Production and purification of the Tyrosinase enzyme from Soil bacteria

Author Name: *Sapna S. Ingle, Chandrahas N. Khobragade
Author Email: Sapna_ingle@rediffmail.com

Abstract

Tyrosinases are a group of bifunctional enzymes that are involved in the hydroxylation of mono phenols to o-diphenols. These enzymes have their potential applications in several domains like Biosensors, biocatalysis in several pathways, and production of L-DOPA from tyrosine. Detection and removal of phenolic compounds from wastewater also involves the use of tyrosinases. In view of the extensive roles played by these enzymes the current work aimed to isolate and identify the bacteria capable of producing this enzyme in the invitro study followed by the enzyme extraction and purification. The work dealt with the maximum production of the enzyme in two different media by bacterial isolates so as to detect the best media for the maximum yield. Once the production was done the crude enzyme has been extracted and is subjected to purification based on different aspects and methodologies. Various purification protocols like Ammonium sulphate precipitation, Dialysis, Ion exchange chromatography, gel filtration etc have been employed for the maximum purification. The enzyme assay and concentration estimations were also performed and Medium A and C7-Pseudomonas geniculata resulted in the maximum yield and activity of the enzyme.

Keywords

Tyrosinases, L-DOPA, Optimization, Guaze Agar, Production Media, Salt Precipitation, Gel Filtration, Ion Exchange Chromatography.

Introduction

Tyrosinases are the enzymes that are majorly involved in the formation of the naturally occurring pigment Melanin. Tyrosinases being involved in the melanin formation have inherent properties like absorption of UV radiation, metals, sound and also have anti-oxidant and semi-conductor properties [1] thus are used in the production of complex biopolymers. Cultures of streptomyces have been used in the commercial scale production of Tyrosinases [2]. The best production yield reported till date for tyrosinase was about 1g/l, which was produced by cultivating the Trichoderma reesei a filamentous fungus for 6days[3]. Immobilized tyrosinase enzymes are used in the production of L DOPA [4]. Several organisms have been used in the production of these enzymes at the commercial scale like Mushrooms, Bacteria, and Fungus etc. a study was undertaken on the production of tyrosinases from Verrucomicrobium spinosum. These organisms were later expressed in was expressed in Escherichia coli and the enzyme production was studied in both batch and fed-batch cultivations [5]. One of the studies was carried out on the biochemical characterizations of human tyrosinase and to provide an unlimited source of the enzyme for further study, an expression plasmid, pHis-Tyrosinase, which contains the entire coding sequence except the signal sequence of a human tyrosinase, was constructed and expressed in Escherichia coli [6]. Some strains of Streptomyces isolated from soil were also used in the invitro production of the tyrosinase enzyme extracellularly The enzyme was purified using eries of column chromatographies on Duolite A-2 and CM-cellulose and gel filtration on Sephadex G-100 [7] In view of the extensive applicability and vital role of the tyrosinase enzymes the current research was focused onto the production of maximum yield from the bacteria followed by the purification and enzyme estimations at various stages of purification.

Conclusion

The current study involved the use of bacteria isolated from the soil near slaughter house in the production of tyrosinase enzymes. From the master plate three cultures were selected for the study and labeled as C4, C7 and C10 and identified as Rhodococcus ruber strain C4, C7-Pseudomonas geniculata strain C7 and C10 as Pseudomonas geniculata strain C10. The cultures were also shown positive test for the tyrosinase enzyme production like melanin formation and pigmentation on gause media. The cultures were further used for the production of enzyme in two different medias namely A and B that differ in their composition. The enzyme assay study proved that the Media A can be the best source for maximum enzyme activity when compared to media B. In which the media A and the Pseudomonas geniculata strain C7 was the best combination for the maximum enzyme activity. The purification of the enzyme was performed using various methodologies and the enzyme produced was evaluated by Lawry’s method at each step of purification. The work concludes that the Pseudomonas geniculata with the media A is the best combination for the maximum production of the enzyme. The work can further be extended for the optimization of the culture conditions for the maximum production of the enzyme.

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