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

Abstract

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX 1 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X 33 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 X 33 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 His 6 -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 His 6 -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 (His 6 -ROLw) and the mutant (His 6 -ROLw-N134H) proteins was carried out.

Rhizopus oryzae Lipase, a Promising Industrial Enzyme: Biochemical Characteristics, Production and Biocatalytic Applications

“…Moreover, as all enzymes, ROL activity is highly influenced by the pH and temperature. Optimum activity pH values of 8 have been principally reported [56,57,73,75,[90][91][92][93][94][95][96]. However, other studies have also stated more acid [74,[97][98][99][100][101][102][103] and basic [68,104,105] optimums.…”

Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

“…Using this yeast provides a number of advantages, including little endogenous protein secretion, the absence of endogenous lipases and esterases, the presence of a powerful, tightly regulated methanol-inducible alcohol oxidase 1 promoter (P AOX1 ), and the ability to grow at high cell densities on defined media [30]. The high suitability of P. pastoris for ROL production is confirmed by the vast amount of literature on its cloning and expression [31][32][33][34][35]. Previous studies have examined the effects of cloning and expressing the whole ROL prosequence (97 amino acids) with the mature sequence in P. pastoris [36,37].…”

Cloning and Over Expression of CVS Rabies Virus Glycoprotein Gene in Pichia pastoris by Multimeriztion

“…The increased in molecular mass weight in our experiment might be due to alpha secretory signal and his tags which adds nearly 12kd protein. Salah et al, 2009 reported the increased size of the Rabies Glycoprotein is due to posttranslational modification of secreted proteins such as O-glycosylation, lipidation or phosphorylation. In our experiment, the Pichia produced G protein is comparable to authentic G protein since it was normally reacted with the confirmation epitope anti-G MAbs.…”