Fig 1.
The map of pUC18-T7 mini vector with E.Coli rrnB transcription termination.
Lac promoter (Blue color), Lac UV 5 Promoter (Blue color), Lac Operator (Blue color), Fusion Gene (purple color), rrnB T1 terminator (Color less), Ampicillin Resistant gene AmpR and pMB1 origin of replication.
Table 1.
The list of the primers used in the present study.
Fig 2.
(A) Agarose gel electrophoresis of protease and chitinase gene amplicon. Lane 1 corresponds to ladder, and Lane 2 and 4 indicate the presence of the protease gene (1.3 kb) and chitinase gene (1.2 kb), respectively; (B) Lane 1 represents DNA ladder, lane 2 to 5 represents overlapping PCR product of 2.9 kb; (C) The pUC18miniTn7FusionX Clone confirmation, Lane1- ladder, Lane 2- Unknown, Lane 3- pUC18miniTn7FusionX linearised for cloning, Lane 4- pUC18 MiniTn7 FusionX digested with BamHI and KpnI showing the insert release of 2.9 kb.
Fig 3.
(A and B) Activity assay of recombinant bacteria having different constructs. A = PRI—Protease, FP- Protease activity in the fusion construct. B = CHI-chitinase, FCH-chitinase activity in fusion construct.
Fig 4.
Native PAGE of fusion proteins expressed by E.coli.
Lane 1- Protein marker (44.3 KDa, 66.4 KDa, 97.2KDa, 116 KDa, and 200 KDa), Lane 2- Sample purified using Ni-NTA column without IPTG induction; Lane 3-Sample purified using Ni-NTA column with IPTG induction; Lane 4- Crude protein without insert. Proteins were resolved on the 12.5% PAGE with standard molecular weight markers. The gel was allowed to run in Tris-glycine buffer at 30 mA constant current for 2.5 h followed by staining with Coomassie Brilliant Blue R-250. The molecular weight of the separated proteins was determined by comparing them with molecular weight standards.
Table 2.
The activity of recombinant proteins expressed in E.coli.
Fig 5.
Activities of the purified proteins at pH 5–9 Values are the average of five replicates and significant at P<0.05 (A); Activities of the purified proteins at 20–50°C. Values are the average of five replicates and significant at P<0.05 (B).
Fig 6.
The effect of various metal ions (5–10 mM) on the activity of the protease and chitinase.
Values are the average of five replicates and significant at P<0.05 (A). The effect of various metal ions (5 mM and 10 mM) on the activity of chitinase. (B). Effect of denaturing agents and organic solvents at 1% and 5% on protease activity C). Effect of denaturing agents and organic solvents (1% and 5% of each) on chitinase activity (D).