• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.207-214
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• 2012

Laccase- and peroxidase-free tyrosinase has commercial importance in the production of L-3, 4-dihydroxyphenylalanine (L-DOPA), which is mainly used in the treatment of Parkinson's disease. In the present study, isolation of an actinomycetes microbial strain capable of producing only tyrosinase is reported. Among all soil isolates, three individual colonies revealed black color around the colony in the presence of tyrosine. Further screening for laccase and peroxidase activities using syringaldazine denoted that one of the isolates, designated as RSP-T1, is laccase and peroxidase negative and produces only tyrosinase. The microbe was authenticated as Streptomyces antibioticus based on 16S ribotyping. Effective growth of this isolate was noticed with the use of medium (pH 5.5) containing casein acid hydrolysate (10.0 g/l), $K_2HPO_4$ (5.0 g/l), $MgSO_4$ (0.25 g/l), L-tyrosine (1.0 g/l), and agar (15 g/l). The scanning electron micrograph depicted that the microbe is highly branched and filamentous in nature. The enzyme production was positively regulated in the presence of copper sulfate. The impact of different fermentation parameters on tyrosinase production depicted that the maximized enzyme titer values were observed when this isolate was grown at 6.5 pH and at $30^{\circ}C$ temperature under agitated conditions (220 rpm). Among all the studied physiological parameters, agitation played a significant role on tyrosinase production. Upon optimization of the parameters, the yield of tyrosinase was improved more than 100% compared with the initial yield.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).