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The specificity of the bound and free forms of Lactobacillus casei subsp.casei IFPL 731 proteinase towards the αs1-casein-(1-23)-fragment has been studied. The use of the chelant agent EDTA for the extraction of the proteinase affects its specificity compared to either the use of lysozyme and mutanolysin or the whole-cell proteinase form. This gives a different pattern of the αs1-casein-fragment hydrolysis, as observed by HPLC. The effect of different chemical agents on the specific activity of the proteinase also varies depending on the method used to release the proteinase.