Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1
Trichoderma sp. is a fungus capable of producing three categories of cellulase for cellulose degradation into glucose; endoglucanase, cellobiohydrolase and β- glucosidase. However, native production of β-glucosidase from fungi is often at low concentrations and takes a longer time. Furthermore, p...
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my-upm-ir.927982022-06-20T03:26:37Z Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1 2019-03 Mohamad Sobri, Mohamad Farhan Trichoderma sp. is a fungus capable of producing three categories of cellulase for cellulose degradation into glucose; endoglucanase, cellobiohydrolase and β- glucosidase. However, native production of β-glucosidase from fungi is often at low concentrations and takes a longer time. Furthermore, product inhibition caused by glucose on β-glucosidase reduces resulting yields, making it the rate limiting enzyme and represents an obstacle for commercial cellulose hydrolysis. Studies on β-glucosidases produced by Trichoderma sp. have elucidated two variants, which have been classified into glycosyl hydrolase (GH) families 1 and 3, with attention given to GH family 1 (Bgl2), owing to its relatively lower sensitivity to glucose inhibition, a desirable character for bioprocess development for efficient lignocellulosic biomass saccharification. As such, using locally isolated Trichoderma asperellum UPM1, this study has sought to characterise the bgl2 gene isolated and following heterologous expression in Escherichia coli, characterise the recombinant enzyme for enzyme activity and glucose tolerance. Trichoderma asperellum bgl2 (Tabgl2) gene sequence isolated was found to be 1398 nucleotides in length, encoding a protein of 465 amino acids in length, with an estimated molecular weight of 52798.31 Daltons. The identity of Trichoderma sp. glycosyl hydrolase family 1 β-glucosidase was affirmed by the presence of N-terminal signature of 15 amino acids in length, cis-peptide bonds at A180-P181 and W417-S418, conserved active site motifs with glutamate (E) residues (‘TFNEP’ and ‘VTENG’), 17 corresponding substrate binding and a lone conserved stabilising tryptophan (W) residue. Automated protein structure homology-modelling revealed the common triosephosphate isomerase (TIM) barrel fold, functioning as a monomer while protein phylogeny analyses positioned the isolated protein to a clade with known Trichoderma sp. β- glucosidases. Intracellular protein localisation was confirmed by the absence of a signal sequence. Suggestive glucose tolerance was inferred from the presence of 14 of 22 consensus residues from known glucose tolerant amino acid residues as well as the presence of corresponding residues L167 and P172, crucial in the retention of the active site’s narrow cavity, found in glucose tolerant Bgl2 from Trichoderma reesei. Characterisation thus proceeded by codon optimisation of the gene followed by transformation and heterologous expression in Escherichia coli using plasmid vector pET-20b(+), targeted for periplasmic expression of recombinant T. asperellum Bgl2 (TaBgl2). Protein expression analysis using SDS-PAGE showed the presence of a ~52 kDA protein in size while the crude enzyme extracts showed a specific activity of 0.0081 U/mg in the periplasmic fraction, 11.6-fold higher than in the periplasmic fraction of the E. coli host without IPTG induction. Glucose tolerance was affirmed with 40% of relative activity retained in a concentration of 0.2 M glucose. Thus, the relatively low sensitivity of TaBgl2 to inhibition by glucose makes this enzyme a potential candidate for further analyses in cellulose hydrolysis. Trichoderma Recombinant proteins Glucosidases 2019-03 Thesis http://psasir.upm.edu.my/id/eprint/92798/ http://psasir.upm.edu.my/id/eprint/92798/1/FBSB%202019%2031%20IR.pdf text en public masters Universiti Putra Malaysia Trichoderma Recombinant proteins Glucosidases Ramli, Norhayati |
| institution |
Universiti Putra Malaysia |
| collection |
PSAS Institutional Repository |
| language |
English |
| advisor |
Ramli, Norhayati |
| topic |
Trichoderma Recombinant proteins Glucosidases |
| spellingShingle |
Trichoderma Recombinant proteins Glucosidases Mohamad Sobri, Mohamad Farhan Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1 |
| description |
Trichoderma sp. is a fungus capable of producing three categories of cellulase
for cellulose degradation into glucose; endoglucanase, cellobiohydrolase and β-
glucosidase. However, native production of β-glucosidase from fungi is often at
low concentrations and takes a longer time. Furthermore, product inhibition
caused by glucose on β-glucosidase reduces resulting yields, making it the rate
limiting enzyme and represents an obstacle for commercial cellulose hydrolysis.
Studies on β-glucosidases produced by Trichoderma sp. have elucidated two
variants, which have been classified into glycosyl hydrolase (GH) families 1 and
3, with attention given to GH family 1 (Bgl2), owing to its relatively lower
sensitivity to glucose inhibition, a desirable character for bioprocess
development for efficient lignocellulosic biomass saccharification. As such, using
locally isolated Trichoderma asperellum UPM1, this study has sought to
characterise the bgl2 gene isolated and following heterologous expression in
Escherichia coli, characterise the recombinant enzyme for enzyme activity and
glucose tolerance.
Trichoderma asperellum bgl2 (Tabgl2) gene sequence isolated was found to be
1398 nucleotides in length, encoding a protein of 465 amino acids in length, with
an estimated molecular weight of 52798.31 Daltons. The identity of Trichoderma
sp. glycosyl hydrolase family 1 β-glucosidase was affirmed by the presence of
N-terminal signature of 15 amino acids in length, cis-peptide bonds at A180-P181
and W417-S418, conserved active site motifs with glutamate (E) residues
(‘TFNEP’ and ‘VTENG’), 17 corresponding substrate binding and a lone
conserved stabilising tryptophan (W) residue. Automated protein structure
homology-modelling revealed the common triosephosphate isomerase (TIM)
barrel fold, functioning as a monomer while protein phylogeny analyses
positioned the isolated protein to a clade with known Trichoderma sp. β-
glucosidases. Intracellular protein localisation was confirmed by the absence of
a signal sequence. Suggestive glucose tolerance was inferred from the presence
of 14 of 22 consensus residues from known glucose tolerant amino acid residues as well as the presence of corresponding residues L167 and P172, crucial in the
retention of the active site’s narrow cavity, found in glucose tolerant Bgl2 from
Trichoderma reesei.
Characterisation thus proceeded by codon optimisation of the gene followed by
transformation and heterologous expression in Escherichia coli using plasmid
vector pET-20b(+), targeted for periplasmic expression of recombinant T.
asperellum Bgl2 (TaBgl2). Protein expression analysis using SDS-PAGE
showed the presence of a ~52 kDA protein in size while the crude enzyme
extracts showed a specific activity of 0.0081 U/mg in the periplasmic fraction,
11.6-fold higher than in the periplasmic fraction of the E. coli host without IPTG
induction. Glucose tolerance was affirmed with 40% of relative activity retained
in a concentration of 0.2 M glucose. Thus, the relatively low sensitivity of TaBgl2
to inhibition by glucose makes this enzyme a potential candidate for further
analyses in cellulose hydrolysis. |
| format |
Thesis |
| qualification_level |
Master's degree |
| author |
Mohamad Sobri, Mohamad Farhan |
| author_facet |
Mohamad Sobri, Mohamad Farhan |
| author_sort |
Mohamad Sobri, Mohamad Farhan |
| title |
Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1 |
| title_short |
Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1 |
| title_full |
Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1 |
| title_fullStr |
Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1 |
| title_full_unstemmed |
Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1 |
| title_sort |
characterisation and expression of recombinant beta-glucosidase 2 from trichoderma asperellum upm1 |
| granting_institution |
Universiti Putra Malaysia |
| publishDate |
2019 |
| url |
http://psasir.upm.edu.my/id/eprint/92798/1/FBSB%202019%2031%20IR.pdf |
| _version_ |
1747813769502785536 |