Pectin extraction from lime pomace by cold-active polygalacturonase-assisted method.

An Antarctic yeast was cultivated to produce and enzymatic extract with polygalacturonase activity, whose biochemical properties were studied. It assisted pectin extraction from lime pomace at 20 °C. The extract produced by Tausonia pullulans 8E had an optimum temperature of 40 °C and optimum pH of 5.0. At 20 °C, it displayed 54% of relative activity. A good pH-stability and thermostability were observed. Hg+2 and Co+2 inhibited its activity in 40 and 11%, respectively. Pectin was obtained from lime pomace at 20 °C, with 15% of yield after 120 min extraction. Uronic acid (46%) and neutral sugars (53%) were determined. Pectin's molecular weight was estimated in the order of 100,000 g mol-1. By anion-exchange chromatography, pectin-linked and free neutral sugars were observed. The high-methoxyl pectin was used to prepare low-calorie gels. It was demonstrated that this cold-active enzyme allows enzymatic-assisted pectin extraction at 20 °C.

The keratinolytic bacteria Bacillus cytotoxicus as a source of novel proteases and feather protein hydrolysates with antioxidant activities.

BACKGROUND: Argentina's geothermal areas are niches of a rich microbial diversity. In 2020, species of Bacillus cytotoxicus were isolated for the first time from these types of pristine natural areas. Bacillus cytotoxicus strains demonstrated the capability to grow and degrade chicken feathers with the concomitant production of proteases with keratinolytic activity, enzymes that have multitude of industrial applications. The aim of this research was to study the production of the proteolytic enzymes and its characterization. Also, feather protein hydrolysates produced during fermentation were characterized. RESULTS: Among the thermotolerant strains isolated from the Domuyo geothermal area (Neuquén province, Argentina), Bacillus cytotoxicus LT-1 and Oll-15 were selected and put through submerged cultures using feather wastes as sole carbon, nitrogen, and energy source in order to obtain proteolytic enzymes and protein hydrolysates. Complete degradation of feathers was achieved after 48 h. Zymograms demonstrated the presence of several proteolytic enzymes with an estimated molecular weight between 50 and > 120 kDa. Optimum pH and temperatures of Bacillus cytotoxicus LT-1 crude extract were 7.0 and 40 °C, meanwhile for Oll-15 were 7.0 and 50 °C. Crude extracts were inhibited by EDTA and 1,10 phenanthroline indicating the presence of metalloproteases. Feather protein hydrolysates showed an interesting antioxidant potential measured through radical-scavenging and Fe3+-reducing activities. CONCLUSION: This work represents an initial approach on the study of the biotechnological potential of proteases produced by Bacillus cytotoxicus. The results demonstrated the importance of continuous search for new biocatalysts with new characteristics and enzymes to be able to cope with the demands in the market.

Debaryomyces hansenii F39A as biosorbent for textile dye removal.

Many industries generate a considerable amount of wastewater containing toxic and recalcitrant dyes. The main objective of this research was to examine the biosorption capacity of Reactive Blue 19 and Reactive Red 141 by the Antarctic yeast Debaryomyces hansenii F39A biomass. Some variables, including pH, dye concentration, amount of adsorbent and contact time, were studied. The equilibrium sorption capacity of the biomass increased with increasing initial dye concentration up to 350mg/l. Experimental isotherms fit the Langmuir model and the maximum uptake capacity (qmax) for the selected dyes was in the range of 0.0676-0.169mmol/g biomass. At an initial dye concentration of 100mg/l, 2g/l biomass loading and 20±1°C, D. hansenii F39A adsorbed around 90% of Reactive Red 141 and 50% of Reactive Blue 19 at pH 6.0. When biomass loading was increased (6g/l), the uptake reached up to 90% for Reactive Blue 19. The dye uptake process followed a pseudo-second-order kinetics for each dye system. As seen throughout this research study, D. hansenii has the potential to efficiently and effectively remove dyes in a biosorption process and may be an alternative to other costly materials.

Bacillus cytotoxicus Isolated from a Pristine Natural Geothermal Area Reveals High Keratinolytic Activity.

Geothermal areas are the niches of a rich microbial diversity that is not only part of the intangible patrimony of a country but also the source of many microbial species with potential biotechnological applications. Particularly, microbial species in geothermal areas in Argentina have been scarcely explored regarding their possible biotechnological uses. The purpose of this work was to explore the proteolytic and keratinolytic enzymatic potential of microorganisms that inhabit in the Domuyo geothermal area in the Neuquén Province. To this end, we did enrichment cultures from two high-temperature natural samples in mineral media only supplemented with whole chicken feathers. After the isolation and the phylogenetic and morphologic characterization of different colonies, we obtained a collection of Bacillus cytotoxicus isolates, a species with no previous report of keratinolytic activity and only reported in rehydrated meals connected with food poisoning outbreaks. Its natural habitat has been unknown up to now. We characterized the proteolytic and keratinolytic capacities of the B. cytotoxicus isolates in different conditions, which proved to be remarkably high compared with those of other similar species. Thus, our work represents the first report of the isolation as well as the keratinolytic capacity characterization of strains of B. cytotixicus obtained from a natural environment.

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.

Development of double emulsion nanoparticles for the encapsulation of bovine serum albumin.

In the present work, a double emulsion was developed for the encapsulation of Bovine Serum Albumin (BSA) as a model protein for the future encapsulation of viral proteins. The first emulsion polydispersity index (PDI) was studied with increasing concentrations of poly (ε-caprolactone) (PCL) as stabilizer (from 16% w/v to 5% w/v) and polyvinyl alcohol (PVA) as the surfactant in the second emulsion at 1.5% w/v. Results suggest that at decreasing concentrations of PCL the PDI of the emulsion also decrease, indicating that viscosity of the emulsion is crucial in the homogeneity of the resultant size distribution of the nanoparticles. When PVA concentration in the second emulsion was increased from 1.5% w/v to 2.5% w/v the PDI also increased. To study the relationship between the structure of the surfactant in the second emulsion and the resultant BSA encapsulation, emulsions were prepared with Pluronic F68 and PVA both at 1.5% w/v and PCL in the first emulsion at 5% w/v. Results indicated that Pluronic F68 was a better stabilizer because at the same experimental conditions encapsulation of BSA was 1.5 higher than PVA. FTIR studies confirmed the presence of BSA in the nanoparticles. SEM and TEM microscopies showed a size distribution of 300nm-500nm size of nanoparticles. Circular dichroism studies demonstrated that the secondary structure of the protein was conserved after the encapsulation into the nanoparticles.

Purification and Biochemical and Kinetic Properties of an Endo-Polygalacturonase from the Industrial Fungus Aspergillus sojae.

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.

Pectinolytic yeasts from cold environments: novel findings of Guehomyces pullulans, Cystofilobasidium infirmominiatum and Cryptococcus adeliensis producing pectinases.

One hundred and three yeasts isolated from soil samples from King George Island and Tierra del Fuego province were screened in relation with their capability to produce pectinolytic enzymes. Although all the yeasts showed well-developed colonies at 20 °C, only eight showed a clear halo around the colony, indicative of pectin degradation. A secondary screening demonstrated that only four yeasts were capable to produce pectinases at low temperatures (8 °C). It could be seen that the selected yeasts were able to grow and produce high levels of polygalacturonase activity when submerged fermentations were performed using pectin-containing fruit wastes as substrates. None of the strains produced neither lyase nor rhamnogalacturonan hydrolase activities. Regarding pectin esterase activity, it was only produced in lower amounts by G. pullulans 8E (0.022 U ml-1). A TLC analysis of the substrate cleavage pattern of the pectinolytic systems was consistent with an endo-type activity. The clarification of apple juice was only accomplished by G. pullulans pectinolytic system, with a clarification of 80% (%T650) using 4 U/ml of enzyme at 20 °C. As far as we concern this work describes for the first time the production of pectinases by the cold-adapted yeasts species Cystofilobasidium infirmominiatum, Cryptococcus adeliensis and G. pullulans.

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.

Studies on PVA pectin cryogels containing crosslinked enzyme aggregates of keratinase.

Polyvinyl alcohol-pectin (PVA-P) films containing enrofloxacin and keratinase were developed to treat wounds and scars produced by burns and skin injuries. However, in order to prevent enzyme inactivation at the interface between the patch and the scars, crosslinked enzyme aggregates (CLEAs) from a crude extract of keratinase produced by Paecilomyces lilacinus (LPSC#876) were synthesized by precipitation with acetone and crosslinking with glutaraldehyde. Soluble vs. CLEA keratinase (K-CLEA) activities were tested in 59% (v/v) hydrophobic (isobutanol and n-hexane) and hydrophilic (acetone and dimethylsulfoxide) solvents mixtures. K-CLEA activity was 1.4, 1.7 and 6.6 times higher in acetone, n-hexane and isobutanol than the soluble enzyme at 37 °C after 1 h of incubation, respectively. K-CLEA showed at least 45% of enzyme residual activity in the 40-65 °C range, meanwhile the soluble biocatalyst was fully inactivated at 65 °C after 1h incubation. Also, the soluble enzyme was completely inactivated after 12 h at pH 7.4 and 45 °C, even though K-CLEA retained full activity. The soluble keratinase was completely inactivated at 37 °C after storage in buffer solution (pH 7.4) for 2 months, meanwhile K-CLEAs kept 51% of their activity. K-CLEA loaded into polyvinyl alcohol (PVA) and PVA-P cryogels showed six times lower release rate compared to the soluble keratinase at skin pH (5.5). Small angle X-ray scattering (SAXS) analysis showed that K-CLEA bound to pectin rather than to PVA in the PVA-P matrix.

Enzymatic hydrolysis of gelatin layers of X-Ray films and release of silver particles using keratinolytic serine proteases from Purpureocillium lilacinum LPS # 876.

Enzymatic decomposition of gelatin layers on used X-ray films and repeated utilization of the enzyme for potential application in silver recovery were investigated using keratinolytic serine proteases from Purpureocillium lilacinum LPS # 876. At pH 9.0, the enzymatic reaction was enhanced by the increase of enzyme concentration or by the increase of the temperature up to 60℃. Under the conditions of 6.9 U/ml, 60℃, and pH 9.0, hydrolysis of the gelatin layers and the resulting release of silver particles were achieved within 6 min. The protective effect of polyols against thermal denaturation was investigated. The presence of glycerol and propylene glycol increased enzyme stability. When the reusability of the enzyme for gelatin hydrolysis was tested, it could be seen that it could be effectively reused for more cycles when glycerol was added, compared with the enzyme without protective agents. The results of these repeated treatments suggested that a continuous process of recycling silver from used X-ray is feasible. Keeping in mind that recycling is (at the present time) needed and imperative, it can be remarked that, in this research, three wastes were successfully used: hair waste in order to produce serine proteases; glycerol in order to enhance enzyme thermal stability; and used Xray films in order to recover silver and PET films.

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.

Aspergillus kawachii produces an inulinase in cultures with yacon (Smallanthus sonchifolius) as substrate

Background: Inulinases have been extracted and characterized from inulin-storing tissues; however, production of microbial inulinases have recently draw much attention as they offer several industrial advantages. Many microorganisms, including filamentous fungi, yeast and bacteria have been claimed as inulinase producers. These hydrolases are usually inducible and their exo-acting forms may hydrolyze fructose polymers (inulin) and oligosaccharides such as sucrose and raffinose. Fungal inulinase extracts are often produced as stable mixture of highly active fructanhydrolases. From a practical prospective, the best known inulinases to date are those produced by species of Penicillium, Aspergillus and Kluyveromyces. Results: The production of extracellular inulinase by A. kawachii in liquid cultures, using either inulin or yacon derived materials as CES as well as inulinase inducers, is reported. In addition, a partial characterization of the enzyme activity is included. Conclusions: Yacon derived products, particularly yacon juice, added to the culture medium proved to be a good CES for fungal growth as well as an inducer of enzyme synthesis. Partial characterization of the enzyme revealed that it is quite stable in a wide range of pH and temperature. In addition, characterization of the reaction products revealed that this enzyme corresponds to an exo-type. These facts are promising considering its potential application in inulin hydrolysis for the production of high fructose syrups.

Immobilized keratinase and enrofloxacin loaded on pectin PVA cryogel patches for antimicrobial treatment.

A keratinase isolated from Paecilomyces lilacinus (LPS #876) was tested against proteins present in the skin but the high enzyme activity was detected on collagen. Keratinase was physically immobilized onto PVA-pectin cryogels and enzyme release was 20.8±2.1%, 63.8±0.2%, 41.5±3.5% and 26.0±3.5% in cryogels containing pectins with esterification degrees (DE) 33.0%, 55.0%, 62.7% and 71.7% respectively at 37°C after 3h incubation. In presence of 0.75 M NaCl, the percentage of enzyme release changed to: 57.5±1.5, 65.8±3.8, 57.3±0.2 and 34.0±4.0 for the four pectins respectively. In-vitro studies of enrofloxacin release from PVA-pectin cryogels at pH close to the human skin (pH=5.5) showed 15.0% free antibiotic following first order kinetic at 37°C after 5h incubation. However, in the presence of keratinase only 6.9% of enrofloxacin was released under the same experimental conditions.

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.

Purification and characterization of a novel alkaline α-L-rhamnosidase produced by Acrostalagmus luteo albus.

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal nonreducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area, with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali-tolerant soil fungus from Argentina. We also present an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In the presence of rhamnose as the sole carbon source, this fungus produces a rhamnosidase with a molecular weight of 109 kDa and a pI value of 4.6, as determined by SDS-PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrofenil-α-L-rhamnopiranoside as substrate, the enzyme activity showed pH and temperature optima of 8.0 and 55°C, respectively. The enzyme exhibited Michaelis-Menten kinetics, with K (M) and V (max) values of 3.38 mmol l(-1) and 68.5 mmol l(-1) min(-1), respectively. Neither divalent cations such as Ca(2+), Mg(2+), Mn(2+), and Co(2+) nor reducing agents such as ß-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn(2+) at a concentration of 0.2 mM. This enzyme showed the capacity to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. Based on these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered a potential new biocatalyst for industrial applications.

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.