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The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor.

Pereira EO, Tsang A, McAllister TA, Menassa R - Biotechnol Biofuels (2013)

Bottom Line: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose.As such, they present the most abundant source for discovery of new enzymes.The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada ; Department of Biology, The University of Western Ontario, London, ON N6A 5B7, Canada.

ABSTRACT

Background: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries.

Results: We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively.

Conclusion: Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the lipA expression cassette. 35S promoter, 35S promoter from Cauliflower Mosaic Virus 35S gene; NOS, nopaline synthase terminator; tCUP, tobacco crypric upstream promoter translation enhancer; Pr1b, tobacco pathogenesis related 1b protein secretory signal peptide; XpressTM, epitope tag (Invitrogen); attB1 and attB2, Gateway® recombination sites; C-myc, detection/purification tag; KDEL, endoplasmic reticulum retrieval tetrapeptide. Schematic not drawn to scale.
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Figure 2: Schematic representation of the lipA expression cassette. 35S promoter, 35S promoter from Cauliflower Mosaic Virus 35S gene; NOS, nopaline synthase terminator; tCUP, tobacco crypric upstream promoter translation enhancer; Pr1b, tobacco pathogenesis related 1b protein secretory signal peptide; XpressTM, epitope tag (Invitrogen); attB1 and attB2, Gateway® recombination sites; C-myc, detection/purification tag; KDEL, endoplasmic reticulum retrieval tetrapeptide. Schematic not drawn to scale.

Mentions: To characterize LipA properties, we attempted to over-express the lipA gene in A. niger, and in the plant Nicotiana benthamiana. We were unsuccessful in obtaining LipA expression in A. niger (data not shown), but obtained high expression in N. benthamiana in transient co-expression of the lipA gene (Figure 2) with the suppressor of posttranscriptional gene silencing p19 [12]. Crude extracts as well as C-myc purified proteins from pooled samples from five different plants were resolved by SDS-PAGE and GelCode™ Blue staining and were confirmed by western blot analysis with monoclonal antibodies against the fused C-myc tag. This analysis showed that the plant-produced LipA protein has a molecular mass of about 56 kDa (Figure 3a and b). The observed band was larger than the predicted theoretical molecular weight of 46 kDa as the tags add about 5 kDa to the 41 kDa protein. As LipA has a putative N-glycosylation site, a deglycosylation experiment was performed by digesting LipA with N-glycosidase F (PNGase F). This led to a small downshift in the band size (Figure 4). However, even with deglycosylation, the molecular mass (46 kDa) predicted from the cDNA was substantially lower than that determined by SDS-PAGE. Since intrinsic charge can lead to anomalous migration on SDS-PAGE [13], this difference may reflect the acidic nature of LipA protein, which has an estimated pI of 4.9.


The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor.

Pereira EO, Tsang A, McAllister TA, Menassa R - Biotechnol Biofuels (2013)

Schematic representation of the lipA expression cassette. 35S promoter, 35S promoter from Cauliflower Mosaic Virus 35S gene; NOS, nopaline synthase terminator; tCUP, tobacco crypric upstream promoter translation enhancer; Pr1b, tobacco pathogenesis related 1b protein secretory signal peptide; XpressTM, epitope tag (Invitrogen); attB1 and attB2, Gateway® recombination sites; C-myc, detection/purification tag; KDEL, endoplasmic reticulum retrieval tetrapeptide. Schematic not drawn to scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750315&req=5

Figure 2: Schematic representation of the lipA expression cassette. 35S promoter, 35S promoter from Cauliflower Mosaic Virus 35S gene; NOS, nopaline synthase terminator; tCUP, tobacco crypric upstream promoter translation enhancer; Pr1b, tobacco pathogenesis related 1b protein secretory signal peptide; XpressTM, epitope tag (Invitrogen); attB1 and attB2, Gateway® recombination sites; C-myc, detection/purification tag; KDEL, endoplasmic reticulum retrieval tetrapeptide. Schematic not drawn to scale.
Mentions: To characterize LipA properties, we attempted to over-express the lipA gene in A. niger, and in the plant Nicotiana benthamiana. We were unsuccessful in obtaining LipA expression in A. niger (data not shown), but obtained high expression in N. benthamiana in transient co-expression of the lipA gene (Figure 2) with the suppressor of posttranscriptional gene silencing p19 [12]. Crude extracts as well as C-myc purified proteins from pooled samples from five different plants were resolved by SDS-PAGE and GelCode™ Blue staining and were confirmed by western blot analysis with monoclonal antibodies against the fused C-myc tag. This analysis showed that the plant-produced LipA protein has a molecular mass of about 56 kDa (Figure 3a and b). The observed band was larger than the predicted theoretical molecular weight of 46 kDa as the tags add about 5 kDa to the 41 kDa protein. As LipA has a putative N-glycosylation site, a deglycosylation experiment was performed by digesting LipA with N-glycosidase F (PNGase F). This led to a small downshift in the band size (Figure 4). However, even with deglycosylation, the molecular mass (46 kDa) predicted from the cDNA was substantially lower than that determined by SDS-PAGE. Since intrinsic charge can lead to anomalous migration on SDS-PAGE [13], this difference may reflect the acidic nature of LipA protein, which has an estimated pI of 4.9.

Bottom Line: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose.As such, they present the most abundant source for discovery of new enzymes.The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada ; Department of Biology, The University of Western Ontario, London, ON N6A 5B7, Canada.

ABSTRACT

Background: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries.

Results: We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively.

Conclusion: Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins.

No MeSH data available.


Related in: MedlinePlus