Bacterial Lipase Research Articles

Pseudomonas nanhaiensis sp. nov., a lipase-producing bacterium isolated from deep-sea sediment of the South China Sea.

A bacterial lipase producing bacterium, designated SCS 2-3, was isolated from deep-sea sediment of the South China Sea. Phylogenetic analysis based on the 16S rRNA sequence revealed that strain SCS2-3 belonged to the genus Pseudomonas and had 98.56% similarity to P. xinjiangensis NRRL B-51270T as the closest relative strain. MLSA using four protein-coding genes (dnaK, gyrA, recA, and rpoB) showed strain SCS 2-3 to form a separate branch. ANI and in silico DDH values between strain SCS 2-3 and related type strains of Pseudomonas were less than 81.51% and 23.80%, respectively. Genome comparison showed that strain SCS 2-3 shared 1875 core gene families with other eight closely related type strains in Pseudomonas, and the number of strain-unique genes was 263. Through gene annotations, genes related to lipase were found in the genome. Furthermore, a combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strain SCS 2-3 represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nanhaiensis sp. nov. is proposed. The type strain is SCS 2-3T (= GDMCC 1.2219T = JCM 34440T).

Elaboration on the architecture of pH-sensitive surface charge-adaptive micelles with enhanced penetration and bactericidal activity in biofilms

BackgroundBiofilm formation is one of the main reasons for persistent bacterial infections. Recently, pH-sensitive copolymers have fascinated incredible attention to tackle biofilm-related infections. However, the proper incorporation of pH-sensitive segments in the polymer chains, which could significantly affect the biofilms targeting ability, has not been particularly investigated. Herein, we synthesized three types of pH-sensitive copolymers based on poly (β-amino ester) (PAE), poly (lactic-co-glycolic acid) (PLA) and polyethylene glycol (PEG), PAE-PLA-mPEG (A-L-E), PLA-PAE-mPEG (L-A-E) and PLA-PEG-PAE (L-E-A) to address this issue.ResultsThe three copolymers could self-assemble into micelles (MA-L-E, ML-A-E and ML-E-A) in aqueous medium. Compared with MA-L-E and ML-A-E, placing the PAE at the distal PEG end of PLA-PEG to yield PLA-PEG-PAE (ML-E-A) was characterized with proper triggering pH, fully biofilm penetration, and high cell membrane binding affinity. Further loaded with Triclosan (TCS), ML-E-A/TCS could efficiently kill the bacteria either in planktonic or biofilm mode. We reasoned that PAE segments would be preferentially placed near the surface and distant from the hydrophobic PLA segments. This would increase the magnitude of surface charge-switching capability, as the cationic PAE+ would easily disassociate from the inner core without conquering the additional hydrophobic force arising from covalent linkage with PLA segments, and rapidly rise to the outermost layer of the micellar surface due to the relative hydrophilicity. This was significant in that it could enable the micelles immediately change its surface charge where localized acidity occurred, and efficiently bind themselves to the bacterial surface where they became hydrolyzed by bacterial lipases to stimulate release of encapsulated TCS even a relatively short residence time to prevent rapid wash-out. In vivo therapeutic performance of ML-E-A/TCS was evaluated on a classical biofilm infection model, implant-related biofilm infection. The result suggested that ML-E-A/TCS was effective for the treatment of implant-related biofilm infection, which was proved by the efficient clearance of biofilm-contaminated catheters and the recovery of surrounding infected tissues.ConclusionsIn summary, elaboration on the architecture of pH-sensitive copolymers was the first step to target biofilm. The ML-E-A structure may represent an interesting future direction in the treatment of biofilm-relevant infections associated with acidity.Graphic abstract

Biomimetic pH/lipase dual responsive vitamin-based solid lipid nanoparticles for on-demand delivery of vancomycin

In this study, ascorbyl tocopherol succinate (ATS) was designed, synthesized and characterized via FT-IR, HR-MS, H1 NMR and C13 NMR, to simultaneously confer biomimetic and dual responsive properties of an antibiotic nanosystem to enhance their antibacterial efficacy and reduce antimicrobial resistance. Therefore, an in silico-aided design (to mimic the natural substrate of bacterial lipase) was employed to demonstrate the binding potential of ATS to lipase (−32.93 kcal/mol binding free energy (ΔGbind) and bacterial efflux pumps blocking potential (NorA ΔGbind: −37.10 kcal/mol, NorB ΔGbind: −34.46 kcal/mol). ATS bound stronger to lipase than the natural substrate (35 times lower Kd value). The vancomycin loaded solid lipid nanoparticles (VM-ATS-SLN) had a hydrodynamic diameter, zeta potential, polydispersity index and entrapment efficiency of 106.9 ± 1.4 nm, −16.5 ± 0.93 mV, 0.11 ± 0.012 and 61.9 ± 1.31%, respectively. In vitro biocompatibility studies revealed VM-ATS-SLN biosafety and non-haemolytic activity. Significant enhancement in VM release was achieved in response to acidified pH and lipase enzyme, compared to controls. VM-ATS-SLN showed enhanced sustained in vitro antibacterial activity for 5 days, 2-fold greater MRSA biofilm growth inhibition and 3.44-fold reduction in bacterial burden in skin infected mice model compared to bare VM. Therefore, ATS shows potential as a novel multifunctional adjuvant for effective and targeted delivery of antibiotics.

Detection lipase gene of Pseudomonas aeruginosa from crude oil contaminated soil

The microbial lipases are industrially more substantial. The bacterial lipase enzymes can be extracellular and intracellular, and are extremely affected by bacterial nutrition and various physicochemical factors like temperature degree, pH, the sources of carbon and nitrogen, inorganic salts and agitation. The objective of current work is isolation and identify of P. aeruginosa from crude oil contaminated soil depending on PCR targeted lipA gene. 20g (15 samples) of crude oil contaminated soil were collected from north oil refineries/Kirkuk/Iraq. The isolates were identified according to microscopically diagnosis and colonial properties, biochemical tests, API-20E system with diagnosis by PCR technique based on gene lip A. PCR products by electrophoresis demonstrated only 8 isolates (53.33%) with positive results with lipA 558 from all isolates of P. aeruginosa.

Light-triggerable and pH/lipase-responsive release of antibiotics and β-lactamase inhibitors from host-guest self-assembled micelles to combat biofilms and resistant bacteria

Major challenges remain in combat with the inevitable rise of intrinsic resistance to antibiotics and biofilm formation. Herein, we explore host–guest self-assembled micelles for light-triggered and stimuli-responsive release of antibiotics and β-lactamase inhibitors to combat biofilms derived from methicillin-resistant Staphylococcus aureus (MRSA). β-Cyclodextrin-capped phenylboronic acid-tetraphenylethylene (PBA-TPE) conjugates are coupled with ampicillin (Amp) via reactive oxygen species (ROS)-cleavable thioketal linkers to obtain cd-PTTA prodrug. Adamantane-capped poly(ethylene glycol)-poly(ε-caprolactone) (PECL-ad) amphiphilic copolymers are linked with cd-PTTA via host–guest complexation and simultaneously self-assembled into PECL@PTTA micelles. The digestion of poly(ε-caprolactone) segments by bacterial lipase destructs micelles and the aggregation-induced emission feature of cd-PTTA leads to ROS generation after light illumination. The produced ROS destroys biofilms and breaks thioketal linkers to release Amp antibiotics, while the pH-responsive removal of β-cyclodextrin activates the PBA β-lactamase inhibitors, affording synergistic actions on MRSA. The light illumination and intrinsic signals of acidic pH and lipase display interactive promotions of Amp release, micelle destabilization, and β-lactamase inhibition. Compared to those of cd-PTTA, the PECL@PTTA micelle treatment under light exposure shows sustained growth inhibition of planktonic MSRA, 2-fold higher elimination rate of biofilms, and 28-fold lower number of live MRSA embedded in biofilms. In an MRSA subcutaneous infection model, the micelle treatment with light exposure could eradicate bacteria and a complete wound closure was observed with normal re-epithelialization and skin morphology. Thus this strategy enables synchronous release of antibiotics and activation of β-lactamase inhibitors for photodynamic destruction of biofilms and restoration of the antibiotic activity to resistant bacteria embedded.

Key mutation sites for improvement of the enantioselectivity of lipases through protein engineering

Lipases can resolve many chiral compounds of commercial interest. However, for some substrates, it has been difficult to find lipases with the necessary chemo-, regio- and enantioselectivities. Searching in nature for new lipases with the desired selectivities is a difficult task and is not always successful. An alternative strategy is to engineer existing lipases. However, until now, there has been no systematic evaluation to identify which positions in the protein molecule should be engineered to obtain variants with the desired selectivities. Such an evaluation is possible because, even though the catalytic clefts of lipases have differing shapes, there is sufficient structural similarity for one to identify the key regions involved. In this review, we analyze representative studies that have engineered bacterial and fungal lipases for increased enantioselectivity for application in racemic resolutions. Based on this analysis, we identify key sites of the catalytic cleft at which mutations should be targeted in attempts to improve the enantioselectivity of lipases. The locations of these key sites depend on whether the chiral substrate is a carboxylic acid or an alcohol.

Bacterial Lipase Neutralized Toxicity of Lipopolysaccharide on Chicken Embryo Cardiac Tissue

It has been shown that near all organs, especially the cardiovascular system, are affected by bacterial lipopolysaccharide via the activation of Toll-like receptor signaling pathways. Here, we tried to find the blunting effect of bacterial lipase on lipopolysaccharide (LPS)-induced cardiac tissue toxicity in chicken embryos. 7-day fertilized chicken eggs were divided randomly into different groups as follows; Control, Normal Saline, LPS (0.1, 0.5 and 1mg/kbw), and LPS (0.1, 0.5 and 1mg/kbw) plus 5mg/ml Lipase. On day 17, the hearts were sampled. The expression of genes such as GATA4, NKX2.5, EGFR, TRIF, and NF-ƙB was monitored using real-time PCR analysis. Using western blotting, we measured NF-ƙB protein level. Total antioxidant capacity, glutathione peroxidase, and Catalase activity were also studied. Microvascular density and anterior wall thickness were monitored in histological samples using H&E staining. High dose of LPS (1mg/kbw) increased the expression of TRIF but not NF-ƙB compared to the control group (p < 0.05). We found a statistically significant reduction in groups that received LPS + Lipase compared to the control and LPS groups (p < 0.05). Western blotting revealed that the injection of Lipase could reduce LPS-induced NF-ƙB compared to the control group (p < 0.05). The expression of GATA4, NKx2.5, and EGFR was not altered in the LPS group, while the simultaneous application of LPS and Lipase significantly reduced GATA4, NKx2.5, and EGFR levels below the control (p < 0.05). We found non-significant differences in glutathione peroxidase, and Catalase activity in all groups (p > 0.05), while total antioxidant capacity was increased in groups that received LPS + Lipase. Anterior wall thickness was diminished in LPS groups and the use of both lipase and LPS returned near-to-control values (p < 0.05). Despite a slight increase in microvascular density, we found statistically non-significant differences in all groups (p > 0.05). Bacterial lipase reduces detrimental effects of LPS on chicken embryo heart induced via Toll-like receptor signaling pathway.

Virulence and Antimicrobial Resistance in Canine Staphylococcus spp. Isolates.

Dogs are reservoirs of different Staphylococcus species, but at the same time, they could develop several clinical forms caused by these bacteria. The aim of the present investigation was to characterize 50 clinical Staphylococcus isolates cultured from sick dogs. Bacterial species determination, hemolysins, protease, lipase, gelatinase, slime, and biofilm production, presence of virulence genes (lukS/F-PV, eta, etb, tsst, icaA, and icaD), methicillin resistance, and antimicrobial resistance were investigated. Most isolates (52%) were Staphylococcus pseudointermedius, but 20% and 8% belonged to Staphylococcus xylosus and Staphylococcus chromogenes, respectively. Gelatinase, biofilm, and slime production were very common characters among the investigated strains with 80%, 86%, and 76% positive isolates, respectively. Virulence genes were detected in a very small number of the tested strains. A percentage of 14% of isolates were mecA-positive and phenotypically-resistant to methicillin. Multi-drug resistance was detected in 76% of tested staphylococci; in particular, high levels of resistance were detected for ampicillin, amoxicillin, clindamycin, and erythromycin. In conclusion, although staphylococci are considered to be opportunistic bacteria, the obtained data showed that dogs may be infected by Staphylococcus strains with important virulence characteristics and a high antimicrobial resistance.

Bioswitchable Antibacterial Coatings Enable Self‐Sterilization of Implantable Healthcare Dressings

AbstractHealthcare‐acquired bacterial infections are a threat to public health. Therefore, development of self‐sterilizing implantable dressing materials is in increasing demand. Herein, a series of quaternized triblock copolymers, namely, QP‐b‐PCL‐b‐QPs, are synthesized and self‐assembled into reverse micelles (RMs) in tetrahydrofuran. The RMs contain biocompatible poly(ε‐caprolactone) (PCL) blocks in the shell to render biosafety and responsiveness, and biocidal quaternary blocks in the core to render antibacterial activity. In the presence of bacterial lipase, the biodegradable PCL blocks are hydrolyzed, resulting in the responsive release of quaternary biocidal agents (QBAs) to enable self‐sterilization. The RMs can be facilely impregnated into commercial gelatin sponge (GS) to fabricate RM2‐coated GS, which impose potent antibacterial activity in the presence of lipase. Compared to Gram‐negative P. aeruginosa, the Gram‐positive S. aureus and B. subtilis are more susceptible to QBAs released from RM2‐coated GS. The in vivo antibacterial assays and histological analyses further confirm the validity of RM2‐coated GS in reducing bacterial infection in mice model. These results, taken together, provide a promising strategy for the development of bioswitchable self‐sterilizing dressing materials to reduce healthcare‐acquired bacterial infections.

Effect of electron beam irradiation on phytochemical composition, lipase activity and fatty acid of quinoa

Electron beam irradiation (EBI) is a new steriNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, Chinalization technology, and research on how EBI can affect the chemical components of quinoa is limited. Therefore, this study focused on the changes in phytochemical composition, total bacteria count, lipase activity, and fatty acid of quinoa after EBI (0, 1, 2, 4, and 8 kGy). Results showed that EBI could inhibit the increase in total bacterial amount, lipase activity, and free fatty acid content during storage. The saponin content in quinoa did not change after EBI, while the phytic acid content decreased by 19% at 8 kGy. Total phenolic content (TPC) of free phenolics (FPs) significantly (p < 0.05) increased by 33% at 8 kGy. Total flavonoid content (TFC) of acid-hydrolysable phenolics (AHPs)significantly (p < 0.05) decreased by 24% at 4 kGy. TFC in AHP and TFC in base-hydrolysable phenolics (BHPs) did not show significant (p > 0.05) changes, while TPC in BHP and TFC in FP improved. The content of polyunsaturated fatty acids increased by 3% at 1 kGy. The value of ω3/ω6 (0.21) increased by 5% after EBI. The results indicated that EBI has strong potential for application in the quinoa industry without damaging the phytochemicals and fatty acid composition.

Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38

A novel cold-active true lipase from Pseudomonas sp. KE38 was cloned, sequencing and expressed in E. coli by degenerate PCR and genome walking technique. The open reading frame of the cloned gene encoded a polypeptide chain of 617 amino acids with a confirmed molecular weight of 64 kD. Phylogenetic analysis of the deduced amino acid sequence of the lipase indicated that it had high similarity with lipases of subfamily Ι.3 of bacterial lipases. Recombinant lipase was purified in denatured form as inclusion bodies, which were then renatured by urea followed by dialysis. Lipase activity was determined titrimetrically using olive oil as substrate. The enzyme showed optimal activity at 25 °C, pH 8.5 and was highly stable in the presence of various metal ions and organic solvents. Low optimal temperature and high activity in the presence of methanol and ethanol make this lipase a potential candidate for transesterification reactions and biodiesel production.

Detergent-compatible fungal cellulases

Detergent enzymes are currently added to all powder and liquid detergents that are manufactured. Cellulases, lipases, amylases, and proteases are used in the detergency to replace toxic phosphates and silicates and to reduce high energy consumption. This makes the use of enzymes in detergent formulation cost effective. Fungi are producers of important extracellular enzymes for industrial use. The fungal and bacterial cellulases maintain the shape and color of the washed garments. There is a high demand for cellulases at the market by detergent industries. With this high demand, genetic engineering has been a solution due to its high production of detergent-compatible cellulases. Fungi are the famous source for detergent-compatible cellulases production, but still, there is a lack of the cost-effective process of alkaline fungal cellulase production. Review papers on detergent-compatible bacterial cellulase and amylase and detergent-compatible fungal and bacterial proteases and lipases are available, but there is no review on detergent fungal cellulases. This review aims to highlight the production, properties, stability, and compatibility of fungal cellulases. It will help other academic and industrial researchers to study, produce, and commercialize the fungal cellulases with good aspects.

A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB.

Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter ‘ragi’ from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 μg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.

DETECTION OF SOME TYPES OF OILS THAT INDUCE LIPASE PRODUCTION IN SOME BACTERIAL ISOLATES

Bacterial lipases are a highly versatile class of hydrolytic enzymes which catalyze the hydrolysis of triglycerol to glycerol and fatty acids. They play an important role in biotechnological and industrial processes, including oleo chemical, food, pharmaceutical detergent formulation, cosmetic, leather, textile, and paper industries applications. Tremendous interest among scientists and industrialists has been arisen in the last decade in microbial lipases due to their versatility and ease of mass production.In the current work, natural oils such as Triglyceride, Tween 20, Tween 80, Olive oil, Sweet almond, Celery oil, Sesame, Rosemary oils were implemented to enhance lipase production by soil and pathogenic bacterial isolates. Results revealed that triglyceride and tween 80 had the best enhancing activity for lipase production in most isolates. It was also shown that Pseudomonas and Bacillus were the most potential lipase producing bacteria.A novel modified tributary agar was prepared by the addition of Sudan blue dye and used for the detection of lipase producers. It was shown to give a very clear zone even in the case of weak producing strains as compared to the original medium.Specific lipase activity had been determined Staphylococcus aureus (in the present research. The highest lipase activity was demonstrated by Pseudomonas (4.5u/ml), while Staphylococcus aureus did not show any lipase activity.

The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system

A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett–Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box–Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.

Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR

Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with Tm values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a Tm value of 68.0°C. Although the increase of Tm was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes.

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Characterization and Lipase Production of Micrococcus sp. L69 Isolated from Palm Oil-contaminated Soil

This research aims to characterise and study the lipase production of Micrococcus sp. L69 isolated from palm oil-contaminated soil. Lipase production was carried out by cultivating the bacteria in the medium containing vegetable oils. The lipase activity was determined by spectrophotometric method toward p-nitrophenyl palmitate as a substrate. The results of this research showed that the bacteria isolate L69 was a unique lipolytic bacteria. Based on sequence of 16S rRNA gene, the bacteria had low similarity level (≤ 93%) to sequences data listed in GenBank. Olive oil and coconut oil are good carbon sources for bacterial growth and lipase production. The highest lipase activity achieved at temperature of 50°C and pH 7.0. The addition of 7 mM Ca2+ and Fe++ enhanced the lipase activity for 1.95- and 2.26-fold, respectively.

PRODUCTION OF LIPASE BY IMMOBILIZED BACILLUS THURINGIENSIS AND LYSINIBACILLUS SPHAERICUS AND THEIR BIODEGRADATION POTENTIAL ON DIESEL

This study reported production of lipase by immobilized Bacillus thuringiensis. Bacteria isolates were screened on Bushnell-Hass Mineral Salt medium containing 1% v/v Diesel for oil degradation. The potent isolates were identified using 16S rRNA as Bacillus thuringiensis. The isolates were immobilized in gelatin matrix and cultured for lipase production in a submerged medium. The crude lipase extracted was used for degradation of Diesel. Optimum degradation of Diesel 41.4% was obtained by lipase from Immobilized Bacillus thuringiensis and 31.6% for Lysinibacillus sphaericus at pH 7 and 35 in 20 days. GC-MS analysis was carried out to show the compounds degraded after 20 days. This study therefore presented the use of immobilized bacterial lipase in degradation of Diesel as a simple and effective approach.