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Results Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. Verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%.
The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1. Conclusions The enzyme preparations produced by recombinant P. Verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes.
The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable to express both heterologous enzymes simultaneously. Citation: Bulakhov AG, Volkov PV, Rozhkova AM, Gusakov AV, Nemashkalov VA, Satrutdinov AD, et al. (2017) Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance. PLoS ONE 12(1): e0170404. Editor: Shihui Yang, National Renewable Energy Laboratory, UNITED STATES Received: November 21, 2016; Accepted: January 4, 2017; Published: January 20, 2017 Copyright: © 2017 Bulakhov et al. This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files, but glaI gene with its promoter part was deposit to GenBank, accession number KY086000. Funding: Support was provided by the Russian Science Foundation, Grant 1, []. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Filamentous fungi from the Ascomycota phylum proved to be efficient producers of highly active extracellular cellulase systems []. They include various species belonging to the genera Trichoderma ( T.
Razrabotany metodiki otsenki otnositel'noy intensivnosti dinamicheskogo nablyudeniya detey v poliklinike i sezonnoy obrashchaemosti, a takzhe metodika analiza povtornykh sluchaev gospitalizatsii. Raschitany doveritel'nye intervaly srednikh znacheniy povtornykh gospitalizatsiy, dlitel'nosti nablyudeniya v zavisimosti ot tipa poseleniya, vozrasta i nozologii. If on the list of references there are references (links) to the sources on the Internet, check their availability! Grafik uborki tualeta v shkole obrazec 10. Information about authors. Present full name (last, first, and patronymic names), academic degree, academic title, position, affiliated institutions of all authors.
Longibrachiatum, T. Harzianum, T. Koningii, etc.), Penicillium ( P. Funiculosum, P.
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Verruculosum, P. Decumbens, etc.), Myceliophthora (M. Thermophila), Chaetomium (C.
Thermophilum) and others [–]. However, quite many fungal species produce multienzyme cocktails having a low level of the β-glucosidase (BGL) activity that is not enough for a fast conversion of cellobiose and other oligosaccharides, formed in cellulose hydrolysis under the action of cellulolytic enzymes (endoglucanases and cellobiohydrolases), to the final product, glucose [,, ]. Cellobiose released during the enzymatic hydrolysis of cellulose rather strongly inhibits cellobiohydrolases, thus reducing the cellulase system performance []. Verruculosum B1-537 is a high-cellulase fungal strain that can also be used as a host to express homologous or heterologous enzymes [, ]. In spite of the high cellulase activity, B1-537 produces relatively low level of the BGL (~3% of the total secreted protein) that is not enough for efficient hydrolysis of cellulosic materials [, ]. On the other hand, it has been shown that extra addition of 40 units of the BGL from Aspergillus niger (AnBGL) to the P.