A novel thiol-dependent serine protease from Neocosmospora sp. N1

Abstract

Alkaline proteases have several industrial applications. In the present study, newly isolated Neocosmospora sp. N1 was screened as hyper producer of serine protease. A multimeric protease of the fungus was purified to homogeneity till 96.78 fold purification with 22.51% recovery. The homogeneity of purified enzyme was checked by native PAGE and its molecular weight was found to be 198.03 kDa by MALDI-TOF. On SDS-PAGE analysis, enzyme was found to be a hetero oligomer of 17.66 kDa and 20.89 kDa subunits. The purified enzyme showed maximum activity with casein as substrate at 60 °C and pH 8.5. The K_m and V_max values were found to be 0.015 mg/ml and 454.45 U/ml, respectively. The enzyme was completely inhibited by PMSF, while the activity was 40% enhanced using β-mercaptoethanol, suggesting that it is a thiol-dependent serine protease. The purified protease was active over an alkaline pH range from 7 to 12 and temperatures from 20 °C to 60 °C. The enzyme exhibited excellent stability, almost 100% towards organic solvents such as toluene, benzene and hexane, surfactants such as Triton X-100, Tween-20, Tween-80 and SDS, as well as commercial detergents. The significant properties of purified enzyme assure that it could be a potential candidate for commercial purposes.

Keywords: Biotechnology; Enzyme kinetics; Enzymology; MALDI-TOF; Microbial biotechnology; Microbiology; Serine protease; Superdex 200.

Fig. 1

(A) Isolate N1 grown in liquid medium and supernatant obtained was loaded on agarose plates containing casein (a), gelatin (b) and soybean (c), respectively. Plates were stained with 0.3% CBB R-250 to observe clear zone of protein hydrolysis and zone diameter was measured subsequently. (B) Morphological characteristics of isolate N1. (a) Growth of isolate N1 on Potato Dextrose Agar. (b) Scanning electron microscopic image of isolate showing fungal hyphae and (c) fascicle arrangement of macroconidia. (C) Phylogenetic tree constructed by the neighbor-joining method (MEGA X software) showing the position of Neocosmospora sp. N1 with the sum of branch length = 3.72569419. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

Fig. 2

Purification of alkaline protease from Neocosmospora sp. N1 (A) Elution profile of enzyme on DEAE cellulose anion exchange column, equilibrated with 25 mM Tris-HCl buffer, pH 8.5. Bound fractions were eluted with the same buffer in a linear salt gradient of NaCl (0–1M) at a flow-rate of 1.0 ml/min. All fractions were assayed for protease activity (⋄-blue fill) and monitored for protein content (□-red fill). (B) Chromatographic profile of purified protease on Sephadex G-200 gel filtration column. Fractions showing protease activity were pooled, concentrated and applied to Sephadex G-200 column, equilibrated with 25 mM Tris-HCl buffer. Elution was performed with the same buffer at a flow rate of 8 ml/h and protease activity (○) and protein content (○) was determined subsequently. (C) Purification profile of enzyme on Superdex 200 increase 30/100 GL column. Active enzyme was injected in AKTA purifier (10 × 300mm) equilibrated with 20 mM Tris-HCl buffer containing 1M NaCl. Protein was eluted with the same buffer at a flow rate of 0.75 ml/min and detected using a UV spectrometric detector at 280 nm. The peak obtained at elution volume of 12.8 ml contains protease activity.

Fig. 3

Electrophoretic profile of purified protease and molecular weight determination. (A) Native-PAGE analysis. Lane 1: standard protein marker; lane 2: enzyme after ammonium sulphate fractionation (0–90%); lane 3: protease fraction after ion exchange chromatography; lane 4: purified enzyme obtained after Sephadex G-200 chromatography; lane 5: zymogram of purified enzyme showing caseinolytic activity. (B) 12% SDS PAGE. M: standard marker; C: crude enzyme; D: purified enzyme run under denaturing conditions; N: purified enzyme run under non-denaturing conditions. (C) MALDI-TOF spectrum of purified enzyme. The mass spectrum shows a series of protonated molecular ions. The molecular mass of the enzyme was found to be 198030.850 Da.

Fig. 4

Multiple sequence alignment of the peptide sequences obtained from protease derived from Neocosmospora sp. N1 with those of subtilisin-like serine protease pepD from Aspergillus niger strain CBS 513.88 (A2QTZ2), subtilisin-like serine protease from Aspergillus niger ATCC 13496 (A0A370BNF1), protease from Aspergillus neoniger CBS 115656 (A0A318ZA13), protease from Aspergillus vadensis CBS 113365 (A0A319BAX9), protease from Aspergillus eucalypticola CBS 122712 (A0A317VKS4). The asterisk (*) sign indicates fully conserved amino acid residue; colon (:) indicates conservation between groups of strongly similar properties and period (.) represents conservation between groups of weakly similar properties. Peptidase S8 domain in the protein sequence is marked by grey color.

Fig. 5

(A) Effect of temperature on protease activity. The reaction mixture was incubated at respective temperatures and assayed by standard assay method. (B) Effect of temperature on protease stability. Enzyme was pre-incubated at respective temperatures viz.40 °C, 50 °C, 60 °C and 70 °C for 4 h and samples were assayed after 15 min interval. Relative activity was calculated against non-heated enzyme assayed at 60 °C (100%). (C) Effect of pH on protease activity. The enzyme activity was measured at a pH range of 6–12 with the interval of 0.5 using three buffer systems. (D) Effect of pH on enzyme stability. The enzyme was pre-incubated with buffers of different pH (5–12) for 24 h at 37 °C and relative activity was determined under standard assay conditions.

Fig. 6

Lineweaver-Burk plot for alkaline protease under varying substrate (casein) concentration (1–20 mg/ml).