Screening and Isolation of Bacillus sp. Producing Thermotolerant Protease from Raw Milk

Kannikar SANTONG; Warangkana CHUNGLOK; Monthon LERTCANAWANICHAKUL; Phuwadol BANGRAK

Authors

Kannikar SANTONG Utilization of Natural Products Research Unit, Walailak University, Nakhon Si Thammarat 80161
Warangkana CHUNGLOK School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat 80161
Monthon LERTCANAWANICHAKUL School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat 80161
Phuwadol BANGRAK School of Science, Walailak University, Nakhon Si Thammarat 80161

Keywords:

Bacillus sp., thermotolerant protease, raw milk

Abstract

Screening and isolation of Bacillus sp. were carried out from raw milk samples using Luria Bertani (LB) supplemented with 2 % skimmed milk as a selective media. Forty-one isolates with a clear zone surrounding a colony were primary selected and identified by using biochemical characteristics, Gram stain, and growth of bacteria at 50 °C. Ten out of 41 isolates showing a clear zone diameter of more than 10 mm were selected and evaluated for the presence of protease activity. BA26 and BA27 gave high levels of protease activity with 12 U/mg protein towards 1.5 % casein at 50 °C for 10 min. Based on the biochemical and physiological characteristics, BA26 and BA27 were classified as Brevibacillus non reactive. However, their 16S rRNA gene sequence showed 99 % identity to that of Bacillus subtilis. The enzymes were more specific to 1 % casein than 1 % gelatin. Moreover, the selected bacteria secreted extracellular protease upon incubation at 50 °C and the enzymes were stable even at high temperatures up to 100 °C and 121 °C. This confirmed that the enzyme protease produced by Bacillus sp. are thermotolerant proteases.

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