English

Jean Bernard Ndayambaje,,Meenakshisundaram S

doi:10.5897/ajb2013.12986

Abstract

Process techniques of the methylotrophic yeast Pichia pastoris for the production and recovery of heterologous phytase proteins has developed in last 20 years. High expression levels using methanol as induction have been made in the quality of recombinant proteins in the fermenter culture and in the quality of the protein product. This allowed rapidly P. pastoris to become the system of choice for the expression of recombinant proteins in yeast. The experimental designs, the methanol/L-alanine co-feeding strategy and optimization of phytase production by P. pastoris supported by the optimum levels of variables and lower temperature expression produced high level of phytase activity which could be scaled up to produce phytase for food additives at industrial level.  An overall phytase activity was 8632 U/ml, this means 332 fold increase compare to the wild type of phytase. This work demonstrates not only the impact of α-factor prepro secretion signal and efficiency of methanol/L-alanine co-induction strategy for phytase production by recombinant P. pastoris Mut+ strains, but also shows new insights for the expression of bioproduct at lower temperature.   Key words: Phytase, gene expression, Pichia pastoris, process optimization, co-feeding strategy, Deglycosylation.

Highlights

The phosphate (Phytic acid) which is released from non ruminant animals becomes pollutant to the environment and as far as the phytase enzyme for its degradation is needed as food additive (Yu et al, 2012; Xiong et al, 2006; Haefner et al 2005)
Ligation PCR was performed with T4 DNA ligase, AOX1 forward and phytase reverse primers were used for the confirmation PCR reaction
Phytase gene 1.3 kb has been sent to synergy lab for sequencing and it was compared with previously isolated phytase genes

Summary

Introduction

The phosphate (Phytic acid) which is released from non ruminant animals becomes pollutant to the environment and as far as the phytase enzyme for its degradation is needed as food additive (Yu et al, 2012; Xiong et al., 2006; Haefner et al 2005). Methanol is used as energy and carbon source and as an inducer of recombinant protein expression (McKinney et al, 2004), and long ago, at high concentrations, it inhibits growth (Zhang et al, 2000). Phase and Glycerol Fed Batch are first fed to the culture to increase biomass concentration and the culture is switched to methanol to increase productivity, cell density and to reduce the induction time. The proteolysis of the secreted products and cell death in the high cell density bioreactor cultures is the main limitations when the enzyme is expressed at higher temperature (Li et al., 2006; Pakkanen et al, 2003; Lee et al, 2005; Porro et al., 2005).

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Conclusion