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Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

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An Erratum to this article was published on 03 January 2010

Abstract

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX1 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X33 and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X33 cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His6-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His6-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His6-ROLw) and the mutant (His6-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.

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Abbreviations

ROLw:

Rhizopus oryzae WPG lipase

RDL:

Rhizopus delemar lipase

RNL:

Rhizopus niveus lipase

ROL29:

Rhizopus oryzae lipase

A:

Amino acids

Asn (N):

Asparagine

His (H):

Histidine

His6 :

6 Histidine

Ser (S):

Serine

D:

Aspartic acid

LB:

Luria-Bertani

GA:

Gum arabic

BSA:

Bovine serum albumin

DTT:

Dithiothreitol

NaDC:

Sodium deoxycholate

PCR:

Polymerase chain reaction

SDS/PAGE:

Sodium dodecyl sulfate/polyacrylamide gel electrophoresis

TC2 :

Triacetin

TC3 :

Tripropionin

TC4 :

Tributyrin

TC8 :

Trioctanoin

TC18 :

Triolein

TAG:

Triacylglycerol

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Acknowledgments

This work is supported financially by « Ministère de la recherche scientifique-Tunisia » through a grant to « Laboratoire de Biochimie et de Génie Enzymatique des Lipases—ENIS ».

We thank JAOUA Mohamed for sequencing DNA, LADJIMI Moncef, BEZZINE Sofiane, SAYARI Adel and MILED Nabil for the considerable collaboration during the preparation of this work.

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Authors and Affiliations

  1. Laboratoire de biochimie et de génie enzymatique des lipases, ENIS, Route de Soukra, “BPW”, 3038, Sfax, Tunisia

    Riadh Ben Salah, Youssef Gargouri & Hafedh Mejdoub

  2. Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, Route de Sidi Mansour, Km 6, P.O. Box « K », 3038, Sfax, Tunisia

    Ali Gargouri

  3. Laboratoire de Lipolyse Enzymatique, UPR 9025, CNRS, Marseille, France

    Robert Verger

Authors
  1. Riadh Ben Salah
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  2. Ali Gargouri
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  3. Robert Verger
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  4. Youssef Gargouri
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  5. Hafedh Mejdoub
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Corresponding author

Correspondence to Riadh Ben Salah.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s11274-009-0279-9

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Ben Salah, R., Gargouri, A., Verger, R. et al. Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization. World J Microbiol Biotechnol 25, 1375–1384 (2009). https://doi.org/10.1007/s11274-009-0024-4

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  • DOI: https://doi.org/10.1007/s11274-009-0024-4

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