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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

Substrate specificity of the lipase from Acremonium alcalophilum towards p-Nitrophenol fatty acid esters with varied acyl chain lengths. Values are means ± SD (n = 3).
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Figure 6: Substrate specificity of the lipase from Acremonium alcalophilum towards p-Nitrophenol fatty acid esters with varied acyl chain lengths. Values are means ± SD (n = 3).

Mentions: As the gene of interest possessed a sequence similar to that of other genes coding for lipases and feruloyl esterases we evaluated the specificity/preference of LipA for a variety of fatty acid and hydroxycinnamate ester substrates. To investigate the substrate specificity of LipA, the hydrolyzing activity of the purified enzyme was determined at its pH and temperature optima of pH 8.0 and 40°C, respectively. The substrates analyzed included pNP esters of various carbon chain lengths, methyl cinnamates and the acetylated compounds oat spelt xylan and xylose tetra-acetate. As shown in Figure 6, LipA exhibited low specificity preference towards short chain pNP ester (C2) and intermediate activity towards long-chain pNP esters (C14 and C16). Although lipases are capable of hydrolyzing long chain fatty acid esters, some lipases have maximum activity towards medium or shorter acyl groups. LipA had a clear preference for medium chain fatty acid ester (pNP caprylate, C8) (Figure 6) demonstrating substrate specificity similar to that obtained with other lipases from microorganisms such as Bacillus stearothermophilus L1 [15] and Aeromonas sp. LPB 4 [16]. The enzyme showed a very broad activity toward pNP esters with the exception of pNP arabinopyranoside, pNP arabinofuranoside and pNP xylopyranoside (Table 1), illustrating its specificity towards the hydrolysis of acyl esters but no activity towards aromatic residues. No activity was found towards methyl cinnamate suggesting that this enzyme has no hydroxycinnamoyl esterase properties (Table 1).


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)

Substrate specificity of the lipase from Acremonium alcalophilum towards p-Nitrophenol fatty acid esters with varied acyl chain lengths. Values are means ± SD (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Substrate specificity of the lipase from Acremonium alcalophilum towards p-Nitrophenol fatty acid esters with varied acyl chain lengths. Values are means ± SD (n = 3).
Mentions: As the gene of interest possessed a sequence similar to that of other genes coding for lipases and feruloyl esterases we evaluated the specificity/preference of LipA for a variety of fatty acid and hydroxycinnamate ester substrates. To investigate the substrate specificity of LipA, the hydrolyzing activity of the purified enzyme was determined at its pH and temperature optima of pH 8.0 and 40°C, respectively. The substrates analyzed included pNP esters of various carbon chain lengths, methyl cinnamates and the acetylated compounds oat spelt xylan and xylose tetra-acetate. As shown in Figure 6, LipA exhibited low specificity preference towards short chain pNP ester (C2) and intermediate activity towards long-chain pNP esters (C14 and C16). Although lipases are capable of hydrolyzing long chain fatty acid esters, some lipases have maximum activity towards medium or shorter acyl groups. LipA had a clear preference for medium chain fatty acid ester (pNP caprylate, C8) (Figure 6) demonstrating substrate specificity similar to that obtained with other lipases from microorganisms such as Bacillus stearothermophilus L1 [15] and Aeromonas sp. LPB 4 [16]. The enzyme showed a very broad activity toward pNP esters with the exception of pNP arabinopyranoside, pNP arabinofuranoside and pNP xylopyranoside (Table 1), illustrating its specificity towards the hydrolysis of acyl esters but no activity towards aromatic residues. No activity was found towards methyl cinnamate suggesting that this enzyme has no hydroxycinnamoyl esterase properties (Table 1).

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