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Mol Biotechnol. 2017 Jul;59(7):271-283. doi: 10.1007/s12033-017-0015-x.

Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus.

Author information

1
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia.
2
Faculty of Medical Sciences, Newcastle University Medicine Malaysia, 79200, Iskandar Puteri, Johor, Malaysia.
3
Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia Bintulu Sarawak Campus, 97008, Bintulu, Sarawak, Malaysia.
4
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia.
5
Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia.
6
Malaysia Genome Institute, Jalan Bangi Lama, 43000, Kajang, Selangor, Malaysia.
7
Department 4 (Materials and Environment), Federal Institute of Materials Research and Testing, Unter den Eichen 87, 12205, Berlin, Germany.
8
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia. fabyff@ukm.edu.my.

Abstract

Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris. Expressed GH1254 had an apparent molecular mass of 42 kDa, but purification was hampered by its low expression levels in shaken flasks. To obtain more of the enzyme, GH1254 was produced in a bioreactor that resulted in a fourfold increase in crude enzyme levels. The purified enzyme was active towards soluble synthetic substrates such as 4-methylumbelliferyl-β-D-cellobioside, 4-nitrophenyl-β-D-cellobioside and 4-nitrophenyl-β-D-lactoside but was non-hydrolytic towards Avicel or carboxymethyl cellulose. GH1254 catalyzed optimally at 35 °C and maintained 70% of its activity at 25 °C. This enzyme is thus potentially useful in food industries employing low-temperature conditions.

KEYWORDS:

Avicel; Biomass; Bioreactor; Cellulase; Saccharification; Symbiont

PMID:
28573450
DOI:
10.1007/s12033-017-0015-x
[Indexed for MEDLINE]

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