High level production of a recombinant acid stable exoinulinase from Aspergillus kawachii.

Exoinulinases-enzymes extensively studied in recent decades because of their industrial applications-need to be produced in suitable quantities in order to meet production demands. We describe here the production of an acid-stable recombinant inulinase from Aspergillus kawachii in the Pichia pastoris system and the recombinant enzyme's biochemical characteristics and potential application to industrial processes. After an appropriate cloning strategy, this genetically engineered inulinase was successfully overproduced in fed-batch fermentations, reaching up to 840 U/ml after a 72-h cultivation. The protein, purified to homogeneity by chromatographic techniques, was obtained at a 42% yield. The following biochemical characteristics were determined: the enzyme had an optimal pH of 3, was stable for at least 3 h at 55 °C, and was inhibited in catalytic activity almost completely by Hg+2. The respective Km and Vmax for the recombinant inulinase with inulin as substrate were 1.35 mM and 2673 µmol/min/mg. The recombinant enzyme is an exoinulinase but also possesses synthetic activity (i. e., fructosyl transferase). The high level of production of this recombinant plus its relevant biochemical properties would argue that the process presented here is a possible recourse for industrial applications in carbohydrate processing.

Plant Growth Promotion Activity of Keratinolytic Fungi Growing on a Recalcitrant Waste Known as "Hair Waste".

Purpureocillium lilacinum (Thom) Samsom is one of the most studied fungi in the control of plant parasitic nematodes. However, there is not specific information on its ability to inhibit some pathogenic bacteria, fungi, or yeast. This work reports the production of several antifungal hydrolytic enzymes by a strain of P. lilacinum when it is grown in a medium containing hair waste. The growth of several plant-pathogenic fungi, Alternaria alternata, Aspergillus niger, and Fusarium culmorum, was considerably affected by the presence of P. lilacinum's supernatant. Besides antifungal activity, P. lilacinum demonstrates the capability to produce indoleacetic acid and ammonia during time cultivation on hair waste medium. Plant growth-promoting activity by cell-free supernatant was evidenced through the increase of the percentage of tomato seed germination from 71 to 85% after 48 hours. A 21-day plant growth assay using tomato plants indicates that crude supernatant promotes the growth of the plants similar to a reference fertilizer (p > 0.05). These results suggest that both strain and the supernatant may have potential to be considered as a potent biocontrol agent with multiple plant growth-promoting properties. To our knowledge, this is the first report on the antifungal, IAA production and tomato growth enhancing compounds produced by P. lilacinum LPSC #876.

Characterization, optimization, and scale-up of cellulases production by trichoderma reesei cbs 836.91 in solid-state fermentation using agro-industrial products.

The application of cellulases in saccharification processes is restricted by its production cost. Consequently, new fungal strains able to elaborate higher cellulases titers and with special activity profiles are required to make the process economical. The aim of this investigation was to find a promising wild-type Trichoderma strain for cellulases production. The Trichoderma reesei strain 938 (CBS 836.91) was selected among twenty strains on the basis of cellulase-agar-plate screening. Evaluation of the selected strain on six solid substrates indicated the highest activities to be obtained from wheat bran. Statistical analyses of the experimental design indicated a significant effect of pH and moisture on the generation of endoglucanase (EGA) and filter-paper (FPA) activity. Furthermore, a central-composite design-based optimization revealed that pH values between 6.4 and 6.6 and moisture from 74 to 94% were optimal for cellulases production. Under these conditions, 8-10 IU gds(-1) of FPA and 15.6-17.8 IU gds(-1) of EGA were obtained. In addition, cultivation in a rotating-drum reactor under optimal conditions gave 8.2 IU gds(-1) FPA and 13.5 IU gds(-1) EGA. Biochemical characterization of T. reesei 938 cellulases indicated a substantially higher resistance to 4 mM Fe(+2) and a slightly greater tolerance to alkaline pH in comparison to Celluclast(®). These results suggest that T. reesei 938 could be a promising candidate for improved cellulases production through direct-evolution strategies.

Evaluation of agro-industrial wastes, their state, and mixing ratio for maximum polygalacturonase and biomass production in submerged fermentation.

The potential of important agro-industrial wastes, apple pomace (AP) and orange peel (OP) as C sources, was investigated in the maximization of polygalacturonase (PG), an industrially significant enzyme, using an industrially important microorganism Aspergillus sojae. Factors such as various hydrolysis forms of the C sources (hydrolysed-AP, non-hydrolysed-AP, hydrolysed-AP + OP, non-hydrolysed-AP + OP) and N sources (ammonium sulphate and urea), and incubation time (4, 6, and 8 days) were screened. It was observed that maximum PG activity was achieved at a combination of non-hydrolysed-AP + OP and ammonium sulphate with eight days of incubation. For the pre-optimization study, ammonium sulphate concentration and the mixing ratios of AP + OP at different total C concentrations (9, 15, 21 g l(-1)) were evaluated. The optimum conditions for the maximum PG production (144.96 U ml(-1)) was found as 21 g l(-1) total carbohydrate concentration totally coming from OP at 15 g l(-1) ammonium sulphate concentration. On the other hand, 3:1 mixing ratio of OP + AP at 11.50 g l(-1) ammonium sulphate concentration also resulted in a considerable PG activity (115.73 U ml(-1)). These results demonstrated that AP can be evaluated as an additional C source to OP for PG production, which in turn both can be alternative solutions for the elimination of the waste accumulation in the food industry with economical returns.

Kinetic modelling of thermal inactivation of a keratinase from Purpureocillium lilacinum LPSC # 876 and the influence of some additives on its thermal stability.

Thermal inactivation of a keratinase produced by Purpureocillium lilacinum LPSC #876 was kinetically investigated using several enzyme inactivation models at the temperature range of 50-65 °C. Among the models studied, the Weibull distribution was the best model that describes the residual activity of P. lilacinum keratinase after heat treatment over the selected temperatures. The stabilising effect of metal ions (Ca2+ or Mg2+, 5 mmol l(-1)) or polyols (propylene glycol and glycerol, 10% v/v) was investigated, showing that the presence of Ca2+ increases the enzyme stability significantly. Conforming to the increased Ca2+ concentration, thermal stability of the enzyme also increased, with 10 mM of Ca2+ being the concentration of metal in which the enzyme retained 100% of its original activity after being incubated for 1 h at 55 °C. The effects of temperature on Weibull equation parameters and on the characteristics of the inactivation curves were evaluated. In the absence of any additives (control), the reliable time (t R) of the keratinase, analogous to D value, ranged from 484.16 to 63.67 min, while in the presence of Ca2+ the t R values ranged from 6,221 to 414.95 min at 50-65 °C. P. lilacinum keratinase is a potentially useful biocatalyst, and therefore, kinetic modelling of thermal inactivation addresses an important topic for its application in various industrial processes.

Study of the production of alkaline keratinases in submerged cultures as an alternative for solid waste treatment generated in leather technology.

Six nonpathogenic fungal strains isolated from alkaline soils of Buenos Aires Province, Argentina (Acremonium murorum, Aspergillus sidowii, Cladosporium cladosporoides, Neurospora tetrasperma, Purpureocillium lilacinum (formerly Paecilomyces lilacinus), and Westerdikella dispersa) were tested for their ability to produce keratinolytic enzymes. Strains were grown on feather meal agar as well as in solid-state and submerged cultures, using a basal mineral medium and "hair waste" as sole sources of carbon and nitrogen. All the tested fungi grew on feather meal agar, but only three of them were capable of hydrolyzing keratin, producing clear zones. Among these strains, P. lilacinum produced the highest proteolytic and keratinolytic activities, both in solid-state and submerged fermentations. The medium composition and culture conditions for the keratinases production by P. lilacinum were optimized. Addition of glucose (5 g/l) and yeast extract (2.23 g/l) to the basal hair medium increased keratinases production. The optimum temperature and initial pH for the enzyme production were 28℃ and 6.0, respectively. A beneficial effect was observed when the original concentration of four metal ions, present in the basal mineral medium, was reduced up to 1:10. The maximum yield of the enzyme was 15.96 Uc/ml in the optimal hair medium; this value was about 6.5-fold higher than the yield in the basal hair medium. These results suggest that keratinases from P. lilacinum can be useful for biotechnological purposes such as biodegradation (or bioconversion) of hair waste, leading to a reduction of the environmental pollution caused by leather technology with the concomitant production of proteolytic enzymes and protein hydrolyzates.

Bioprocessing of "Hair Waste" by Paecilomyces lilacinus as a Source of a Bleach-Stable, Alkaline, and Thermostable Keratinase with Potential Application as a Laundry Detergent Additive: Characterization and Wash Performance Analysis.

Paecilomyces lilacinus (Thom) Samson LPS 876, a locally isolated fungal strain, was grown on minimal mineral medium containing "hair waste," a residue from the hair-saving unhairing process, and produced a protease with keratinolytic activity. This enzyme was biochemically characterized. The optimum reaction conditions, determined with a response surface methodology, were 60°C and pH 6.0. It was remarkably stable in a wide range of pHs and temperatures. Addition of Ca(2+), Mg(2+), or sorbitol was found to be effective in increasing thermal stability of the protease. PMSF and Hg(2+) inhibited the proteolytic activity indicating the presence of a thiol-dependent serine protease. It showed high stability toward surfactants, bleaching agents, and solvents. It was also compatible with commercial detergents (7 mg/mL) such as Ariel, Skip, Drive, and Ace, retaining more than 70% of its proteolytic activity in all detergents after 1 h of incubation at 40°C. Wash performance analysis revealed that this protease could effectively remove blood stains. From these properties, this enzyme may be considered as a potential candidate for future use in biotechnological processes, as well as in the formulation of laundry detergents.

Evaluación de la capacidad de solubilización de pectina de cáscada de limón usando protopectinasa-SE / Evaluation of pectin solubilization capability from lemon peel using protopectinase-SE

Se ha desarrollado un protocolo para la producción y masificación de células de achiote en suspensión, a partir de callos friables obtenidos de tejidos de hojas, como estrategia para la obtención de metabolitos antiofídícos, especialmente compuestos fenólicos, y para ello se ha evaluado el efecto de las concentraciones de inóculo, glucosa, fósforo y nitrógeno sobre la cinética de crecimiento celular, en el medio ½MS+2,4-D (5 ppm)+BAP (1 ppm), almacenados a 25º C, en oscuridad y a 140 rpm, utilizando un diseño factorial completamente aleatorizado de cuatro factores y dos niveles, con evaluación a los 20 y 40 días de establecimiento. El tratamiento que presenta la mayor producción de biomasa de células de achiote en suspensión tiene una concentración inicial de biomasa 4 g/l, 20 g/l de glucosa, 0.13 g/l de fósforo y 2.52 g/l de nitrógeno. La cinética de crecimiento de las células de achiote en suspensión, a las condiciones de cultivo de este tratamiento, presenta una fase exponencial bien definida de 25 días; a partir de allí se establece una fase estacionaria hasta el tiempo final de la evaluación (40 días). Se comparan los contenidos de fenoles totales entre el material obtenido in vitro y el material vegetal proveniente de plantas crecidas ex-vitro, como criterio válido para justificar posteriores trabajos de producción metabólica in-vitro en esta especie vegetal.