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Bacteria isolated from a limestone cave and its ability to produce industrially important enzymes


Journal of Bacteriology & Mycology: Open Access
Muhammad Afzal,1,2,3 Ihsan-Ud-din,4 Muhammad Rafiq,2 Sahib Zada,2 Naveed Hussain,3 Muhammad Irfan,2 Niaz Muhammad,5 Aamer Ali Shah,2 Fariha Hasan2

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Abstract

Caves have extreme environment with diverse microbial flora. In the present study a bacterial isolates, KC5- MRL, AF-3, AF-6 and AF-23 isolated from Kashmir Smast(cave), a limestone cave in PirsaiMardan,, KPK, Pakistan, was found to produce three industrially important enzymes; lipase, protease and amylase. The bacterial isolates were identified as Lysinibacillussphaericus KC5-MRL, Bacillussubtilis AF-3, Bacillus subtilis AF-6 and Bacillus subtilis AF-23(Accession No. KF010827, MH027600, MH027601 and MH027602), respectively. Optimum pH for the growth of Lysinibacillussphaericus KC5- MRL, was around 7 and grew best at 35ºC. The optimum activities of lipase and amylase was observed at 30°C after 24 hr of incubation and pH 5 (42.23 U/ml) and (15U/ml) respectively. Maximum lypolytic activity (181.93 U/ml)was observed when 8% inoculum was used. Proteolytic activity of L. sphaericus KC5-MRL was found to be 59 U/ml, after 48 hr at 30°C.Highest stability of lipase, amylase and protease was observed at pH 10, 7 and 8 respectively. L. sphaericus KC5-MRL lipase, protease and amylase were stable at 35ºC. Triton X-100 and sodium dodecyl sulfate (SDS) stimulated the lipase and protease activities, whereas, Triton X-100 and T-80 stimulatedamylolyticactivity. Lipase activity was stimulated by Mg++, NH4+ and Ca++. Amylase activity was stimulated by Na+ and Ca++.Protease activity was stimulated by Na+ and Mg+. Chloroform, formaldehyde, methanol and benzene stimulated amylase activity. Nitrobenzene, methanol, benzene, and acetone stimulated protease activity. Ethylenediaminetetraacetic acid (EDTA) showed stimulatory effect on lipase. EDTA, Trisodium citrate TSC, mercaptoethanol, Phenyl acetaldehyde (PAA) and PMSF reduced amylase activity. All the modulators reduced protease activity except TSC and PMSF which stimulated its activity. The study concludes that these enzymes can be used for different purposes in various industries such as food and detergent.

Keywords

KC5- MRL, Kashmir smast, enzymes, limestone, Pakistan

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