Dataset Information

Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon.

ABSTRACT: Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8-70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70°C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12) endo-1,4-?-glucanase, termed Cel12E. The enzyme shares 45% sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92°C and was active on a variety of linear 1,4-?-glucans like carboxymethyl cellulose, ?-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble ?-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving ?-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential.

SUBMITTER: Leis B

PROVIDER: S-EPMC4486847 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon.

Leis Benedikt B Heinze Simon S Angelov Angel A Pham Vu Thuy Trang VT Thürmer Andrea A Jebbar Mohamed M Golyshin Peter N PN Streit Wolfgang R WR Daniel Rolf R Liebl Wolfgang W

Frontiers in bioengineering and biotechnology 20150701

Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8-70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70° ...[more]

PMID: 26191525

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Project description:BackgroundAmylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases.ResultAll tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi.ConclusionsAlmost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.

Project description:Hyperthermophiles, living in environments above 80°C and usually coupling with multi-extreme environmental stresses, have drawn great attention due to their application potential in biotechnology and being the primitive extant forms of life. Studies on their survival and adaptation mechanisms have extended our understanding on how lives thrive under extreme conditions. During these studies, the "cross-stress" behavior in various organisms has been observed between the extreme high temperature and other environmental stresses. Despite the broad observation, the global view of the cross-stress behavior remains unclear in hyperthermophiles, leaving a knowledge gap in our understanding of extreme adaptation. In this study, we performed a global quantitative proteomic analysis under extreme temperatures, pH, hydrostatic pressure (HP), and salinity on an archaeal strain, Thermococcus eurythermalis A501, which has outstanding growth capability on a wide range of temperatures (50-100°C), pH (4-9), and HPs (0.1-70 MPa), but a narrow range of NaCl (1.0-5.0 %, w/v). The proteomic analysis (79.8% genome coverage) demonstrated that approximately 61.5% of the significant differentially expressed proteins (DEPs) responded to multiple stresses. The responses to most of the tested stresses were closely correlated, except the responses to high salinity and low temperature. The top three enriched universal responding processes include the biosynthesis and protection of macromolecules, biosynthesis and metabolism of amino acids, ion transport, and binding activities. In addition, this study also revealed that the specific dual-stress responding processes, such as the membrane lipids for both cold and HP stresses and the signal transduction for both hyperosmotic and heat stresses, as well as the sodium-dependent energetic processes might be the limiting factor of the growth range in salinity. The present study is the first to examine the global cross-stress responses in a piezophilic hyperthermophile at the proteomic level. Our findings provide direct evidences of the cross-stress adaptation strategy (33.5% of coding-genes) to multiple stresses and highlight the specific and unique responding processes (0.22-0.63% of coding genes for each) to extreme temperature, pH, salinity, and pressure, which are highly relevant to the fields of evolutionary biology as well as next generation industrial biotechnology (NGIB).

Dataset Information

Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon.

Publications

Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon.

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