Biochemical characterization of a new thermostable lipase from Bacillus pumilus strain

Abstract

Objective: Research and characterization of new thermostable lipases from bacterial strains isolated from tannery waters in the old medina of Fez. Methods: Gene which encodes the 16S ribosomal RNA for a bacterial species was amplified via PCR and sequenced (Bacillus pumilus HF544325). The extracellular lipase from B. pumilus is purified by gel filtration (Sephacryl S-200) and cation exchange chromatography (Mono S sepharose cation exchanger). The N-terminal sequences of purified Bacillus pumilus lipase were determined by automated Edman's degradation, using an Applied Biosystems 470 A protein sequencer equipped with PTH 120A analyser. The activity of lipase was examined within the pH range of 6.0-10.0 and the effect of pH on lipase stability was determined by incubating the lipase fraction in various buffer solutions ranging from 3.0 to 10.0 for 24 h at room temperature. Results:The results showed that Bacillus pumilus is a strain that produce non-inducible lipase. This enzyme has a molecular weight of 27 kDa and presents a maximal activity at pH 8 and 45°C. The 18 N-terminal amino acid residues showed a high degree of homology with other Bacillus lipase sequences. After treatment in 100°C for 5 min, the thermostable enzyme maintains 60% of its activity, which is greater than that those founded in previous works. The enzyme retained 100% of its activity after 30 min incubation at 70°C. Conclusion: This newly isolated lipase is thermostable and it has a significant difference which was observed when the biochemical properties of the Bacillus pumilus lipase were compared to others microbial lipases. The Bacillus pumilus lipase can be considered as a good candidature for industrial and biotechnological applications. Key Words: Bacillus pumilus, lipase, purification, thermostable Conflict of Interest: The authors have no conflict of interest. ÖZETAmaç: Çalışmanın amacı Fez şehrinde bulunan tabakhane sularından izole edilen bakteri suşla-rından, sıcaklığa dayanıklı yeni bir lipaz enzimini saflaştırarak karakterize etmektir. Metod: Bakteri türleri için 16S ribosomal RNA'yı kodlayan gen PCR ile çoğaltılarak sekanslanmıştır (Bacillus pumilus HF544325). Ekstraselüler lipaz jel filtrasyonu (Sefakril S-200) ve katyon değiştirici kromatografi kullanılarak (Mono S Sefaroz katyon değiştirici) B. pumilus'den saflaştırılmıştır. Saflaştırılan Bacillus pumilus lipazının N-terminal dizisi Edman degredasyon yöntemi (Applied Biosystems 470 A protein sekanslama cihazı ile PTH120A analizör) ile saptanmıştır. Lipaz aktivitesi 6.0-10.0 pH aralığında incelenmiş, pH'nın lipaz enziminin dayanık-lılığına olan etkisi lipaz fraksiyonunu pH'sı 3.0 ile 10.0 arasında değişen çeşitli tampon çözel-tilerinde 24 saat oda sıcaklığında inkübe ederek araştırılmıştır. Bulgular: Sonuçlar Bacillus pumilus suşunun indüklenmeyen lipaz enzimini ürettiğini gös-termektedir. Lipaz enzimi 27 kDa molekül ağırlığına sahip olup, en yüksek aktiviteyi pH 8 ve 45°C'de göstermektedir. Amino ucundaki 18 amino asit diğer Bacillus lipaz dizileri ile yüksek oranda ...

Purification of Bacillus licheniformis Lipase and its Application as an Additive in Detergent for Destaining

“…The Sephacryl S‐200 column chromatography was very effective for lipase purification due to the increase of the fold purification to 97.43. In accordance with our results, lipase from B. pumilus purified by four purification steps (ammonium sulfate precipitation, heat treatment, Sephacryl S‐200, and Mono‐S chromatography) reached 210‐fold with enzyme yield of 36% (Faouzi et al, 2015). Our results proved that the overall yield of 22.07% was higher than lipases extracted from different Bacillus strains, such as B. licheniformis SCD11501 (8.4%) (Sharma and Kanwar, 2017) and B. subtilis NS 8 (16%) (Olusesan et al, 2011), but approximately similar to B. licheniformis MTCC‐10498 (24%) (Sharma and Kanwar, 2012).…”

“…Generally, bacterial lipases require an alkaline or neutral pH for their metabolic processes (Bharathi and Rajalakshmi, 2019). Similar results have been reported where Bacillus subtili s (Saraswat et al, 2017) and B. pumilus HF544325 (Faouzi et al, 2015) showed maximum production of lipase at pH 8.0. However, a high lipase productivity has been yielded by culture media with at pH 10.0 and 11.0 for B. subtilis 168 (Lesuisse and Schanck, 1993) and Bacillus flexus XJU‐1 (Niyonzima and More, 2015), respectively.…”

“…The great stability of LBL at elevated temperatures (50–70 °C) suggested that the enzyme is thermotolerant. In the same context, other studies reported purified lipases from B. thermoamylovorans BHK67 (retained 50% of the initial activity at 40–60 °C) (Sharma et al, 2018), B. pumilus (retained 60% of initial activity at 50–60 °C) (Faouzi et al, 2015), B. licheniformis M13 (retained 98% of initial activity at 50–60 °C) (Kaur et al, 2016), and B. amyloliquefaciens E1PA (retained 75% of initial activity at 30–45 °C) (Saengsanga et al, 2016). In fact, our findings marked clearly the potentiality of a thermostable LBL to be utilized in detergent industry.…”

Electro-fermentative production of thermotolerant lipases by electrogenic bacteria isolated from palm oil mill effluents

“…S1E29, was shown at 45oC (263.8 U/ ml), 50oC (263.8 U/ ml), 50 o C (473 U/ ml), and 55 o C (419.8 U/ ml), respectively ( Fig 6). The result obtained is similar to the report of Lima et al, (2004) who observed maximum activity of lipase produced by B. megaterium at 55 o C. Similarly, Faouzi et al, (2015) reported that lipase produced by B. pumilus exhibited optimum activity at 45 o C. The previous study of Lima et al, (2012) recorded that the optimum lipase activity has been found between 30-60 o C. Devi and Chary, (2018) attributed the higher enzyme activity observed at elevated temperatures to the increased kinetics of the enzymatic reactions. The thermostable lipases are very promising for industrial applications such as detergent formulations and other bio-transformations, since processes performed at high temperatures increase the reaction rate.…”

Lipases: Sources, immobilization techniques, and applications

“…Bacterial lipases are remarkably numerous and frequently described as thermo-stable and substrate non-specific (Dhake et al, 2013;Vaquero et al, 2016). Recent studies highlight the thermal stability and industrial potential of bacterial lipases, such as those from Thermophilic Bacterium, Bacillus licheniformis lipases (Rashid et al, 2013), Bacillus pumilus lipase isolated from tannery waters (Laachari, El Bergadi, et al, 2015), and extracellular lipase of Bacillus licheniformis (Rashid et al, 2013). For instance, the lipase Serratia marcescens N3 is used to hydrolyze several types of edible oils, and it is most active when applied to Gingily oil (Zaki & Saeed, 2012).…”