Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India.
A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4-37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg(-1)). The maximum specific enzyme activity (398 U mg(-1)) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg(-1). The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10-60°C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures.
Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Sanatnagar, Srinagar 190 005, India.
Iram
I
Saba
Parvaiz H
PH
Qazi
Shabir A
SA
Rather
Refaz A
RA
Dar
Qurrat A
QA
Qadri
Nasier
N
Ahmad
Sarojini
S
Johri
Subash C
SC
Taneja
Sami
S
Shawl
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015373:Bacterial Typing Techniques; D002244:Carbon / Q000378:metabolism; D003080:Cold Temperature; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007194:India; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009584:Nitrogen / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D020588:Stenotrophomonas / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D002244:Carbon; D010450:Endopeptidases; C087822:alkaline protease; D009584:Nitrogen