Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

Szijártó, Nóra; Siika‐aho, Matti; Tenkanen, Maija; Alapuranen, Marika; Vehmaanperä, Jari; Réczey, Kati; Viikari, Liisa

doi:10.1016/j.jbiotec.2008.05.010

Cited by 65 publications

(32 citation statements)

References 24 publications

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“…The recombinant fusion protein was expressed in T. reesei, resulting in an approximate 2-fold increase in the percentage of solubilized avicel compared to the wild-type M. albomyces enzyme expressed in T. reesei. A similar result was obtained by Szijártó et al [65], though the improvement was less significant. Moreover, a fusion protein containing the catalytic modules of fungal CBHs and bacterial EGs, connected with a CBH linker, was developed in a Trichoderma host for increased cellulase enzyme yield and effectiveness in the synergistic degradation of crystalline cellulose [82].…”

Section: Other Fusion Strategiessupporting

confidence: 88%

“…As CBH1 can compose more than half of the total secreted protein from T. reesei, cbh1 is a very strong inducible promoter [61]. Such strong inducible promoters homologous to the expression host have been used for the heterologous expression of T. reesei, P. funiculosum, Aspergillus aculeatus or Melanocarpus albomyces cellobiohydrolase (cbh) genes [42,[62][63][64][65][66][67]. In general, homologous strong promoters appear to result in higher expression yields than heterologous promoters [34,58].…”

Section: Promoters For Driving the Heterologous Expression Of Cellobimentioning

confidence: 99%

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Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives

Zoglowek

Lübeck

Ahring

et al. 2015

Process Biochemistry

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Section: Other Fusion Strategiessupporting

confidence: 88%

Section: Promoters For Driving the Heterologous Expression Of Cellobimentioning

confidence: 99%

Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives

Zoglowek

Lübeck

Ahring

et al. 2015

Process Biochemistry

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“…The highest rate of substrate conversion was detected in the high-solids loading assays, possibly because of the lack of a CBM, which results in more association events due to reduced diffusion limitations (14,29). A similar value for PASC hydrolysis ratio was reported for HjCel7A and Thermoascus aurantiacus Cel7A (assayed with CBMs) as well as Melanocarpus albomyces Cel7B (which naturally lacks a CBM), whereas LqCel7B showed lower Avicel conversion ratios compared with the same cellobiohydrolases without CBMs (30,31). Also, the main product from PASC and Avicel hydrolysis was cellobiose (G2), followed by glucose (G1) and trace cellotriose (G3).…”

Section: Resultssupporting

confidence: 65%

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Kern

McGeehan

Streeter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

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Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.gribble | carbohydrate degrading enzymes

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“…Given the multivariate nature of cellulose hydrolysis, it is desirable to have diverse cellulases to choose from to optimize enzyme formulations for different applications and feedstocks. Recent studies have documented the superior performance of cellulases from thermophilic fungi relative to their mesophilic counterparts in laboratory scale biomass conversion processes (1,2), where enhanced stability leads to retention of activity over longer periods of time at both moderate and elevated temperatures. Fungal cellulases are attractive because they are highly active and can be expressed in fungal hosts such as Hypocrea jecorina (anamorph Trichoderma reesei) at levels up to 40 g/L in the supernatant.…”

mentioning

confidence: 99%

A family of thermostable fungal cellulases created by structure-guided recombination

Heinzelman

Snow²,

Wu³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

245

171

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SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63°C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63°C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15°C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated. biofuels ͉ cellobiohydrolase ͉ cellulose hydrolysis ͉ Trichoderma reesei ͉ CBH II T he performance of cellulase mixtures in biomass conversion processes depends on many enzyme properties including stability, product inhibition, synergy among different cellulase components, productive binding versus nonproductive adsorption and pH dependence, in addition to the cellulose substrate physical state and composition. Given the multivariate nature of cellulose hydrolysis, it is desirable to have diverse cellulases to choose from to optimize enzyme formulations for different applications and feedstocks. Recent studies have documented the superior performance of cellulases from thermophilic fungi relative to their mesophilic counterparts in laboratory scale biomass conversion processes (1, 2), where enhanced stability leads to retention of activity over longer periods of time at both moderate and elevated temperatures. Fungal cellulases are attractive because they are highly active and can be expressed in fungal hosts such as Hypocrea jecorina (anamorph Trichoderma reesei) at levels up to 40 g/L in the supernatant. Unfortunately, the set of documented thermostable fungal cellulases is small. In the case of the processive cellobiohydrolase class II (CBH II) enzymes, Ͻ10 natural thermostable gene sequences are annotated in the CAZy database (www.cazy.org). This limited number, combined with the difficulty of using directed evolution to generate diverse thermostable...

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Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

Cited by 65 publications

References 24 publications

Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives

Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

A family of thermostable fungal cellulases created by structure-guided recombination

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