Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate

Zarafeta, Dimitra; Kissas, Dimitrios; Sayer, C.; Gudbergsdottir, Soley; Ladoukakis, Efthymios; Isupov, Michail N.; Chatziioannou, Aristotelis; Peng, Xu; Littlechild, Jennifer A.; Σκρέτας, Γεώργιος; Kolisis, Fragiskos N.

doi:10.1371/journal.pone.0146454

Cited by 67 publications

(46 citation statements)

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“…Moreover, Cel5R shows activity in the presence of high salt concentration. Recently, it has been reported that the halophilic cellulase isolated from Icelandic hot spring showed decreased activity in the presence of increasing concentration of NaCl compared to control reaction with no salt19. On the other hand, the thermophilic GH5 endoglucanase isolated from Thermoanaerobacter tengcongensis MB4 retains less than 15% of its activity after 12-hours of pre-incubation in 4 M NaCl55.…”

Section: Discussionmentioning

confidence: 99%

“…The Cel5R shows thermostability up to 58 °C and pH stability from 5–9. Surprisingly, the Cel5R shows halotolerance and extreme halostability in 4 M NaCl, 3 M LiCl and 2 M KCl which is higher than other known halostable cellulases1819. Thus, the combination of extreme halostability with moderate thermal and pH stability makes Cel5R a potential candidate for industrial applications.…”

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confidence: 94%

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Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome

Garg

Srivastava

Brahma

et al. 2016

Sci Rep

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Cellulase catalyzes the hydrolysis of β-1,4-linkages of cellulose to produce industrially relevant monomeric subunits. Cellulases find their applications in pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. These industries always have great demand for cellulases that can work efficiently even in harsh conditions such as high salt, heat, and acidic environments. While, cellulases with high thermal and acidic stability are already in use, existence of a high halotolerant cellulase is still elusive. Here, we report a novel cellulase Cel5R, obtained from soil metagenome that shows high halotolerance and thermal stability. The biochemical and functional characterization of Cel5R revealed its endoglucanase activity and high halostability. In addition, the crystal structure of Cel5R determined at 2.2 Å resolution reveals a large number of acidic residues on the surface of the protein that contribute to the halophilic nature of this enzyme. Moreover, we demonstrate that the four free and non-conserved cysteine residues (C65, C90, C231 and C273) contributes to the thermal stability of Cel5R by alanine scanning experiments. Thus, the newly identified endoglucanase Cel5R is a promising candidate for various industrial applications.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 94%

Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome

Garg

Srivastava

Brahma

et al. 2016

Sci Rep

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“…However, there are reports of obtaining a highly thermostable enzyme with activity at low pH values. Zarafeta et al (2016) characterized thermostable (70 °C) and highly halotolerant cellulases derived from a hot spring isolate. Fig.…”

Section: The Characteristics Of the Biopreparationmentioning

confidence: 99%

Characterization and Influence of a Multi-enzymatic Biopreparation for Biogas Yield Enhancement

Oszust¹,

Pawlik²,

Janusz³

et al. 2017

BioResources

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A multi-enzymatic biopreparation of Trichoderma atroviride G79/11 origin was characterized. The fungus showed relatively high cellulase production in a soybean flour-cellulose-lactose medium. Subsequently, based on its post-culture liquid, the biopreparation of the enzyme mixture was developed and characterized. The liquid form of the enzyme mixture reached 22 U cm -3 of cellulolytic activity and its lyophilisate exhibited 1.09 U cm -3 at pH 5.1 and 50 °C. The enzyme mixture was characterized by the following activities: xylanase, β-glucosidase, carboxymethyl cellulase, polygalactouronase, pectinesterase, amylase, lactase, and protease. A method for an efficient conditioning process of organic waste (fruit processing waste, dairy sewage sludge, corn silage, and grain broth) for biogas yield enhancement using the enzyme mixture was proposed. The enzyme mixture increased the efficacy of biogas production by 30% when the lyophilizate (0.5 mg g -1 d.m.) was applied prior to fermentation. A method for conducting the enzymatic conditioning process of organic waste using the enzyme mixture as a pretreatment was proposed. This was part of the optimization of the methane fermentation process to increase the biogas yield. Consequently, after application of the biopreparation, the efficiency of anaerobic digestion of organic waste was improved.

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“…Such organisms are encountered in all types of terrestrial and marine hot environments, and are represented only by bacterial and archaeal species. Hyperthermostable enzymes encoded in such genomes can be identified by screening genomic or metagenomic material, either bioinformatically (Levisson et al, 2007; Graham et al, 2011; Zarafeta et al, 2016) or through functional genomic screening (Ewis et al, 2004; Tirawongsaroj et al, 2008; Peng et al, 2011). …”

Section: Introductionmentioning

confidence: 99%

EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability

et al. 2016

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Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure to elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring following in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retains high levels of catalytic activity after exposure to temperatures as high as 95°C for several hours. Furthermore, it exhibits very good stability against exposure to high concentrations of a variety of organic solvents. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modeling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold that seems to include a “subdomain insertion”, which is similar to the one present in its closest homolog of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a range of organic solvents and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications.

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Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate

Cited by 67 publications

References 64 publications

Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome

Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome

Characterization and Influence of a Multi-enzymatic Biopreparation for Biogas Yield Enhancement

EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability

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