Physicochemical properties of proteinases from selected psychrotrophic bacteria

Abstract

The physicochemical properties of eight extracellular proteinases secreted by psychrotrophic bacteria of dairy origin have been studied. Seven of these proteinases were able to withstand ultra heat treatment (UHT) with D values at 140 °C ranging from 2 to 300 s. The six Pseudomonas fluorescens proteinases were glycoproteins of mol. wt 47000-49500. The two Serratia marcescens proteinases, of mol. wt of 51000, did not contain carbohydrate but in other respects were similar to the Pseudomonas proteinases. The proteinases were inhibited by various metal chelators and all contained Ca and Zn in similar proportions. Their amino acid compositions were similar, with alanine as the N-terminal group, cysteine completely absent and very low levels of methionine. Isoelectric points ranged from 5-10 to 8-25. Their physical and chemical properties enabled them to be classified as alkaline metalloendopeptidases. A similarity index (SAn) was used to predict sequence homology between ten proteinases of known amino acid composition. Comparisons of SAn of these proteinases showed only minor sequence differences except for those of Ps. fluorescens MC60. Heat resistance could not be related wholly to similarities in protein sequence, but could be related both to the strength of stabilizing Ca 2+ -protein interactions and to the randomness inherent within the folding of the peptide chain.

Purification and Characterization of a Metalloprotease fromChryseobacterium indologenesIx9a and Determination of the Amino Acid Specificity with Electrospray Mass Spectrometry

“…No activity was lost after heating at 50°C in SMS buffer for up to 30 min. This increased thermostability of proteases in the presence of SMS buffer was also reported by Stepaniak and Fox (2) and Mitchell et al (9). In practice this means that the naturally occurring minerals in milk might add to the thermostability of the proteases.…”

“…Zn 2ϩ was the most effective restorer of activity and a , and Mg 2ϩ ions were used effectively to restore the activity of the apoenzymes of most of the psychrotrophic proteases (7). However, Mn 2ϩ (4,14), Ba 2ϩ and Sr 2ϩ (3,15), Ni 2ϩ (4), and Fe 2ϩ (9,10) were also able to restore activity partially. Only in two cases were the proteases irreversibly inhibited by EDTA: the protease from P. fluorescens NCDO 2085 (12) and one of the proteases from Micrococcus GF (6).…”

“…Mitchell and Ewings (9) found that the protease from six P. fluorescens and two Serratia marcescens strains were all inhibited to different extents by phosphoramidon at a concentration of 20 g ⅐ ml Ϫ1 . The highest inhibition occurred with the protease from P. fluorescens where only 32% activity was left after incubation with phosphoramidon.…”

Age Gelation, Sedimentation, and Creaming in UHT Milk: A Review

“…However, some proteinases are heat‐stable and are not completely inactivated by the heat treatment conditions used in UHT processing. Some proteinases display exceptional heat stability and are capable of withstanding even very severe heating processes such as those used in retort sterilization (Law, ; Mitchell, Ewings, & Bartley, ; Richardson & Newstead, ; Richardson & Te Whaiti, ; Stoeckel et al., ). Some heat stable proteinases, such as those from the plasmin system, are indigenous to the milk system, while others, such as the enzymes produced by certain psychrotrophic bacteria are exogenous and thus not naturally found in milk.…”

Effects of plasmin and plasminogen on textural biochemical and microbiological properties of clotted cream

“…Number of psychrophilic bacteria in clotted cream samples produced in the study were found to be below 1 log cfu/g during the first 10 days of storage (Table ). Proteolytic enzymes formed by psychrotroph bacterial contaminants found in raw milk have a negative effect both on processing properties of raw milk and quality of heat processed milk and dairy products (Mitchell, Ewings, & Bartley, ). Psychrotroph bacteria which are mostly isolated from dairy products can develop at 3–7 °C and consist both gram‐negative (such as Pseudomonas , Achromobacter , Aeromonas , Serratia , Alcaligenes , Chromobacterium , and Flavobacterium spp.)…”