Isolation and Characterization of Mannanolytic Thermophilic Bacteria From Palm Oil Shell and Their Mannanase Enzyme Production Properties

Abstract

A mannanolytic thermophilic bacterium (L-07) was isolated from palm oil shell after 2 days of enrichment in liquid medium supplemented with 1% palm kernel meal as mannan source. Sequence analysis of 16S-rRNA indicated that L-07 was similar (98%) to Geobacillus stearothermophilus, a species of thermophilic aerobic bacteria. We found that G. stearothermophilus L-07 produced extracellular β-1,4-mannanases, but no β-manosidase and α-galactosidase activities. The growth of L-07 reached its maximum (3.0 x 10 6 cell/ml) at 12-20 hours, while the highest β-mannanase activity (0.52 U/ml) was observed in culture medium after 36 hours of cultivation at 60 o C. The medium containing locust bean gum was the best for producing extracellular β-1,4-mannanases compared with kolang kaling, konjak, and palm kernel meal. SDS-PAGE and zymogram analysis demonstrated that crude mannanase complex of L-07 from locust bean gum containing medium comprised three active bands with molecular weight of 85, 73 and 50 kDa.

Halotolerant Bacillus sp. for Mannan Degradation Isolated from Mangrove Ecosystem at Hanura Beach Lampung

“…Observations were made included mannanolytic index and bacterial staining. Selected colony with highest clear zones was tested using enzymatic assay with DNS method to determine the length of incubation time for enzyme production 15 . salt tolerance test Mannanolytic isolates were further tested for their resistance to salt content.…”

“…Bacteria were grown on SWC agar with the addition of salt as much as 0%, 3%, and 6% NaCl. The size of the colony was observed after an incubation period of 24 hours at room temperature 15 . ph tolerance test Mannanolytic isolates were tested for their resistance to media pH stress.…”

“…Bacteria were grown on SWC agar media with pH 4, 7, and 10 whereas 1 N HCl and 1 N NaOH were utilized respectively. After an incubation period of 24 hours at room temperature, growth and size of the colonies were observed and recorded 15 .…”

“…The starter was incubated at room temperature for 48 hours using an orbital shaker. Furthermore, the starter was inoculated as much as 5 mL into 45 mL of liquid SWC media with the addition of 0.5% locust bean gum substrate and incubated in the shaker orbital (120 rpm) 15 .…”

“…Enzyme activity was determined using 3.5-dinitrosalicylic acid (DNS) method and the absorbance was read at a wavelength of 540 nm. One unit (IU) of -mannanase activity was defined as the amount of protein producing 1 μmol/L of reducing sugar per minute (e.g., mannose) under standard conditions 15 .…”