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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

Effect of pH and temperature on LipA enzyme activity. (A) The influence of pH on activity was determined using 1 mM pNP-caprylate in 100 mM buffers (pH 4.0-5.0 acetate buffer, pH 6.0-9.0 phosphate buffer, pH 10.0-11.0 carbonate buffer) at 40°C for 30 min. (B) The influence of temperature was determined in a range from 20-90°C using 1 mM pNP caprylate in 100 mM phosphate buffer pH 7.0 for 30 min. The test was performed in triplicate and the average values were transformed in the relative activity (%).
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Figure 5: Effect of pH and temperature on LipA enzyme activity. (A) The influence of pH on activity was determined using 1 mM pNP-caprylate in 100 mM buffers (pH 4.0-5.0 acetate buffer, pH 6.0-9.0 phosphate buffer, pH 10.0-11.0 carbonate buffer) at 40°C for 30 min. (B) The influence of temperature was determined in a range from 20-90°C using 1 mM pNP caprylate in 100 mM phosphate buffer pH 7.0 for 30 min. The test was performed in triplicate and the average values were transformed in the relative activity (%).

Mentions: As the goal of this study was to identify and characterize alkaliphilic enzymes for potential application in industrial processes, temperature and pH optima are important characteristics for the efficient usage of this enzyme. Purified plant-produced LipA was used to study the effect of pH and temperature using p-nitrophenyl caprylate as substrate. The optimal pH of purified LipA under the standard assay conditions was found to be pH 8.0 (Figure 5a). The enzyme showed maximum lipase activity at 40°C with broad thermostability ranging from 20 C to 50°C (Figure 5b).


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)

Effect of pH and temperature on LipA enzyme activity. (A) The influence of pH on activity was determined using 1 mM pNP-caprylate in 100 mM buffers (pH 4.0-5.0 acetate buffer, pH 6.0-9.0 phosphate buffer, pH 10.0-11.0 carbonate buffer) at 40°C for 30 min. (B) The influence of temperature was determined in a range from 20-90°C using 1 mM pNP caprylate in 100 mM phosphate buffer pH 7.0 for 30 min. The test was performed in triplicate and the average values were transformed in the relative activity (%).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of pH and temperature on LipA enzyme activity. (A) The influence of pH on activity was determined using 1 mM pNP-caprylate in 100 mM buffers (pH 4.0-5.0 acetate buffer, pH 6.0-9.0 phosphate buffer, pH 10.0-11.0 carbonate buffer) at 40°C for 30 min. (B) The influence of temperature was determined in a range from 20-90°C using 1 mM pNP caprylate in 100 mM phosphate buffer pH 7.0 for 30 min. The test was performed in triplicate and the average values were transformed in the relative activity (%).
Mentions: As the goal of this study was to identify and characterize alkaliphilic enzymes for potential application in industrial processes, temperature and pH optima are important characteristics for the efficient usage of this enzyme. Purified plant-produced LipA was used to study the effect of pH and temperature using p-nitrophenyl caprylate as substrate. The optimal pH of purified LipA under the standard assay conditions was found to be pH 8.0 (Figure 5a). The enzyme showed maximum lipase activity at 40°C with broad thermostability ranging from 20 C to 50°C (Figure 5b).

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