A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum : Production, Purification, and Characterization
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A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization Fabiane Cristina dos Santos 1 & Marco Aurelio Schuler de Oliveira 1 & Flavio Augusto Vicente Seixas 2 & Ione Parra Barbosa-Tessmann 1 Received: 28 August 2019 / Accepted: 12 March 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
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 β-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme. Keywords Penicillium digitatum . Cellobiohydrolase I . Purification . Characterization
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003307-9) contains supplementary material, which is available to authorized users.
* Ione Parra Barbosa-Tessmann [email protected] Extended author information available on the last page of the article
Applied Biochemistry and Biotechnology
Introduction The global market for industrial enzymes is expected to reach nearly 6.2 billion dollars by 2020 [1]. Microbial products prevail, and hydrolases are the most commonly employed class of industrial enzymes, which includes amylases, proteases, cellulases, and lipases [1, 2]. Cellulases are sold in a significant volume and are engaged in the following industries: food, animal feeding, starch processing, fruit juice and vegetal pulp extraction, paper, brewery and wine, textile and laundry, as well as in agriculture and research [3]. More recently, there has been an interest in the use of cellulases to convert cellulose-containing biomass to fermentable sugars to prod
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