Method for the preparation of k-carrageenase

Abstract

Halophilic marine gram negative bacteria, isolated from decayed algae, hydrolyzed κ-carrageenan. To maximize κ-carrageenase production in the cost effective manner, a novel medium was defined having minimum components and their optimum concentration by statistical optimization method. Hence there evolved a novel medium composition for enhanced κ-carrageenase production by a new halotolerant marine bacterium, Pseudomonas spp.

Description

Claims

1. A method for the preparation of κ-carrageenase, wherein the said method comprising the steps of: (i) growing the halotolerant marine bacterium, Pseudomonas sp. with accession number MTCC 5261 in a liquid medium comprises carrageenan, yeast extract, sodium chloride, K2HPO4 and KH2PO4 in the ratio ranging from 0.1:1.0:50:1:0.5 to 3.0:1.0:15.0:0.5:0.05 respectively for 16-72 hours at 15-50° C. to obtain culture;(ii) centrifuging the culture obtained from step (i) at 5000-8000 rpm for 20-40 min to obtain cell free extract as supernatant;(iii) purifying the crude enzyme from supernatant obtained from step (ii) by treating it with ammonium sulphate at a temperature in the range of 3-15° C. and aging the mixture for a period in the range of 12-36 hours followed by centrifugation at 5000-8000 rpm at 3-15° C. to get the pellet containing κ-carrageenase;(iv) suspending the pellets obtained from step (iii) in Tris-HCl buffer solution; and(v) dialyzing the suspension obtained in step (iv) for removing the adhered ammonium sulphate followed by gel filtration to get desired purified κ-carrageenase.

2. A method as claimed in claim 1 wherein, the said bacterium, Pseudomonas sp. have the following characteristics: a) halophilic;b) gram negative, exhibits gram variability;c) motile aerobic rods;d) it degrades algal polysaccharide i.e. κ-carrageenan.

3. A method as claimed in claim 1, wherein the specific activity of said κ-carrageenase is in the range of 10-200 galactose units/mg protein.

4. A method as claimed in claims 1, wherein the said κ-carrageenase have the molecular weight 128 KDa.

5. A method for the preparation of κ-carrageenase substantially as herein described with reference to the examples and drawings accompanying this specification.