Produção de quimosina B de Bos taurus em Pichia pastoris

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in κ casein by an aspartic protease. Bovine chymosin B is commonly used at the industrial level for milk coagulation because it combines a strong clotting activity with a low general proteolytic activity. In the present study, we report the expression, purification and characterization of recombinant chymosin B expressed in the methylotrophic yeast Pichia pastoris. To achieve high levels of production, the cDNA fragment coding for prochymosin was designed and synthesized based on codon usage optimization for expression in P. pastoris. The synthetic gene (CHYMb) was cloned into expression vectors based on the PGK1 promoter (constitutive expression) and AOX1 promoter (inducible expression). The resulted constructs were integrated in the yeast genome. Transformant clones producing high levels of enzyme activity from each system (P1 and A3, respectively) were analyzed in a simultaneous culture for comparison of enzymatic production. High levels of chymosin activity were observed in the clone controlled by the PGK1 promoter which showed high production levels throughout the culture. Optimization for enzymatic production was developed through factorial design. The influence of the initial cellular density, methanol and nitrogen source concentration were accessed for enzymatic production from clone A3. The interaction of the initial cellular density and methanol concentration was responsible for doubling enzyme production. The influence of different concentrations of nitrogen and carbon sources (soy extract and inverted sugar, respectively) was also observed to increase enzymatic production in clone P1. Statistic analysis demonstrated that higher concentrations of each factor increased enzyme production. The profile of enzymatic activity was evaluated in fermentors under the conditions determined during optimizations. In this case, the activities observed in clones A3 and P1 were superior to those found when theses clones were grown in shake flasks. The recombinant chymosin was purified by a single molecular exclusion chromatographic step. Purified enzyme derived from supernatant fraction was shown to be present in its processed form and a fraction of glycosylated protein was also detected. The specificity for casein κ was shown to be similar to that from commercial recombinant chymosin produced by Aspergillus niger (Chr. Hansen). The high production and high specificity of recombinant chymosin derived from P. pastoris renders this expression system as an attractive alternative for large scale industrial production of this enzyme.

ASSUNTO(S)

enzimas industriais biologia molecular pichia pastoris coagulação do leite quimosina

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