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Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme.
Protein Expr Purif. 2009 Jun; 65(2):185-9.PE

Abstract

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of approximately 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.

Authors+Show Affiliations

Departamento de Biologia, Faculdade de Filosofia Ciências e Letras, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19073263

Citation

de Almeida, Fabiana M., et al. "Heterologous Expression in Escherichia Coli of Neurospora Crassa Neutral Trehalase as an Active Enzyme." Protein Expression and Purification, vol. 65, no. 2, 2009, pp. 185-9.
de Almeida FM, Bonini BM, Beton D, et al. Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme. Protein Expr Purif. 2009;65(2):185-9.
de Almeida, F. M., Bonini, B. M., Beton, D., Jorge, J. A., Terenzi, H. F., & da Silva, A. M. (2009). Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme. Protein Expression and Purification, 65(2), 185-9. https://doi.org/10.1016/j.pep.2008.11.010
de Almeida FM, et al. Heterologous Expression in Escherichia Coli of Neurospora Crassa Neutral Trehalase as an Active Enzyme. Protein Expr Purif. 2009;65(2):185-9. PubMed PMID: 19073263.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme. AU - de Almeida,Fabiana M, AU - Bonini,Beatriz M, AU - Beton,Daniela, AU - Jorge,João A, AU - Terenzi,Héctor F, AU - da Silva,Aline M, Y1 - 2008/12/06/ PY - 2008/10/31/received PY - 2008/11/21/revised PY - 2008/11/24/accepted PY - 2008/12/17/entrez PY - 2008/12/17/pubmed PY - 2009/8/25/medline SP - 185 EP - 9 JF - Protein expression and purification JO - Protein Expr. Purif. VL - 65 IS - 2 N2 - Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of approximately 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. SN - 1096-0279 UR - https://www.unboundmedicine.com/medline/citation/19073263/Heterologous_expression_in_Escherichia_coli_of_Neurospora_crassa_neutral_trehalase_as_an_active_enzyme_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(08)00319-7 DB - PRIME DP - Unbound Medicine ER -