Ana Claudia Vici

Screening of filamentous fungi for lipase production: Hypocrea pseudokoningii a new producer with a high biotechnological potential

Abstract Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40°C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0–8.0 at room temperature. The intracellular lipase was thermostable up to 60°C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.

Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis

Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

Increase of the phytase production by Aspergillus japonicus and its biocatalyst potential on chicken feed treatment

Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phosphorus. The phytase production by Aspergillus japonicus was optimized using Plackett–Burman designs (PBD), composite central rotational designs (CCRD), and response surface methodology from standard Czapek medium. The enzyme was applied in broiler chicken and laying hen foods. Analysis from PBD showed that KH2PO2, MgSO4 · 7H2O, and yeast extract had significant influences on phytase secretion (p < 0.05). The best results from the CCRD experiments were obtained using (A) 0.040% KH2PO4, (B) 0.050% MgSO4 · 7H2O, and (C) 0.040% yeast extract, enhancing in 49–53 U mg−1 protein. The determination coefficient (R2) was 0.92 and Fcalc was 7.48 times greater than Flisted. Thus, the reduced coded model: Y (U mg−1)=50.29+4.30A−3.35(A)2−4.80(B)2+5.62C−4.26(C)2

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.

Enzyme System from Aspergillus in Current Industrial Uses and Future Applications in the Production of Second-Generation Ethanol

Abstract Multiple fungal glycoside hydrolases can be used in biorefinery processes in the conversion of biomass into biofuels. The main polymers of plant biomass are cellulose and hemicellulose, which, together with lignin, constitute the most abundant organic compounds present in nature. Cellulose and hemicellulose are hydrolyzed by cellulolytic and hemicellulolytic enzymatic systems to monomers as glucose and xylose, respectively, which may be fermented by yeasts into second-generation ethanol. Aspergillus is a distinguished fungal genus, important in the production of enzymes that are able to degrade plant cell wall, which is a key step in the bioconversion of sugarcane biomass. This chapter will describe several species of Aspergillus as excellent producers of fibrolytic enzymes as well as current advances in the understanding of glycoside hydrolases, auxiliary activities, and important properties for the conversion of biomass into second-generation ethanol.