Two isolates of Trichoderma, which reduce the incidence of witches'broom disease caused in cocoa by Crinipellis perniciosa, were evaluated for their potential to produce hydrolases in liquid medium. Very low or no hydrolytic activity was produced in the absence of any substrate. The activities of chitinase, N-acetylglucosaminidase, β-1,3-glucanase, total cellulase, endoglucanase, aryl-β-glucosidase, β-glucosidase, protease and amylase increased dramatically within 72-120 h of growth in the presence of specific substrates. Except for N-acetylglucosaminidase and β-glucosidase Trichoderma harzianum isolate 1051 produced the largest amounts of hydrolases. The possible involvement of these enzymes in the antagonistic interaction between Trichoderma and C. perniciosa is discussed.

ABSTRACT

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 ∼60 kDa. 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 58675 Da 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 −20°C and 4°C.

Crude glycerol, also known as glycerin, is the main byproduct of the biodiesel industry. It has been estimated that up to 40,000 tons of glycerin will be produced each year by 2020. This study evaluated the value-added use of crude glycerol derived from soybean biodiesel preparation as a carbon source for heterologous protein production using the yeast Pichia pastoris. Eleven glycerin samples were obtained by methanolysis of soybean oil using different acids or bases as catalysts. Cell growth experiments showed that crude glycerol containing either potassium or sodium hydroxide resulted in 1.5-2 times higher final cell densities when compared to glycerol P.A. Finally, crude glycerol containing sodium hydroxide was successfully utilized for constitutive heterologous α-amylase production in P. pastoris. This study demonstrated that crude glycerol without any purification steps may be directly used as carbon source for protein production in P. pastoris.

BackgroundA 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.ResultsA 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0715-8) contains supplementary material, which is available to authorized users.