Purification and characterization of thermoactive serratiopeptidase from Serratia marcescens AD-W2

Abstract

Serratiopeptidase is a proteolytic enzyme extensively used as an anti-inflammatory and analgesic drug. Present work reports a thermoactive serratiopeptidase from Serratia marcescens AD-W2, a soil isolate from the North-Western Himalayan region of India. The extracellular metalloprotease has been purified by a simple two-step procedure resulting in a specific activity of 20,492 Units/mg protein with 5.28-fold purification. The molecular mass of the metalloprotease, as determined by SDS-PAGE was ~ 51 kDa. The purified serratiopeptidase presented optimum activity at pH 9.0, temperature 50 °C and stability in wide pH and temperature range. Critical temperature of 50 °C confirmed the thermoactivity of the purified serratiopeptidase. The kinetic studies of the purified serratiopeptidase revealed V_max and K_m of 57,256 Units/mL and 1.57 mg/mL, respectively, for casein. The purified serratiopeptidase from S. marcescens AD-W2 was found to be 100% identical to serralysin from Serratia marcescens ATCC 21074/E-15. The catalytic domain comprising of Zn coordinated with three histidine residues (His192, His196, His202), along with glutamate (Glu193) and tyrosine (Tyr232) residues, further confirmed that the purified protein is identical to serralysin.

Keywords: Homology modelling; Metalloprotease; Serralysin; Serratia marcescens; Serratiopeptidase.

Fig. 1

a Morphology of pure isolate AD-W2 on Nutrient Agar plate. b Microscopic image of pure isolate AD-W2 after Gram staining. c Phylogenetic tree of AD-W2 with closely related 16S rRNA gene sequences of Type strains

Fig. 2

Time course profile of S. marcescens AD-W2 for serratiopeptidase production

Fig. 3

The protein elution profile during Ion exchange chromatography using MonoQ 5/50 GL column

Fig.4

a Casein Zymogram and b SDS-PAGE of serratiopeptidase from S. marcescens AD-W2

Fig. 5

Effect of various reaction conditions and stability of serratiopeptidase from S. marcescens AD-W2: a pH; b temperature (Arrhenius plot shown in inset); c inhibitors d substrate concentration; e pH stability; f temperature stability at 50 °C

Fig. 6

Multiple amino-acid sequence alignment of the serratiopeptidase from S. marcescens AD-W2, PRZN_SERME serralysin from S. marcescens (E-15) with 100% sequence identity, PRZN_SERMA Serralysin from S. marcescens with 98.6% sequence identity, 0A240AD01_SERFI Serralysin from S. ficaria with 92.6% sequence identity, D4E064_SEROD Serralysin from S. odorifera DSM 4582 with 82.8% sequence identity and E0SC28_DICD3 Secreted protease A from Dickeya dadantii with 64.8% sequence identity. The protein sequence from serratiopeptidase was aligned to see the similarities, differences, and conservation of various amino acid residues using multiple sequence alignment. The alignment was carried out by using Clustal W platform in Seaview software

Fig. 7

Cartoon view of the overall three-dimensional structure of Serratiopeptidase, a ribbon diagram showing the positions of the secondary structure elements was built using online SWISS-MODEL software (https://swissmodel.expasy.org/) and colored according to secondary-structure element. β-Strands are depicted as arrows, the zinc ion is shown by a grey ball, and calcium ions by black balls

Fig. 8

Ramachandran plot of the chimeric protein. The Ramachandran plot analysis (PROCHECK server); Laskowski et al. (1993), revealed that the modelled structure projected 91.0% of amino acid residues in most favoured regions of the Ramachandran plot, 8.5% are in additional allowed regions, 0.5% residues in generously allowed regions and 0.0% residues in disallowed regions

Fig. 9

Stereoview of the zinc-binding site in the Serratiopeptidase. The zinc ion is shown as a purple ball. The active site of serratiopeptidase from S. marcescens AD-W2 shows the zinc ion coordinated as Zn.1, His.192, His.196, His.202, Glu.193, and Tyr.232 is forming a distorted trigonal bipyramidal catalytic active center