Purification and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458

Smacchi, E.

doi:10.1016/s0378-1097(98)00562-x

Cited by 3 publications

(7 citation statements)

References 15 publications

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“…Hydrophobic interaction chromatography has been a successful technique for the purification of many alkaline proteases. However, on previous occasions, it has been used in combination with other chromatographic techniques [25,45]. The AH-6 protease was a monomeric protein with a molecular mass of 40 kDa as revealed by native and SDS-PAGE (Fig.…”

Section: Enzyme Purification By Hydrophobic Interaction Chromatographymentioning

confidence: 99%

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

Dodia¹,

Rawal²,

Bhimani³

et al. 2007

J Ind Microbiol Biotechnol

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An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8-13, optimally at 9-11. It was stable with 0-4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 degrees C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25-80 degrees C (optimum at 50 degrees C), the purified enzyme had temperature optimum at 37 degrees C, which shifted to 80 degrees C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K(cat)). The enzyme was also calcium dependent and with 2 mM Ca(+2), the activity reached to maximum at 50 degrees C. The crude enzyme was highly thermostable (37-90 degrees C); however, the purified enzyme lost its stability above 50 degrees C and its half life was enhanced by 30 and sevenfold at 60 degrees C with 1 M NaCl and 50 mM Ca(+2), respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.

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Section: Enzyme Purification By Hydrophobic Interaction Chromatographymentioning

confidence: 99%

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

Dodia¹,

Rawal²,

Bhimani³

et al. 2007

J Ind Microbiol Biotechnol

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“…Moreover, tolerance to or activation by NaCl is a prerequisite for the activity of bacterial enzymes in the smear of the surface-ripened cheeses. Similar to the proteinases from A. nicotianae [17] and of B. linens [24], the proline iminopeptidase from A. nicotianae 9458 tolerated NaCl concentrations up to 7.5%. Specialized peptidases capable of hydrolyzing Pro-containing sequences have been postulated to be important for the degradation of casein-derived peptides because of the high content of proline in these molecules [1].…”

Section: Discussionmentioning

confidence: 73%

“…Like other enzymes from smear cheese micro£ora, such as coryneforms, micrococci and yeasts [17,24,25], the proline iminopeptidase from A. nicotianae 9458 had an alkaline pH optimum but showed considerable activity at the pH of the smear of surface-ripened cheese, i.e. 6.0^7.0, which are still compatible with cell growth in cheese surface.…”

Section: Discussionmentioning

confidence: 97%

“…has probably been underestimated in comparison to B. linens and lactic acid bacteria. To date, only two extracellular serine proteinases have been puri¢ed from the dairy A. nicotianae 9458 [17].…”

Section: Discussionmentioning

confidence: 99%

“…Apart from few studies on the enzymology of soil Arthrobacter spp. [14^16], to our knowledge only extracellular proteinases from dairy Arthrobacter have been puri¢ed and characterized [8,17].…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

Smacchi

1999

FEMS Microbiology Letters

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An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37³C and 8.0, respectively, was completely inactivated by heating for 1 min at 80³C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7,5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe 2 , Sn 2 , Cu 2 , Zn 2 , Hg 2 , Co 2 and Ni 2 . The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheeses and may contribute to proteolysis of these cheeses during ripening. ß

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Purification and characterization of an extracellular proline iminopeptidase from Corynebacterium variabilis NCDO 2101

et al. 2001

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Aims: To screen the extracellular proteolytic and lipolytic activities of Corynebacterium variabilis NCDO 2101 and to purify and characterize a proline iminopeptidase enzyme in order to investigate the role of the major component of the smear of bacterial surface-ripened cheeses. Methods and Results: Four chromatographic steps were used to purify the enzyme and a three-factor, ®ve-level Central Composite Design was used to study the interactive effects of cheese-related values of pH, NaCl and temperature. The proline iminopeptidase showed some biochemical properties different from the same enzyme puri®ed from lactic acid bacteria and other smear bacteria. It tolerated NaCl concentrations up to 7á5% and was sensitive to low values of pH especially when they were combined with low temperature. Conclusions: The proline iminopeptidase of C. variabilis NCDO 2101 may have a role in proteolysis during ripening of smear surface-ripened cheeses. Signi®cance and Impact of the Study: The ®ndings of this work contribute to the knowledge of the enzymology of smear bacteria in order to improve the ripening of bacterial surface-ripened cheeses.

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Purification and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458

Cited by 3 publications

References 15 publications

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

Purification and characterization of an extracellular proline iminopeptidase from Corynebacterium variabilis NCDO 2101

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