Jani and Patel

The keratinolytic microorganisms and technologies

48 h. This actively growing culture was used as inoculum

developed for feather degradation are not only to remove

for the protease production (O.D. 0.75 at 660 nm).

the waste feathers efficiently from the nature but also for

making the by-products of the process as valuable protein

3. Enzyme production.

supplement. The protein rich, concentrated feather meal

The Saccharomonospora viridis SJ-21 JX 262282 , was

can also be used for organic farming as semi-slow release,

inoculated in the optimized medium , having composition:

nitrogen fertilizer (Kaul, S. and Sumbali, G. 1999),

casein, 250mg%; peptone, 200mg%; K2HPO 20mg%,

4

Friedrich, A.B., and Antranikian, G. 1996). In this study

KH PO 20mg%, MgSO ,10mg%, CaCl 10mg% , and

2

4

4

2

we emphasized on keratinolytic activity of thermophilic

Glycerol 0.5ml in 100 ml of Distilled water,( pH 9.5.)

actinomycetes Saccharomonospora viridis SJ-21 and its

(28); under shaking conditions (100 rpm) , for 96 h at

protease.

55°C. The culture broth was centrifuged at 10,000 × g

for 20 min at 4ºC and the culture supernatant was used as

Materials and Methods

a source of protease. Activity of enzyme was measured in

1. Isolation and Identification of S. viridis SJ-21

terms of unit (μg/ml/min). One unit of enzyme is defined

as the quantity of enzyme required to release 1μg of

Samples collected from hot water spring, soil and Areetha

tyrosine per minute, under the standard assay conditions

extract were serially diluted using sterile distilled water to

(Hagihara, B. (1958).

spread evenly on skim milk agar medium of pH 8.5 and

incubated at 55ºC for 3-4 days to allow the organisms

Protein content was measured by Lowry’s method with

to grow. The well isolated colonies showing zone of

bovine serum albumin (BSA) as a standard protein.

casein hydrolysis were marked and colony characters

4. Ammonium sulphate Precipitation and Dialysis of

and morphological characters were noted at an interval

enzyme:

of 24 h. Diameter of zone of clearance due to hydrolysis

of casein was recorded which provided a measure of

250 ml of the cell free filtrate was subjected for

their proteolytic activity. On the basis of zone of casein

precipitation by different concentrations of (20- 100%)

hydrolysis, potent isolates were selected for further

solid ammonium sulphate (Dixon, M. and Webb, E.

study.

G., 1964). The precipitated protein was obtained by

centrifugation at 10000 rpm for 15 minutes at 4ºC. Both

The isolate SJ-21 showing zone of casein hydrolysis on

enzyme activity and protein content were determined for

milk agar plates was preserved on Nutrient casein agar

each separate fraction (Khalil B.Q. and Gupt, R. (2003).

slants and sent to Gujarat State Biotechnology Mission

The concentrated enzyme was collected in glycine NaOH

(GSBTM, Gandhinagar) for 16S rRNA sequencing and

buffer and dialyzed overnight against the same buffer till

the BLAST match was used to confirm identification

the ammonium sulphate was completely removed.

of the isolate. The sequences obtained from GSBTM,

Gandhinagar were analyzed at NCBI server (www.ncbi.

The caseinolytic activity of this partially purified

nim.nih.gov) using BLAST tool and have been submitted

enzyme preparation was determined by Hagihara method

to Gene Bank. The phylogenetic tree of the isolate was

(Hagihara, B. 1958).

constructed with MEGA version 4.0 using the neighbor

joining method (Tamura, K. et al., 2007).

5. Characterization of the protease by determination of

molecular weight and type of enzyme

2. Micro-organisms and inoculum preparation

SDS-polyacrylamidegelelectrophoresis(SDS-PAGE)was

Fresh growth of S. viridis SJ-21 was obtained on milk

preformed according to method of Laemmli (Laemmli,

agar medium (pH 8.5) and transferred in to 25 ml broth

1970) as modified by Studier (1973). This method was

containing glycerol 0.5 ml, peptone 1 gm, yeast extract

used to determine the molecular weight of the partially

0.5 ml, NaCl 0.5 gm in 100 ml distilled water and pH 8.5

purified protease. Molecular weight of purified alkaline

and incubated at 50ºC on a rotary shaker at 100 rpm for

protease was measured using a series of protein with

standard molecular weight markers (Sigma, USA)

480