Taxonomy of lipase producing moderately halophilic bacteria
Authors:   , Daroonpunt Rungsima 
Published: November 2023

One hundred and thirty eight of lipase-producing halophilic bacteria from fish sauce, fermented fish, shrimp paste and Poo-chem collected from the factories and the markets were isolated. Seventy isolates showed lipolytic activity on lipolytic agar using Tween (20, 40, 60 or 80) as substrates. Thirty-six isolates that showed more than 1 unit/ml of lipase activity in liquid medium were collected for identification. On the basis of phenotypic characteristics and 16S rRNA sequences, thirty strains were identified as Virgibacillus dokdonensis (3 isolates), Virgibacillus alimentarius (1 isolate), Virgibacillus halodenitrificans (2 isolates), Lentibacillus juripiscarius (1 isolate), Oceanobacillus  iheyensis (1 isolate), Alkalibacillus almallahensis (1 isolate), Halobacillus trueperi (1 isolate), Bacillus amyloliquefaciens subsp. plantarum (1 isolate), Bacillus altitudinis (1 isolate), Bacillus seohaeanensis (1 isolate), Bacillus zhangzhouensis (1 isolate), Corynebacterium falsenii (2 isolates), Corynebacterium variabile (2 isolates), Brevibacterium sediminis (2 isolate), Proteus penneri (1 isolate), Staphylococcus saprophyticus subsp. bovis (2 isolates), Staphylococcus saprophyticus subsp. saprophyticus (1 isolate), Staphylococcus nepalensis (1 isolate), Salinicoccus salsiraiae (1 isolate), Salinicoccus siamensis (3 isolates), Vibrio alginolyticus (4 isolates), Shewanella indica (1 isolate). Bacillus strain NR1-3-2 isolated from fish sauce and Virgibacillus strain KN3-8-4 isolated from shrimp paste (ka-pi) were novel species based on polyphasic taxonomy. Strain NR1-3-2 contained anteiso-C15:0, iso-C15:0 and anteiso-C17:0 as major cellular fatty acids and had diphosphatidyl glycerol (DPG), phosphatidyl glycerol (PG) and one glycolipid as polar lipids. DNA G+C content was 44.2 mol%. The 16S rDNA sequence analyses indicated that strain NR1-3-2 highest similarity with Bacillus iranensis DSM 23995T (97.4%). Strain NR1-3-2T exhibited low DNA -DNA relatedness (39.8%) with Bacillus iranensis DSM 23995Tand was proposed as Bacillus piscicola sp. nov. Strain KN3-8-4 contained anteiso-C15:0, anteiso-C17:0 and iso-C15:0 as major cellular fatty acids and had PG, DPG, two unknown phospholipids and one glycolipid as polar lipids. The DNA G+C content was 43.58 mol%.The 16S rDNA sequence analyses indicated that strain KN3-8-4 closely related to Virgibacillus olivae JCM 30551T (97.85%). This strain showed low DNA -DNA relatedness with Virgibacillus olivae JCM 30551T (28.8%) and was proposed as Virgibacillus kapii sp. nov.

Strain KN3-8-4 selected for lipase purification produced the maximum lipase  at stationary phase and could be achieved when casamino acids was replaced by 0.5% palm oil (w/v) in a modified JCM no. 377 medium with 1% NaCl (w/v), pH 8.5 and incubated at 40°C for 36 h. The KN3-8-4 lipase was purified by cold acetone precipitation, gel filtration and anion exchange chromatography with 18.66-fold purification. The purified lipase from KN3-8-4 was monomeric protein with the molecular mass of about 19.5 kDa by gel filtration and 19 kDa by SDS-PAGE. The enzyme had a maximal activity in the presence of 7% w/v NaCl, pH 8.0 at 40°C. The enzyme exhibited a variable specificity activity towards various p-nitrophenyl esters especially p-nitrophenyl butyrate (C4).