Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2012-10-23

AUTHORS

Neha Garg, Nora Bieler, Tenzin Kenzom, Meenu Chhabra, Marion Ansorge-Schumacher, Saroj Mishra

ABSTRACT

BackgroundLaccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase.ResultsIn this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac.ConclusionLaccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L-1 in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation. More... »

PAGES

75

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1472-6750-12-75

DOI

http://dx.doi.org/10.1186/1472-6750-12-75

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1042959819

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/23092193


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77 finger printing
78 fungi
79 glycosylation
80 glycosylation patterns
81 goal
82 guaiacol
83 high homology
84 high levels
85 homogeneity
86 homology
87 improvement
88 individual enzymes
89 interest
90 investigation
91 investigation of properties
92 isozymes
93 kinetic parameters
94 laccase
95 laccases
96 levels
97 levels of laccase
98 low levels
99 mass finger printing
100 medium
101 multi-copper oxidases
102 native fungi
103 native laccase
104 non-phenolic compounds
105 oxidase
106 oxidation
107 oxidation of phenolics
108 parameters
109 pastoris
110 pattern of glycosylation
111 patterns
112 peptide mass finger printing
113 peptides
114 phenolics
115 position
116 potential
117 presence
118 print analysis
119 printing
120 procedure
121 production medium
122 promoter
123 properties
124 protein
125 pyrogallol
126 rLac
127 recombinant enzyme
128 recombinant laccase
129 redox enzymes
130 redox potential
131 region
132 salt tolerance
133 sequence
134 sequence analysis
135 signal peptide
136 solvent tolerance
137 specific activity
138 study
139 sulfate
140 supernatant
141 synthesis
142 system
143 thermostability
144 three-step procedure
145 tolerance
146 tripeptide
147 versicolor
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