Comparative studies of extracellular and#946; galactosidase enzyme from psychrophilic bacteria isolated from Kafnu and Sangla valley of Himachal Pradesh India

Abstract

newline Abstract newlineCold adapted and extracellular and#946;-galactosidase with high specific activity have potential application in food industry. In order to isolate psychrophilc/psychrotolerant bacteria from soil samples of Kafnu valley and Sangla valley of Himachal Pradesh, only 14 bacterial isolates were purified by enriching in 1% lactose medium at 4º C. Amongst them, only 4 bacterial isolates namely A1, A5-2, B1 and B8 showed growth at low temperature ranging between 4 to 25º C and did not grow above 30º C, hence classified as psychrotolerant. From these 14 bacterial isolates only A5-2 and B8 isolates showed the highest extracellular and#946;-galactosidase activity and were observed to be constitutive as well inducible in nature. The extracellular and#946;-galactosidase activity of A5-2 isolate was observed to be three folds higher than intracellular activity, whereas negligible intracellular activity was observed in B8 isolate. On the other hand, B8 isolate showed two folds higher extracellular and#946;-galactosidase activity in comparison to A5-2 isolate. On the basis of biochemical tests and 16s rDNA sequencing, both A5-2 and B8 isolates were classified as S. quinivorans A5-2 and S. quinivorans B8 and have been submitted in NCBI genebank having accession numbers KJ 176660 and KJ 176661 respectively. The optimal growth of S. quinivorans A5-2 was observed at 20º C and pH 7, whereas S. quinivorans B8 showed optimal growth at 25º C and pH 7. The and#946;-galactosidase activity of S. quinivorans A5-2 was induced 2 folds in nutrient medium supplemented with 1% lactose, which was calculated as 9000 U/mg protein after 90 h of incubation. Whereas, in S. quinivorans B8 the and#946;-galactosidase activity was induced 3 folds and calculated as 23000 U/mg after 120 h of incubation in nutrient medium supplemented with 1% lactose. The optimal temperature of the and#946;- galactosidase activity was observed 50º C for S. quinivorans A5-2 and 60º C for S. quinivorans B8. The optimal pH of and#946;-galactosidase activity was 7 for both isolates. The microbial growth was stimulated by glucose, galactose and sucrose; but only galactose enhanced the and#946;- galactosidase activity in both the isolates, whereas glucose and sucrose inhibited the enzyme activity in both the isolates. The and#946;- galactosidase activity was metal dependent, in which metal ions such as Fe2+, Ba2+ enhanced the activity and Hg2+, Zn2+ completely inhibited the activity in both isolates. Major milk metal ions Ca2+ and Na+ ions and sugars, glucose, galactose and lactose did not show any effect on the and#946;- galactosidase activity. A5-2 and B8 isolates showed growth in heavy metals like Ba2+ at 50 mM concentration. The highest substrate affinity of and#946;- galactosidase for ONPG was observed at 50and#730; C newlinex newlinefor A5-2 isolate with km value of 79 nM. The km for B8 isolate was 200 nM at optimal temperature of 60and#730; C. The rate of reaction determined the substrate enzyme saturation after 10 minutes in both the isolates. SDS-PAGE and Native-PAGE determined the molecular weight of and#946;-galactosidase as ~100 KDa for A5-2 isolate and ~70 KDa for B8 isolate.