Isolation characterization and immobilization of and#914; galactosidase produced by bacterial isolates of dairy industry

Abstract

newline ABSTRACT newlineand#946; galactosidase has been used in the dairy industry for the hydrolysis of lactose. The aim of the study was to isolate and#946; galactosidase producing bacterial isolates from raw milk and curd samples collected from some dairies of Punjab and Himachal Pradesh, India. Bacterial isolate designated as MH2 was selected on the basis of qualitative and quantitative screening analysis for and#946; galactosidase activity. The isolation was performed by plating on nutrient agar medium containing chromogenic substance X gal and ONPG as substrate to determine the and#946; galactosidase activity of bacterial isolate. The bacterial isolate MH2 was Gram s negative and colony morphology of MH2 was creamish white on nutrient agar medium and were circular in form. This bacterial isolate was positive for Catalase test, MR test, Motility test and negative for VP test, Simmon s citrate test, Gelatin hydrolysis and Urease test. On the basis of morphological and biochemical characterization by the criteria of Bergey s Manual of Systematic Bacteriology, the isolate MH2 was tentatively identified as Escherichia coli. The isolate MH2 was confirmed as Escherichia coli. On the basis of 16S rDNA sequencing and showing 99 % similarity with other Escherichia coli strains and therefore designated as Escherichia coli MH2. A 1168 bp of 16S rDNA nucleotide sequence of MH2 isolate was submitted to NCBI under the accession number KX443778. To know the phylogeny of the isolated strain Escherichia coli MH2, a phylogenetic tree was constructed by Molecular Evolutionary Genetic Analysis 7 (MEGA 7.0) using the neighbour joining method. From the phylogenetic analysis it was inferred that the strain MH2 belongs to Escherichia coli. The present study is the first report on the isolation of Escherichia coli from the raw milk as a source for production of and#946; galactosidase. The temperature, pH, substrate concentration and time required for optimum production of and#946; galactosidase enzyme by Escherichia coli MH2 was found to be at 24 h, 37 °C, pH 7.0 and 1 % of lactose, respectively. Xylose was found to be the best carbon source. On the other hand, yeast extract was found to be ideal nitrogen source for and#946; galactosidase production. The and#946; galactosidase activity was inhibited at 10 mM concentration of Fe2+, Mg2+ and Zn2+ respectively in MH2 isolate. On the other hand and#946; galactosidase activity was enhanced in the presence of Ca2+, Cu2+ and Na+. Overall, FeSO4, MgCl2 and ZnSO4 showed more enzyme inhibition as compared to CaCl2, CuSO4 and NaCl metal salts at 10 mM concentration. newlineviii newlineThe and#946; galactosidase of free and immobilized state was compared as a function of thermal stability, pH, incubation time and storage stability. Immobilized state of the enzyme over free state of the enzyme showed increase in optimal temperature from 37 °C to 40 °C for maximum and#946; galactosidase activity of Escherichia coli MH2. For both free and immobilized state of and#946; galactosidase, optimal pH was observed at pH 7.0 and storage stability of free and immobilized enzyme for a period of 30 days at 4 °C was studied. Free enzyme lost its activity after storage period of 15 days whereas enzyme activity of immobilized and#946; galactosidase remained stable upto 25 days. newlineIn order to study the evolution and adaptation at molecular basis and#946; galactosidase was studied by in silico approach to gain better understanding of working of and#946; galactosidase enzyme. Docking study provides molecular insights such as their mode of ligand binding with target. Fifteen and#946; galactosidase proteins of Escherichia coli were analyzed and multiple sequence alignment of and#946; galactosidase showed large number of amino acid residues to be conserved. Hydropathy plot showed that and#946; galactosidase of Escherichia coli is hydrophilic in nature. Docking studies revealed that maximum interaction was found with lactose in Escherichia coli k-12 and Escherichia coli 53638 with total interaction enery (Etotal) of (-240.35) and (-257.69) respectively. Phylogenetic analysis showed divergence among selected and#946; galactosidase of Escherichia coli strains and was found highly conserved. These results can lead to understanding of the evolution and adaptation of and#946; galactosidase enzyme at molecular basis which can further be used in wetlab for further experimentation. newlineFrom the present study it is concluded that and#946; galactosidase present in Escherichia coli MH2 could be useful for hydrolysis of lactose present in milk and its products and can be efficiently used in the dairy industry as well as for the production of galactooligosaccharides.