Isolation of Cellulose Degrading Fungi from Decaying Banana Pseudostem and Strelitzia alba

Cellulases are a group of hydrolytic enzymes that break down cellulose to glucose units. These enzymes are used in the food, beverage, textile, pulp, and paper and the biofuel industries. The aim of this study was to isolate fungi from natural compost and produce cellulases in submerged fermentation (SmF). Initial selection was based on the ability of the fungi to grow on agar containing Avicel followed by cellulase activity determination in the form of endoglucanase and total cellulase activity. Ten fungal isolates obtained from the screening process showed good endoglucanase activity on carboxymethyl cellulose-Congo Red agar plates. Six of the fungal isolates were selected based on high total cellulase activity and identified as belonging to the genera Trichoderma and Aspergillus. In SmF of synthetic media with an initial pH of 6.5 at 30°C Trichoderma longibrachiatum LMLSAUL 14-1 produced total cellulase activity of 8 FPU/mL and endoglucanase activity of 23 U/mL whilst Trichoderma harzianum LMLBP07 13-5 produced 6 FPU/mL and endoglucanase activity of 16 U/mL. The produced levels of both cellulases and endoglucanase by Trichoderma species were higher than the levels for the Aspergillus fumigatus strains. Aspergillus fumigatus LMLPS 13-4 produced higher β-glucosidase 38 U/mL activity than Trichoderma species.

Acetylcholinesterases from Leaf-Cutting antAtta sexdens: Purification, Characterization, and Capillary Reactors for On-Flow Assays

Acetylcholinesterase (AChE) is responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh) leading to acetate and choline (Ch) release. The inhibition of AChE produces a generalized synaptic collapse that can lead to insect death. Herein we report for the first time the isolation of two AChEs fromAtta sexdenswhich were purified by sulphate ammonium precipitation followed by ion exchange chromatography. AsAChE-A and AsAChE-B enzymes have optimum pH of 9.5 and 9.0 and higher activities in 30/50°C and 20°C, respectively, using acetylthiocholine (ATCh) as substrate. Immobilized capillary enzyme reactors (ICERs) were obtained for both enzymes (AsAChE-A-ICER and AsAChE-B-ICER) and their activities were measured by LC-MS/MS through hydrolysis product quantification of the natural substrate ACh. The comparison of activities by LC-MS/MS of both AChEs using ACh as substrate showed that AsAChE-B (free or immobilized) had the highest affinity. The inverse result was observed when the colorimetric assay (Elman method) was used for ATCh as substrate. Moreover, by mass spectrometry and phylogenetic studies, AsAChE-A and AsAChE-B were classified as belonging to AChE-2 and AChE-1 classes, respectively.

Lipolytic Enzymes with Hydrolytic and Esterification Activities Produced by Filamentous Fungi Isolated from Decomposition Leaves in an Aquatic Environment

Microbial lipases are prominent biocatalysts able to catalyze a wide variety of reactions in aqueous and nonaqueous media. In this work, filamentous fungi isolated from leaves decomposed in an aquatic environment were screened for lipase production with hydrolytic activity and esterification. Agar plates with Tween 20 and Rhodamine B were used for selection, while submerged cultures with olive oil were subsequently used to select 38 filamentous fungi. Trichoderma harzianum, Fusarium solani, Trichoderma harzianum F5, and Penicillium sp. F36 were grown in six different culture media. F. solani presented the highest lipase production (2.37 U/mL) with esterification activity of 0.07 U/mL using medium composed of (g.L−1) KH2PO4 1.00, MgSO4 H2O 1.123, and CuSO4 0.06. Supplementation of this culture medium with organic nitrogen sources increased lipase production by 461.3% using tryptone and by 419.4% using yeast extract. Among the vegetable oils from the Amazon region, degummed cotton oil induced lipase production up to 8.14 U/mL. The lipase produced by F. solani F61 has great potential to application in conventional processes and biodiesel production by transesterification of vegetable oils, as well as food industries in the production of fatty acid esters by hydrolysis and esterification.

Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale

Spirosoma linguale is a free-living nonpathogenic organism. Like many other bacteria, S. linguale produces a cell-associated α-N-acetylgalactosaminidase. This work was undertaken to elucidate the nature of this activity. The recombinant enzyme was produced, purified, and examined for biochemical attributes. The purified enzyme was ~50 kDa active as a homodimer in solution. It catalyzed hydrolysis of α-N-acetylgalactosamine at pH 7. Calculated KM was 1.1 mM with kcat of 173 s−1. The described enzyme belongs to the GH109 family.

Thermostable Cellulases from the Yeast Trichosporon sp.

Objectives. Identification of cellulolytic microorganisms is of great interest to the hydrolysis of cellulosic biomass. This study focuses on the identification of cellulolytic yeasts and the optimization of cellulase activities produced by the best performing isolate. Results. 30 cellulolytic yeast isolates were selected. Enzymes produced by an isolate from the Trichosporon genus showed the property to hydrolyze different substrates: carboxymethyl cellulose (CMC), cellulose fiber, and filter paper (FP). The optimum measured temperature was 55°C for CMCase and 60°C for FPase. The optimal pH was 5 for CMCase and 4 to 6 for FPase. The effect of the substrates concentration showed that the best activities were obtained at 100 mg/mL CMC or FP. The highest activities were 0.52 for the CMCase and 0.56 for the cellulase fiber at 10 min incubation, 0.44 IU/mL at 15 min incubation, and 24 h FPase preincubation. Conclusion. Cellulases produced by the studied yeast are capable of hydrolyzing soluble and insoluble substrates at elevated temperatures and at a wide pH range. They are considerable interest in the production of fermentable sugars from lignocellulosic substrates.

Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR)

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

A Midgut Digestive Phospholipase A2 in Larval Mosquitoes, Aedes albopictus and Culex quinquefasciatus

Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophospholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PLA2 was characterized using a microplate PLA2 assay. The enzyme showed substantial activities at 6 and 8 μg/μl of protein concentration with optimal activity at 20 and 25 μg/μl of substrate concentration in Aedes albopictus and Culex quinquefasciatus, respectively. PLA2 activity from both mosquitoes increased in a linear function up to 1 hour of the reaction time. Both enzymes were sensitive to pH and temperature. PLA2 showed higher enzyme activities in pH 8.0 and pH 9.0 from Aedes albopictus and Culex quinquefasciatus, respectively, at 40°C of incubation. The PLA2 activity decreased in the presence of 5 mM (Aedes albopictus) and 0.5 mM (Culex quinquefasciatus) site specific PLA2 inhibitor, oleyloxyethylphosphorylcholine. Based on the migration pattern of the partially purified PLA2 on SDS-PAGE, the protein mass of PLA2 is approximately 20–25 kDa for both mosquitoes. The information on PLA2 properties derived from this study may facilitate in devising mosquitoes control strategies especially in the development of inhibitors targeting the enzyme active site.

Optimization of Xylanase Production from Aspergillus foetidus in Soybean Residue

Enzymatic hydrolysis is an important but expensive step in the process to obtain enzyme derived products. Thus, the production of efficient enzymes is of great interest for this biotechnological application. The production of xylanase by Aspergillus foetidus in soybean residues was optimized using 2×23 factorial designs. The experimental data was fitted into a polynomial model for xylanase activity. Statistical analyses of the results showed that variables pH and the interaction of pH and temperature had influenced the production of xylanase, with the best xylanase production level (13.98 U/mL) occurring at fermentation for 168 hours, pH 7.0, 28°C, and 120 rpm.

Extracellular Pectinase from a Novel Bacterium Chryseobacterium indologenes Strain SD and Its Application in Fruit Juice Clarification

Pectinase is one of the important enzymes of industrial sectors. Presently, most of the pectinases are of plant origin but there are only a few reports on bacterial pectinases. The aim of the present study was to isolate a novel and potential pectinase producing bacterium as well as optimization of its various parameters for maximum enzyme production. A total of forty bacterial isolates were isolated from vegetable dump waste soil using standard plate count methods. Primary screening was done by hydrolysis of pectin. Pectinase activity was determined by measuring the increase in reducing sugar formed by the enzymatic hydrolysis of pectin. Among the bacterial isolates, the isolate K6 exhibited higher pectinase activity in broth medium and was selected for further studies. The selected bacterial isolate K6 was identified as Chryseobacterium indologenes strain SD. The isolate was found to produce maximum pectinase at 37°C with pH 7.5 upon incubation for 72 hours, while cultured in production medium containing citrus pectin and yeast extract as C and N sources, respectively. During enzyme-substrate reaction phase, the enzyme exhibited its best activity at pH of 8.0 and temperature of 40°C using citrus pectin as substrate. The pectinase of the isolate showed potentiality on different types of fruit juice clarification.

Characterization of the Catalytic Structure of Plant Phytase, Protein Tyrosine Phosphatase-Like Phytase, and Histidine Acid Phytases and Their Biotechnological Applications

Phytase plays a prominent role in monogastric animal nutrition due to its ability to improve phytic acid digestion in the gastrointestinal tract, releasing phosphorus and other micronutrients that are important for animal development. Moreover, phytase decreases the amounts of phytic acid and phosphate excreted in feces. Bioinformatics approaches can contribute to the understanding of the catalytic structure of phytase. Analysis of the catalytic structure can reveal enzymatic stability and the polarization and hydrophobicity of amino acids. One important aspect of this type of analysis is the estimation of the number of β-sheets and α-helices in the enzymatic structure. Fermentative processes or genetic engineering methods are employed for phytase production in transgenic plants or microorganisms. To this end, phytase genes are inserted in transgenic crops to improve the bioavailability of phosphorus. This promising technology aims to improve agricultural efficiency and productivity. Thus, the aim of this review is to present the characterization of the catalytic structure of plant and microbial phytases, phytase genes used in transgenic plants and microorganisms, and their biotechnological applications in animal nutrition, which do not impact negatively on environmental degradation.