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Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris

Woon JS, Mackeen MM, Mahadi NM, Illias RM, Abdul Murad AM, Abu Bakar FD

Biotechnol Appl Biochem, 2016 Sep;63(5):690-698.
PMID: 26265428 DOI: 10.1002/bab.1431

The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic(®) CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
Production of an oligosaccharide-specific cellobiohydrolase from the thermophilic fungus Thielavia terrestris

Woon JS, Mackeen MM, Sudin AH, Mahadi NM, Illias RM, Murad AM, et al.

Biotechnol Lett, 2016 May;38(5):825-32.
PMID: 26830095 DOI: 10.1007/s10529-016-2045-z

To express and determine the hydrolytic activity of a cellobiohydrolase (TTCBH6B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris.
Erratum to: Production of an oligosaccharide-specific cellobiohydrolase from the thermophilic fungus Thielavia terrestris

Woon JS, Mackeen MM, Sudin AH, Mahadi NM, Illias RM, Murad AM, et al.

Biotechnol Lett, 2016 May;38(5):833.
PMID: 26914687
Fulltext Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches

Woon JS, Mackeen MM, Illias RM, Mahadi NM, Broughton WJ, Murad AMA, et al.

PeerJ, 2017;5:e3909.
PMID: 29038760 DOI: 10.7717/peerj.3909

BACKGROUND: Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger.
METHODS: In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic(®) CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment.
RESULTS: Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel(®) and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic(®) CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples.
DISCUSSION: Since CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic(®) CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic(®) CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.
Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus

Woon JS, King PJH, Mackeen MM, Mahadi NM, Wan Seman WMK, Broughton WJ, et al.

Mol Biotechnol, 2017 Jul;59(7):271-283.
PMID: 28573450 DOI: 10.1007/s12033-017-0015-x

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.