & purificação , Humanos , Mutagênicos , Neoplasias/metabolismo
20.
Pestic Biochem Physiol ; 155: 15-25, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30857623

RESUMO

Gossypol is a polyphonic toxic compound that is present in cotton plants. The P450 cytochromes CYP6AE14 and CYP9A12 of Helicoverpa armigera are highly induced by gossypol and have been reported to be possibly involved in gossypol degradation. To determine whether the candidate H. armigera CYP6AE14 and CYP9A12 enzymes could metabolize gossypol in vitro, functional recombinant H. armigera CYP6AE14 and CPR (CYP9A12 and CPR) enzymes were successfully expressed in Pichia pastoris (P. pastoris). UPLC-QTOF/MS demonstrated the following results: (1) Free gossypol was spontaneously degraded to the gossypol metabolites G1 (m/z 265) and G2 (m/z 293) without the addition of any enzyme. (2) Free gossypol was observed following the addition of the endogenous or recombinant H. armigera P450 cytochrome CYP6AE14/CYP9A12 enzyme: in the first pathway, free gossypol was dehydroxylated and decarboxylated to G3 (m/z 453), and in the second pathway, the aldehyde group of gossypol and its metabolite were covalently bound with the amine products to form G4 (m/z 437) and G5 (m/z 783). (3) In addition to the gossypol binding pathways, the recombinant H. armigera CPR and CYP9A12 enzymes was found that could further decarboxylate the gossypol intermediate demethylated reduction of gossypolonic acid (m/z 294) and demethylated gossic acid (m/z 265) to G0 (m/z 209) and G0' (m/z 249) respectively.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Gossipol/metabolismo , Mariposas/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Pichia/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Mariposas/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Pichia/genética
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