Recombinant expression, purification, and characterization of an α-amylase from Massilia timonae.

This work reports the amy1 gene cloning from Massilia timonae CTI-57, and its successful expression in Escherichia coli Rosetta™ (DE3) from the pTRCHis2B plasmid. The recombinant AMY1 protein had 47 kDa, and its modeled structure showed a monomer composed of three domains. An N-terminal domain with the characteristic (ß/α)8-barrel structure of α-amylases, which contained the catalytic amino acid residues. The second domain was small, and the C-terminal domain was similar to those found in the barley α-amylase. A phylogenetic analysis demonstrated a high sequence identity of the studied protein with bacterial and plant α-amylases from the GH13_6 subfamily. This is the first characterized bacterial α-amylase from this glucoside hydrolase subfamily. Besides starch, the enzyme was also active against maltodextrin, amylopectin, and blocked p-nitrophenyl α-d-maltoheptaoside, but could not use ß-cyclodextrin or 4-nitrophenyl α-d-glucopyranoside. The K M for highly pure grade soluble starch from potato and V max values were 0.79 mg/mL and 0.04 mg/min, respectively. The calcium ion showed to be essential for the purified enzyme's activity, while EDTA, molybdenum, cobalt, and mercury were strong inhibitors. The enzyme was almost fully active in SDS presence. The enzyme's optimal pH and temperature were 6.0 and 60 °C, respectively, and its denaturation T m was 79 °C. A TLC analysis revealed that glucose and maltose are products of the enzyme's action on starch. In conclusion, this work described the M. timonae GH13_6 subfamily α-amylase, which showed to be thermostable and anionic detergent-resistant.

A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization.

A new cellulase producer strain of Penicillium digitatum (RV 06) was previously obtained from rotten maize grains. This work aim was to optimize the production and characterize this microorganism produced cellulase. A CMCase maximum production (1.6 U/mL) was obtained in stationary liquid culture, with an initial pH of 5.0, at 25 °C, with 1% lactose as carbon source, and cultured for 5 days. The produced enzyme was purified by ammonium sulfate precipitation and exclusion chromatography. The purified enzyme optimal temperature and pH were 60 °C and 5.2, respectively. The experimental Tm of thermal inactivation was 63.68 °C, and full activity was recovered after incubation of 7 h at 50 °C. The purified 74 kDa CMCase presented KM for CMC of 11.2 mg/mL, Vmax of 0.13 µmol/min, kcat of 52 s-1, and kcat/KM of 4.7 (mg/mL)-1 s-1. The purified enzyme had a high specificity for CMC and p-nitrophenyl cellobioside and released glucose and cellobiose as final products of the CMC hydrolysis. The enzyme trypsin digestion produced peptides whose masses were obtained by MALDI-TOF/TOF mass spectrometry, which was also used to obtain two peptide sequences. These peptide sequences and the mass peak profile retrieved a CBHI within the annotated genome of P. digitatum PD1. Sequence alignments and phylogenetic analysis confirmed this enzyme as a CBHI of the glycoside hydrolase family 7. The P. digitatum PD1 protein in silico structural model revealed a coil and ß-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme.

Fungi Isolated from Maize (Zea mays L.) Grains and Production of Associated Enzyme Activities.

Filamentous fungi produce a great variety of enzymes, and research on their biotechnological potential has recently intensified. The objective of this work was to identify, at the species level, using DNA barcoding, 46 fungal isolates obtained from maize grains with rot symptoms. We also analyzed the production of extracellular amylases, cellulases, proteases and lipases of 33 of those fungal isolates. The enzymatic activities were evaluated by the formation of a clear halo or a white precipitate around the colonies in defined substrate media. The found fungi belong to the genera Talaromyces, Stenocarpella, Penicillium, Phlebiopsis, Cladosporium, Hyphopichia, Epicoccum, Trichoderma, Aspergillus, Irpex, Fusarium, Microdochium, Mucor and Sarocladium. In the genus Fusarium, the species Fusarium verticillioides was predominant and this genus presented the highest diversity, followed by the genera Aspergillus. The best genera for lipase production were Cladosporium and Penicillium; while Cladosporium, Aspergillus and Penicillium were best for cellulase activity; Hyphopichia, Aspergillus and Irpex for amylase activity; and Cladosporium and Sarocladium for proteases activity. In conclusion, a collection of fungi from maize seeds presenting rotten symptoms were obtained, among which exist important producers of hydrolases.