A simple, efficient and rapid screening technique for differentiating nitrile hydratase and nitrilase producing bacteria

Abstract

HighlightsA rapid dye based plate screening method for nitrile hydrolyzing enzymes.Method identifies the end products of nitrile hydrolyzing enzymes.The method differentiates between nitrile hydratase and nitrilase producing bacteria.A potential NHase producing bacterial strain was identified as Rhodococcus rhodochrous.

Response surface methodology: An effective optimization strategy for enhanced production of nitrile hydratase (NHase) by Rhodococcus rhodochrous (RS-6)

“…The strain Rhodococcus rhodochrous (RS-6) used for present investigation was isolated in our laboratory from soil [ 22 ]. The bacterial strain was streaked onto nutrient agar (NA) slants and sub-cultured at regular intervals on the same medium and stored under cold conditions (4 °C).…”

“…The cell pellets were transferred into fresh tubes and washed two times with K 2 PO 4 buffer 0.1 M and pH 7.2. The pellets were again suspended in the same buffer and stored under cold conditions until future use [ 22 ].…”

“…The M2 culture medium used for optimization was used for optimization studies. The pre culture was prepared with 50 ml of M2 medium, a loop full of R. rhodochrous (RS-6) culture was inoculated and incubated in temperature controlled shaking conditions (160 rpm) at 30 °C for 24 h. To 50 ml of production medium, CoCl 2 (0.01 g/l) and 1 % acrylonitrile as substrate (inducer) was supplemented and the pre culture (2 ml) was inoculated and incubated in temperature controlled shaking conditions (160 rpm) at 30 °C for 36 h. The cell pellets were obtained by the above mentioned methods [ 22 ].…”

“…The activity of NHase was estimated by spectrophotometric method [ 22 ]. The nitrile hydratase activity one unit is defined as the quantity of the enzyme required to produce one μ mole of acrylamide from acrylonitrile per min under standard assay condition.…”

“…This method also analyzes the data obtained from the experiments and predicts their relationship [ 21 ]. Previously, we have reported that a strain of Rhodococcus rhodochrous (RS-6) isolated by us produces appreciable amount of NHase [ 22 ]. The present study was taken up for optimizing the cultural conditions and nutritional factors for enhancing NHase production from Rhodococcus rhodochrous (RS-6) using RSM with a central composite design.…”