Purification and Characterization of levansucrase from newly isolated strain of Zymomonas mobilis

Abstract

In all stages of biochemical and metabolism reactions, the enzymes play major role as biocatalysts. In industrial utilization, microbial enzymes are predominant as compared with other natural and synthetic enzymes. Isolation, production, characterization as well as applications of several microbial enzymes have continuously progressed in bioindustry. The current study covers the hyper production and purification of levansucrase from Zymomonas mobilis KIBGE-IB14. This bacterial isolate could be a plausible candidate for different biotechnological and industrial processes. Preliminary step presented the screening of bacterial isolate. Production of maximum levansucrase resulted in the selection of Z. mobilis KIBGE-IB14 among different microbial species. The manipulation of different fermentation parameters enhanced the yield of levansucrase and higher titers of enzyme was achieved by using sucrose (15%) as a substrate with pH-6.5 at 30°C for 24 hours. In case of chemical parameters, yeast extract (1.0%), peptone (0.2%), K2HPO4 (1.5%) and CaCl2.2H2O (0.01%) were found to be the most suitable macro and micronutrients.

Different precipitating agents including ammonium sulphate, ethanol and PEG 4000 were used for the partial purification of levansucrase. Ammonium sulphate was selected as a suitable precipitating agent among all precipitants. Gradient precipitation by ammonium sulphate (50%) resulted in 1.6 times increase in purification of levansucrase. After desalting, the final purifcation of levansucrase was achieved using Sepharose CL-6B chromatographic system which purified the enzyme upto 12.13 fold with 1.77% yield. SDS-PAGE electrophoresis and zymography revealed that the apparent molecular weight of purified levansucrase from Z. mobilis KIBGE-IB14 was approximately 130.0 kDa.

The catalytic performance of levansucrase during kinetic analysis was observed in

100.0 mM sodium phosphate buffer (pH-6.0) after 5.0 minutes of incubation with a Vmax and Km values of 21381 U mg -1 and 0.02308 moles L -1 , respectively. Levansucrase quite stable at 25°C and 35 °C as the enzyme retained its activity approximately 83% and 92% for 120 minutes, respectively. Storage stability of levansucrase suggested that the enzyme was stable at -80°C up to 180 days with the residual activity of 94%. Some of the metal ions including K + , Na + , Cs + , Ba +2 , Ca +2 , Cu +2 , Mg +2 and Mn +2 accelerated the activity of levansucrase at 1.0 mM concentration whereas, other metal ions including Co +2 , Hg +2 , Fe +3 and Al +3 showed an inhibitory effect on enzyme activity. In the case of organic solvents, isopropanol (1.0 mM) acted as an activator while, at 10.0 mM concentration of DMSO, formaldehyde and chloroform were found to be inhibitors. Non-ionic surfactants (Triton X-100, Tween-20 and Tween-80) did not have any significant effect on levansucrase activity whereas, anionic detergent (SDS) exhibited a slight inhibition. Amino acid analysis of levansucrase from KIBGE-IB14 revealed that this enzyme is a combination of non-polar (Proline, Alanine and Valine), polar (Glycine and Tryptophan) and basic amino acids (Lysine and Arginine).

To summarize the whole research study, it makes evident that an improved kinetic analysis and successful purification system for levansucrase has been developed. The present investigation provides amino acid composition of levansucrase which could be used as a preliminary data for its molecular characterization. Thus, it is concluded that the titer and performance of levansucrase biosynthesized by Z. mobilis KIBGE-IB14 has been enhanced and it could be used for a wider range of applications in many industrial bioprocesses.