Functional Characterization of Novel Thermostable Cellulases from Compost Metagenomes
DOI:
https://doi.org/10.5281/zenodo.22687822Keywords:
thermostable cellulase, compost metagenomics, functional screening, lignocellulose degradation, biofuel enzymes, GH5 glycoside hydrolaseAbstract
Thermostable cellulases that remain active at the elevated temperatures used in industrial lignocellulose
pretreatment are in persistent demand for biofuel and biorefinery applications, and self-heating compost piles,
which routinely exceed 60 degrees C during the thermophilic phase of decomposition, represent an
underexploited source of such enzymes. We constructed and functionally screened a metagenomic fosmid library
of 18,432 clones from thermophilic-phase compost collected at three municipal green-waste facilities, using a
chromogenic carboxymethyl-cellulose overlay assay to identify cellulolytic clones, followed by shotgun sequencing
of positive fosmid inserts and heterologous expression of candidate open reading frames in Escherichia coli.
Screening identified 47 cellulase-positive clones, from which sequence analysis recovered eleven distinct,
full-length glycoside hydrolase genes representing families GH5, GH9 and GH48, none of which shared more than
74% amino-acid identity with any characterised sequence in the CAZy database. Five candidates were
successfully expressed and purified, and biochemical characterisation identified one enzyme, designated CelA7
(family GH5), with an optimal temperature of 75 degrees C, retention of over 80% activity after 24 hours of
incubation at 70 degrees C, and specific activity on carboxymethyl cellulose of 142 U/mg, comparable to or
exceeding several previously reported thermostable fungal and bacterial cellulases used as reference
benchmarks. CelA7 additionally showed measurable activity on pretreated wheat straw and retained more than
60% of maximal activity in the presence of 1M NaCl, suggesting tolerance to the ionic conditions common in some
pretreatment liquors. These results demonstrate that thermophilic-phase compost metagenomes remain a
productive and comparatively underexploited resource for discovering industrially relevant thermostable cellulases
with sequence novelty sufficient to expand the functionally characterised diversity of glycoside hydrolase family
GH5.
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