ABSTRACT
Metallo-[beta]-lactamases [MBLs] are zinc ion dependent enzymes that are responsible for the emergence and spread of [beta]-lactam resistance among bacterial pathogens. There are uncharacterized putative MBLs in the environment and their emergence is major interference in the generation of universal MBL inhibitors so it is important to identify and characterize novel MBLs. In this study two novel MBLs from Luteimonas sp. J29 and Pseudoxanthomonas mexicana were identified using B3 MBLs as query in BLAST database search. 3D models of putative MBLs generated by SWISSMODEL server taking AIM-1 as a structural template were verified using web based structure assessment and validation programs. Multiple sequence alignment revealed that residues important for substrate binding were conserved and loop region residues [156-162 and 223-230] important for catalysis are variable in these novel MBLs. Homology models showed typical MBL [alpha]/[beta]/[beta]/[alpha] sandwich fold containing six [alpha] helices, twelve [beta] strands and metal interacting residues are conserved in similar way as with other B3 MBLs. We report promising putative B3 MBLs with some variations and substrate docking studies revealed that novel MBLs have attributes close to acquired B3 MBLs
ABSTRACT
Several strains of beta-hemolytic Streptococci produce streptokinase enzyme that can bind and activate human plasminogen to plasmin. Streptokinase degrades the fibrin lump by its explicit lysine joining site and so it is applied as a remedy in thrombolytic therapy. The purpose of the study was to subject wild strain of Streptococcus equisimilis to strain development technique, using random mutagenesis by UV irradiation for enhanced production of streptokinase. To evaluate the hyper production of streptokinase after mutagenesis of wild Streptococcus equisimilis by means of UV irradiation. Randomized study. 2012-2014. Enzyme Biotechnology Laboratory, Department of Biochemistry, University of Agriculture, Faisalabad-Pakistan. UV lamp [TUP 40w lamp which has about 90% of its radiation at 2540-2550 A[0]] was used for the mutation of Streptococcus equisimilis cells [1x 10[7] cells mL[-1]] for enhanced production of streptokinase. 10 mL fresh inoculum was transferred to sterile petri plates, which were exposed to UV light for 30, 60, 90, 120, 150, 180, 210, 240 and 270 minutes. The exposure was carried out at distance of 20cm from the centre of lamp. A dose producing 87% killing was selected as optimum dose, after preparing kill curve. The kill/ survival curve was prepared and time of exposure giving [210 minutes] 3 log kill was selected for mutation of the Streptococcus equisimilis for hyper production of streptokinase enzyme. Enzyme assay was performed for both wild and mutant strains. Dose of 210 minutes was selected as best dose which was followed by the selection using triton X-100. Finally the selected strain S. equisimilis EBL-UV-210 showed 480 U mL[-1] of streptokinase activity in quantitative blood clot liquefaction test, which is quite higher than wild strain [370 U mL[-1]]. This maximum yield of streptokinase was obtained after 24h, at CSL 4%, pH 7.5, 37[degree sign] C, KH[2]PO[4] 0.04%, K[2]HPO[4] 0.05%, MgSO[4]. 7H[2]O 0.04%, NaHCO[3] 0.15%, CaCO[3] 0.004%, CH[3]COONa. CH[3]CO 0.10%, FeSO[4]. 7H[2]O 0.04%, MnCl[2]. 4H[2]O 0.02%, glucose 2%, yeast extract 3% and 5% inoculum size in liquid state fermentation. Results showed that mutated strain gave enhanced streptokinase activity in comparison to the wild strain. Our current study focused on streptokinase production from this UV mutated streptococcus equisimilis species and purification of this enzyme by ammonium sulfate precipitation, Ion exchange and gel filtration chromatography. The activity of streptokinase was determined by using quantitative blood clot liquefaction method
ABSTRACT
To investigate the inhibitory effect of Captopril on level of glycation [in vivo]. [2] To study glycation inhibition in vivo. Study Case study. Period: Sep. 2006 to March. 2008. One year seven months. Department of Biochemistry University of Agriculture, Faisalabad. Different parameters like fluorescence, total proteins, TBA [thiobarbituric acid] method, periodate borohydride assay were used to check the effect of inhibitor on glycation. Thirty two combinations were made and all these combinations were placed at 37°C, at same time for five weeks. 3mL of blood sample was drawn after 1st, 3rd and 5th week of incubation to perform the experiments for glycation and glycation inhibition. Along with the same temperature [37°C], different combinations of glucose and inhibitor were used. Effective concentration of inhibitor helped to decrease the level of glycation. All concentrations of glucose [G, G and G] showed 1 2 3 glycation with protein. The inhibitor Captopril [all concentrations] showed variations in inhibition of glycation at one temperature [37°C with different parameters [Fluorescence, TBA and Periodate] but the most effective concentration of inhibitors at each condition is I [1mM] but I [10 3 1 mM] and I [5 mM] were also equally effective after I. Periodate borohydride Assay is more effective for glycation determination than 2 3 thiobarbituric acid assay. Captopril can be used as glycation inhibitor in future. As it enhances the activity of transketolase, it can produce 3DG compound which can block the AGEs. However, more experimentations should be done on animal or on large scale before its application in diabetic patients