Avaliação do efeito do sal volatil carbamato de amonio sobre a estabilidade de enzimas / Evaluation of the effect of ammonium carbamate on the stability of enzymes

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Salting-out is a precipitation method frequently found in downstream processing schemes of proteins. However, the need for post-precipitation treatment for salt recovery and final purification of the protein may lead precipitation with conventional salts to be costly and limited for environmental questions. Precipitation with Volatile salts is an alternative to conventional precipitation, presenting as main advantage the easy salt removal from precipitates and liquid effluents by a temperature increase or a pressure reduction. Protein salting-out induction with these salts was pioneered by our research group. However, there was evidence of some denaturation, although efficient precipitation was verified for all proteins studied. To address this question, the objective of this work was to evaluate the effect of ammonium carbamate volatile salt on the specific activity of five enzymes (an a-amylase, a lysozyme, a cellulase complex, a peroxidase and a lipase). Experiments were made with enzymes kept at 4.0ºC for 60 min in solutions 1.0 to 5.0 molal of this salt. After this salt removal, the dried masses were dissolved in appropriate buffers and these solutions had their protein concentration and enzymatic activity measured. Similar experiments with conventional salts were also carried-out which the desalination made by gel permeation columns as well as precipitation assays with ammonium carbamate at ionic strength from 5.0 to 9.0 molal. a-Amylase, lysozyme and lipase did not undergo activity loss in salt solutions from 1.0 to 5.0 molal, whereas cellulase and peroxidase showed an average activity loss of 55% and 44%, respectively. Analysis of similar experiments with ammonium sulfate and sodium sulfate led to the conclusion that these two enzymes denatured with volatile salt ammonium carbamate. The precipitation assays did not show enzyme denaturation or separation of phases for a -amylase and lipase while celullase and peroxidase precipitated with some activity reduction. Mass and activity balances determined for these cases did not allow a definitive conclusion about the effect of ammonium carbamate on these two enzymes during the precipitation

ASSUNTO(S)

estabilidade precipitação (quimica) enzimas eletrolitos lolatile electrolytes biosseparation precipitation (chemistry) stability

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