12 research outputs found
Purification and characterization of a beta-Glucanase produced by Trichoderma harzianum showing biocontrol potential
Uma beta-1,3-glucanase foi produzida por Trichoderma harzianum em cultura contendo quitina como fonte de carbono. Duas proteĂnas com atividade de beta-1,3-glucanase foram purificadas atravĂ©s de cromatografia de interação hidrofĂłbica. As massas moleculares destas proteĂnas foram de 29 kDa e 36 kDa. A proteĂna de 36 kDa foi caracterizada quanto Ă influĂȘncia das condiçÔes de pH e temperatura. A atividade mĂĄxima foi encontrada em pH 5,0 e temperatura de 50ÂșC. A proteĂna purificada mostrou-se muito sensĂvel Ă temperatura. Aproximadamente 60% da atividade original foi perdida por incubação da proteĂna a 45ÂșC, 50ÂșC e 60ÂșC, por 30 min. O K M aparente e a Vmax para hidrĂłlise de laminarina em pH 5,0 Ă 37ÂșC, foram de 0,099 mg de açĂșcar redutor/mL e 0,3 mg de açĂșcar redutor/min.mL, respectivamente. Esta enzima mostrou-se insensĂvel a compostos orgĂąnicos e Ăons metĂĄlicos, exceto Ăon fĂ©rrico o qual em uma concentração de 1 mM, inibiu em aproximadamente 100% a atividade da enzima. Ao contrĂĄrio de outras enzimas hidrolĂticas (quitinase e protease) produzidas pelo mesmo isolado 1051 de T. harzianum, a beta-1,3-glucanase descrita aqui nĂŁo afetou a integridade da parede celular do fitopatĂłgeno Crinipellis perniciosa.A beta-1,3-glucanase was produced by Trichoderma harzianum in cultures containing chitin as the sole substrate. Two proteins showing beta-1,3-glucanase activity were purified to apparent homogeneity by hydrophobic chromatography. The molecular masses of these proteins were 29 and 36 kDa. The 36 kDa protein was further characterized. It was active on a broad pH range, and maximal activity was detected at pH 5.0. The optimum temperature of the 36 kDa beta-1,3-glucanase was 50ÂșC, but the purified enzyme was very sensitive to temperature. It lost about 60% or more of the activity after incubation for 30 min at 45, 50 and 60ÂșC. The apparent K M and Vmax for hydrolysis of laminarin at pH 5.0 and 37ÂșC, were 0.099 mg of reducing sugar/mL and 0.3 mg of reducing sugar/min.mL, respectively. The enzyme was insensitive to organic compound and metal ions, except for the ferric ion which inhibited about 100% of the original activity at the concentration of 1 mM. In contrast to other hydrolytic enzymes (a chitinase and a protease) produced by the same T. harzianum isolate (1051), the beta-1,3-glucanase showed no effect on the cell wall of the phytopathogenic fungus Crinipellis perniciosa
Production of enzymes for the diagnosis of CoVid-19
Os coronavĂrus representam um grupo de vĂrus cujos genomas sĂŁo baseados em RNA fita simples de sentido positivo (+)ssRNA sendo causadores de diversas infecçÔes respiratĂłrias em humanos, incluindo a COVID-19. O diagnĂłstico rĂĄpido e preciso deste vĂrus Ă© fundamental para nortear o tratamento da doença. Atualmente, os mais importantes testes de diagnĂłstico deste vĂrus sĂŁo baseadas em imunoensaios (ELISA) ou em testes moleculares (PCR). Por se tratar de um genoma de RNA, a detecção do coronavĂrus se dĂĄ pela RT-PCR que envolve o uso de duas enzimas: transcriptase reversa e Taq DNA polimerase
Cloning, Purification, and Partial Characterization of Bacillus subtilis Urate Oxidase Expressed in Escherichia coli
Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of âŒ60âkDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675âDa which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37°C, respectively, and retained 90% of its activity after 72 hours of incubation at â20°C and 4°C
Engineering Zymomonas mobilis for the production of xylonic acid from sugarcane bagasse hydrolysate
Sugarcane bagasse is an agricultural residue rich in xylose, which may be used as a feedstock for the production of high-value-added chemicals, such as xylonic acid, an organic acid listed as one of the top 30 value-added chemicals on a NREL report. Here, Zymomonas mobilis was engineered for the first time to produce xylonic acid from sugarcane bagasse hydrolysate. Seven coding genes for xylose dehydrogenase (XDH) were tested. The expression of XDH gene from Paraburkholderia xenovorans allowed the highest production of xylonic acid (26.17 ± 0.58 g Lâ1) from 50 g Lâ1 xylose in shake flasks, with a productivity of 1.85 ± 0.06 g Lâ1 h â1 and a yield of 1.04 ± 0.04 gAX/gX. Deletion of the xylose reductase gene further increased the production of xylonic acid to 56.44 ± 1.93 g Lâ1 from 54.27 ± 0.26 g Lâ1 xylose in a bioreactor. Strain performance was also evaluated in sugarcane bagasse hydrolysate as a cheap feedstock, which resulted in the production of 11.13 g Lâ1 xylonic acid from 10 g Lâ1 xylose. The results show that Z. mobilis may be regarded as a potential platform for the production of organic acids from cheap lignocellulosic biomass in the context of biorefineries
Engineered fluorescent strains of cryptococcus neoformans : a versatile toolbox for studies of host-pathogen interactions and fungal biology, including the viable but nonculturable state
Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr
] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans
Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker
Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii
Construction and characterization of centromeric plasmids for Komagataella phaffii using a color-based plasmid stability assay.
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
Use of an on/off tetracycline riboswitch to control protein production in Komagataella phaffii
Abstract The methylotrophic yeast Komagataella phaffii is one of the most important microbial platforms to produce recombinant proteins. Despite its importance in the context of industrial biotechnology, the use of synthetic biology approaches in K. phaffii is hampered by the fact that few genetic tools are available for precise control of gene expression in this system. In this work, we used an RNA aptamer activated by tetracycline to modulate protein production at the translational level. Using lacZ as gene reporter, we have demonstrated significant reduction of the heterologous protein upon addition of tetracycline. Furthermore, this genetic control device was applied for the control of Ku70p. This protein is involved in non-homologous recombination and the control of its production paves the way for the development of strains exhibiting higher rates of homologous recombination
Applications of the Methylotrophic Yeast <i>Komagataella phaffii</i> in the Context of Modern Biotechnology
Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts