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1.
PLoS One ; 15(7): e0235532, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614905

RESUMO

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.


Assuntos
Centrômero/genética , Colorimetria/métodos , Pichia/genética , Plasmídeos/análise , DNA Fúngico/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
2.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 959-968, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567279

RESUMO

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.


Assuntos
L-Lactato Desidrogenase , Lactobacillus casei , Ácidos Fenilpirúvicos , Pichia , L-Lactato Desidrogenase/genética , Lactobacillus casei/enzimologia , Lactobacillus casei/genética , Ácidos Fenilpirúvicos/metabolismo , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Food Chem ; 328: 127110, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32464557

RESUMO

In the present work we aimed to demonstrate the influence of inoculum starter in support high quality fermentation. Cocoa fermentations were performed in wooden boxes and eight yeasts strains were used in separated fermentations of fine cocoa, type Scavina, as starter inoculum. Temperature, pH, titirable acidity, reducing sugar and free amino acids were evaluated during or after fermentation. The influence of starters yeasts on the decrease of acidity, sugar concentration and free amino acids was significant. The strains Candida parapsilosis, Torulaspora delbrueckii and Pichia kluyveri showed greater changes in the reducing sugar and free amino acids in fermented cocoa beans. These results indicate the ability of yeast used as inoculum starter to modify the end condition and further enhance the quality of fine cocoa beans.


Assuntos
Cacau , Microbiologia de Alimentos/métodos , Leveduras , Aminoácidos/análise , Aminoácidos/metabolismo , Cacau/química , Cacau/metabolismo , Candida parapsilosis/genética , Candida parapsilosis/metabolismo , Chocolate , Fermentação , Concentração de Íons de Hidrogênio , Pichia/genética , Pichia/metabolismo , Sementes/química , Sementes/microbiologia , Temperatura , Torulaspora/genética , Torulaspora/metabolismo , Leveduras/genética , Leveduras/metabolismo
4.
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
5.
J Biosci Bioeng ; 130(1): 29-35, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32171656

RESUMO

Saccharomyces cerevisiae can obtain xylose utilization capacity via integration of heterogeneous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes into its metabolic pathway, and XYL2 which encodes the XDH plays an essential role in this process. Herein, we reported that two hypothetical XYL2 genes from the multistress-tolerant yeasts of Issatchenkia orientalis and Torulaspora delbrueckii were cloned, and they encoded two XDHs, IoXyl2p and TdXyl2p, respectively, with the activities for oxidation of xylitol to xylulose. Comparative studies demonstrated that IoXyl2p and TdXyl2p, like the SsXyl2p from Scheffersomyces stipitis, were probably localized to the cytoplasm and strictly dependent on NAD+ rather than NADP+ as the cofactor for catalyzing the oxidation reaction of xylitol. IoXyl2p had the highest specific activity, maximum velocity (Vmax), affinity to xylitol (Km), and catalytic efficiency (kcat/Km) among the three XDHs. The optimum temperature for oxidation of xylitol were at 45 °C by IoXyl2p and at 35 °C by TdXyl2p and SsXyl2p, and the optimum pH of IoXyl2p, TdXyl2p and SsXyl2p for oxidation of xylitol was 8.0, 8.5 and 7.5, respectively. Mg2+ promoted the activities of IoXyl2p and TdXyl2p, but slightly inhibited the activity of SsXyl2p. Most metal ions had much weaker inhibition effects on IoXyl2p and TdXyl2p than SsXyl2p. IoXyl2p displayed the strongest salt resistance among the three XDHs. To summarize, IoXyl2p from I. orientalis and TdXyl2p from T. delbrueckii characterized in this study are considered to be the attractive candidates for the construction of genetically engineered S. cerevisiae for efficiently fermentation of carbohydrate in lignocellulosic hydrolysate.


Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pichia/enzimologia , Torulaspora/enzimologia , Clonagem Molecular , D-Xilulose Redutase/química , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/química , Cinética , Pichia/genética , Pichia/metabolismo , Torulaspora/genética , Torulaspora/metabolismo , Xilitol/metabolismo , Xilose/metabolismo
6.
Enzyme Microb Technol ; 134: 109481, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044028

RESUMO

The recombinant endoglucanase gene (EG I) from Trichoderma reesei was successfully expressed in Pichia pastoris for the purpose of producing oligosaccharides from various biomass-derived substrates. Interestingly, the recombinant endoglucanase I (ReEG I) showed the catalytic activity towards both cellulose and xylan hydrolysis, yet it was more efficient with xylans. Among various glucans and xylans substrates (paper pulp, carboxymethylated cellulose, oat spelt xylan, birchwood xylan), birchwood xylan displayed a higher yield of xylooligosaccharides (XOS) (69.5 % after optimization). Eventually, it was observed that ReEG I could simultaneously produce XOS and COS, when the alkali-extracted corncob residues were used as substrate. This is the first report on simultaneous production of XOS and COS by recombinant endoglucanase I from Trichoderma reesei expressed in Pichia pastoris, where a novel application of genetically engineered enzymes is proposed to provide an attractive application for high value utilization of biomass.


Assuntos
Biomassa , Celulase/genética , Oligossacarídeos/biossíntese , Trichoderma/enzimologia , Celulase/metabolismo , Celulose/metabolismo , Hidrólise , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Trichoderma/genética , Xilanos/metabolismo , Zea mays/metabolismo
7.
Microb Cell Fact ; 19(1): 35, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070347

RESUMO

Porcine growth hormone (pGH) is a class of peptide hormones secreted from the pituitary gland, which can significantly improve growth and feed utilization of pigs. However, it is unstable and volatile in vitro. It needs to be encapsulated in liposomes when feeding livestock, whose high cost greatly limits its application in pig industry. Therefore we attempted to express pGH as intracellular soluble protein in Pichia pastoris and feed these yeasts with partial wall-breaking for swine, which could release directly pGH in intestine tract in case of being degraded in intestinal tract with low cost. In order to improve the intracellular soluble expression of pGH protein in Pichia pastoris and stability in vitro, we optimized the pGH gene, and screened molecular chaperones from E. coli and Pichia pastoris respectively for co-expressing with pGH. In addition, we had also explored conditions of mechanical crushing and fermentation. The results showed that the expression of intracellular soluble pGH protein was significantly increased after gene optimized and co-expressed with Ssa1-Sis1 chaperone from Pichia pastoris. Meanwhile, the optimal conditions of partial wall-breaking and fermentation of Pichia pastoris were confirmed, the data showed that the intracellular expression of the optimized pGH protein co-expressed with Ssa1-Sis1 could reach 340 mg/L with optimal conditions of partial wall-breaking and fermentation. Animal experiments verified that the optimized pGH protein co-expression with Ssa1-Sis1 had the best promoting effects on the growth of piglets. Our study demonstrated that Ssa1-Sis1 could enhance the intracellular soluble expression of pGH protein in Pichia pastoris and that partial wall-breaking of yeast could prevent pGH from degradation in vitro, release targetedly in the intestine and play its biological function effectively. Our study could provide a new idea to cut the cost effectively, establishing a theoretical basis for the clinic application of unstable substances in vitro.


Assuntos
Proteínas Fúngicas/metabolismo , Hormônio do Crescimento/biossíntese , Chaperonas Moleculares/metabolismo , Pichia/metabolismo , Suínos/crescimento & desenvolvimento , Animais , Clonagem Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Pichia/genética , Proteínas Recombinantes/biossíntese
8.
Nature ; 577(7792): 711-716, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969704

RESUMO

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes1. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expression.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Pichia , Regiões Promotoras Genéticas/genética , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/química , Transativadores/metabolismo , Sítios de Ligação , DNA Fúngico/química , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Pichia/química , Pichia/genética , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/química , Fator de Transcrição TFIIA/química , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/metabolismo
9.
Sci Rep ; 10(1): 1383, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992813

RESUMO

Small heat shock proteins (sHSPs) have been thought to function as chaperones, protecting their targets from denaturation and aggregation when organisms are subjected to various biotic and abiotic stresses. We previously reported an sHSP from Oryza sativa (OsHSP20) that homodimerizes and forms granules within the cytoplasm but its function was unclear. We now show that OsHSP20 transcripts were significantly up-regulated by heat shock and high salinity but not by drought. A recombinant protein was purified and shown to inhibit the thermal aggregation of the mitochondrial malate dehydrogenase (MDH) enzyme in vitro, and this molecular chaperone activity suggested that OsHSP20 might be involved in stress resistance. Heterologous expression of OsHSP20 in Escherichia coli or Pichia pastoris cells enhanced heat and salt stress tolerance when compared with the control cultures. Transgenic rice plants constitutively overexpressing OsHSP20 and exposed to heat and salt treatments had longer roots and higher germination rates than those of control plants. A series of assays using its truncated mutants showed that its N-terminal arm plus the ACD domain was crucial for its homodimerization, molecular chaperone activity in vitro, and stress tolerance in vivo. The results supported the viewpoint that OsHSP20 could confer heat and salt tolerance by its molecular chaperone activity in different organisms and also provided a more thorough characterization of HSP20-mediated stress tolerance in O. sativa.


Assuntos
Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP20 , Microrganismos Geneticamente Modificados/metabolismo , Oryza/genética , Pichia/metabolismo , Proteínas de Plantas , Multimerização Proteica , Tolerância ao Sal , Escherichia coli/genética , Proteínas de Choque Térmico HSP20/biossíntese , Proteínas de Choque Térmico HSP20/genética , Microrganismos Geneticamente Modificados/genética , Pichia/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Estresse Salino/genética
10.
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
11.
Protein Expr Purif ; 169: 105572, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31972264

RESUMO

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.


Assuntos
Tripsina/biossíntese , Tripsinogênio/biossíntese , Antígenos/biossíntese , Western Blotting/métodos , Clonagem Molecular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoensaio/métodos , Mutação/genética , Pichia/genética , Pichia/metabolismo , Isoformas de Proteínas/genética , Proteínas Recombinantes/imunologia , Tripsina/genética , Tripsina/imunologia , Tripsinogênio/genética , Tripsinogênio/imunologia
12.
Microbiol Res ; 232: 126372, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31759230

RESUMO

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos Tipo Acasalamento/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos Tipo Acasalamento/fisiologia , Haploidia , Proteínas de Homeodomínio/genética , Família Multigênica , Pichia/genética , Pichia/fisiologia , Proteínas Repressoras/genética , Reprodução/genética , Reprodução/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia
13.
Food Chem ; 310: 125970, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31838375

RESUMO

Glucose oxidases are widely used in various industrial processes, including bread baking. In this study, a novel glucose oxidase gene, CngoxA, from Cladosporium neopsychrotolerans SL16, was cloned and expressed in Pichia pastoris. Recombinant CnGOXA exhibited maximal activity at 20 °C and pH 7.0, and was stable at 30 °C and pH 6.0-9.0 for 1 h, with a half-life of 15 min at 40 °C. Compared with CnGOXA, the half-life of its mutant CnGOXA-M1 (Y169C-A211C), at 40 °C increased approximately 48-fold, and was stable at 30 °C and pH 3.0-12.0 for 1 h. The kcat and catalytic efficiency of CnGOXA-M1 were enhanced 0.7- and 1.6-fold, respectively. Both enzymes were cold-adapted and highly resistant to SDS. Furthermore, CnGOXA-M1 had a more significant effect on bread volume than that of GOX from Aspergillus niger. These favorable enzymatic properties of CnGOXA-M1 make it a potentially useful enzyme for many industrial applications.


Assuntos
Pão , Cladosporium/enzimologia , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Aspergillus niger/enzimologia , Catálise , Cladosporium/genética , Estabilidade Enzimática , Microbiologia de Alimentos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose Oxidase/genética , Concentração de Íons de Hidrogênio , Cinética , Mutação , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Microbiologia do Solo , Temperatura
14.
J Biosci Bioeng ; 129(2): 150-154, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31492608

RESUMO

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80-100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-ß-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30-35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2-8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.


Assuntos
Aspergillus oryzae/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Glucana 1,4-alfa-Glucosidase/genética , Regiões Promotoras Genéticas , Aspergillus oryzae/genética , Biocatálise , Hidrolases de Éster Carboxílico/genética , Eletroforese em Gel de Poliacrilamida , Cinética , Peso Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Biosci Biotechnol Biochem ; 84(3): 463-470, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31752618

RESUMO

Lycopene is a highly valued carotenoid with wide applications in various industries. The market demand for lycopene promotes research in metabolic engineering of heterologous hosts for lycopene. In this study, Pichia pastoris strain GS115 was genetically engineered to produce lycopene by integrating the heterologous lycopene biosynthesis genes from Corynebacterium glutamicum ATCC13032. The resulting strain, L1, produced 0.115 mg/g cell dry weight (DCW) lycopene. Through optimization by promoter selection, improving the precursor supply and expanding the Geranylgeranyl diphosphate (GGPP) pool, ultimately, the lycopene yield of the final optimal strain was 6.146 mg/g DCW with shake flask fermentation and 9.319 mg/g DCW (0.714 g/L) in a 3 L fermenter. The lycopene yield in this study is the highest yield of lycopene in P. pastoris reported to date, which demonstrated the potential of P. pastoris in lycopene synthesis and as a candidate host organism for the synthesis of other high value-added terpenoids.


Assuntos
Licopeno/metabolismo , Engenharia Metabólica , Pichia/genética , Reatores Biológicos , Corynebacterium glutamicum/genética , Fermentação
16.
Enzyme Microb Technol ; 133: 109447, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874680

RESUMO

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.


Assuntos
Celulose , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Saccharum/metabolismo , Vapor , Celulose/metabolismo , Hidrólise , Penicillium/enzimologia , Penicillium/genética , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Artif Cells Nanomed Biotechnol ; 48(1): 259-265, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31851845

RESUMO

A metal-resistant engineered Pichia pastoris was developed here to fulfil the metal bioleaching in aqueous conditions. Parent and recombinant yeasts were grown in YPD medium containing different concentrations of ion metals. XRD, electron microscopy and particle size analyser were used for the characterisation and the nanoparticle analyses. The nanoparticle production kinetics were studied by ICP-OES. The cytotoxicity of nanoparticles was assayed against human cell lines. Media colours changed to a range from purplish-brown to grey during early fermentation stages. The maximum biosorption capacities were recorded 81.23 and 493.35 mg/g for gold and palladium in batch conditions, respectively. Various physical investigations proved monodispersed spherical nanoparticles around 100 nm in size. Pure palladium nanoparticles and PdCl2 represented the least cytotoxic potency towards T47D and EPG85.257 cells. The results demonstrated that the genetically modified yeast is a cost-effective, high-throughput, robust, and facile system for metal biosorption.


Assuntos
Ouro/química , Ouro/metabolismo , Nanopartículas Metálicas , Paládio/química , Paládio/metabolismo , Pichia/genética , Pichia/metabolismo , Biotecnologia , Linhagem Celular , Cor , DNA Recombinante/genética , Ouro/toxicidade , Cinética , Organismos Geneticamente Modificados , Paládio/toxicidade , Pichia/crescimento & desenvolvimento
18.
Protein Expr Purif ; 166: 105503, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31550499

RESUMO

The N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetanus toxin fragment C (TetC). Unaltered TetC (wild-TetC) and eight mutants, in which different numbers and locations of N-glycosylation sites were altered, were expressed in P. pastoris GS115. The recombinant target proteins presented different levels of N-glycosylation. The wild Tet-C and 4 mutations sites of putative N-glycosylation (4Gly mutant: N280Q) had the highest level of secreted protein, while 1 mutation of putative N-glycosylation sites (1Gly mutant: N39/64/85/205Q) had the highest level of intracellular, non-secreted heterologous protein. Reducing the number of native N-glycosylation sites decreased the level of glycosylation, as well as the level of secretion. Introduction of a N-glycosylation site at position 320, however, also reduced the level of expression and secretion of recombinant protein. These results indicate that the number and location of N-glycosylation sites jointly have an effect on the expression and secretion of heterologous glycoproteins in P. pastoris.


Assuntos
Glicoproteínas/genética , Fragmentos de Peptídeos/genética , Pichia/genética , Proteínas Recombinantes/genética , Toxina Tetânica/genética , Sequência de Aminoácidos , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Glicoproteínas/química , Glicosilação , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Pichia/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Toxina Tetânica/química , Transfecção
19.
Protein Expr Purif ; 166: 105509, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31604114

RESUMO

Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 106 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained.


Assuntos
Interferon-alfa/genética , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Albumina Sérica Humana/genética , Sequência de Aminoácidos , Clonagem Molecular , Fermentação , Interferon-alfa/química , Peso Molecular , Peptídeos/química , Pichia/química , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Albumina Sérica Humana/química
20.
Protein Expr Purif ; 166: 105519, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31629955

RESUMO

Chitosanase (EC 3.2.1.132) is an important chitosan-degrading enzyme involved in industrial applications. In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 µg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2-8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of BbCSN-1 was significantly enhanced by Mn2+ but inhibited by Co2+ and Cu2+. These results indicated that BbCSN-1 from B. bassiana could be easily expressed in P. pastoris, which provided a basis for further study on its application.


Assuntos
Beauveria/genética , Glicosídeo Hidrolases/genética , Pichia/genética , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Cátions Bivalentes/química , Clonagem Molecular , Cobalto/química , Cobre/química , Expressão Gênica , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Manganês/química , Pichia/enzimologia , Ligação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Temperatura , Termodinâmica
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