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Purification of Serine Protease Fermentation Concentrate via Heat Treatment

IP.com Disclosure Number: IPCOM000168822D
Publication Date: 2008-Mar-28
Document File: 3 page(s) / 34K

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Abstract

Fermentation of serine protease from Bacillus species includes production of the serine protease plus minor levels of other proteins. Here we show that through a heat treatment step of a formulated protease concentrate, the minor proteins are degraded or “cleaned up” by the protease giving a more pure form of the protein concentrate of interest.

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Purification of Serine Protease Fermentation Concentrate via Heat Treatment

Abstract

Fermentation of serine protease from Bacillus species includes production of the serine protease plus minor levels of other proteins.  Here we show that through a heat treatment step of a formulated protease concentrate, the minor proteins are degraded or “cleaned up” by the protease giving a more pure form of the protein concentrate of interest.

Introduction

Serine proteases are very effective and widely used in many industrial applications from key ingredients in household detergents (laundry and cleaning) to ingredients in cosmetic products.  Bacillus is one of the well known microorganisms exploited for its capability of producing serine protease at an industrial scale.  It is known however that throughout the fermentation of serine protease from Bacillus sp. various other proteins are produced as well. 

Heat treatment has proved to be an effective means of purifying proteases [1], and we have identified conditions that show when exposing a final formulated protease to high heat for various lengths of time, the side proteins become hydrolyzed by the protease action giving a more purified serine protease product.

Experimental

Serine protease was produced in Bacillus sp. at an industrial scale.  The fermentation broth was harvested and put through a recovery process that separated out cellular debris and then concentrated the retained liquid.  The protease was then stabilized with various formulation excipients.

Three aliquots (20 ml each) of the formulated concentrate were measured into 50 ml conical tubes with caps and set into a water bath that was pre-heated to 60oC.  The first tube was removed after 1 hour incubation at 60oC and immediately cooled in an ice bath.  Tubes 2 and 3 were removed from the heated bath at 1.5 hours and 2 hours respectively and cooled in the ice bath as well.

Protein purity was determined by analyzing the protein bands of the heat treated samples and the parent concentrate (no heat treatment) on SDS-PAGE gels by densitometry.  The samples were also assayed for protease and amylase activity to check for degradation of both proteins.  Expression of minor levels of amylase side activity is common in Bacillus fermentations.
Results and Discussion

SDS-PAGE analysis (Coomassie stain) showed that as the time of heat exposure increased from 0 to 2 hours, the minor proteins expressed in the serine protease concentrate were clearly less intense in the heat-treated samples than in the formulated concentrate that had no heat treatment.  All of the protease samples were inactivated via a hydrochloric acid/low pH treatment in order to prevent autolysis in the SDS preparation.  The data was...