Microbial Lipases Research Articles

Biodiesel production from microalgae oil by lipase from Pseudomonas aeruginosa displayed on yeast cell surface

Yeast surface display has become a powerful technology in recent decades and one of the promising areas in this field is the biodiesel synthesis by microbial lipases. Hence, in this study the optimized lipase A (Lip A) gene from Pseudomonas aeruginosa was fused to GPI-anchored protein Gcw61 and successfully displayed on the surface of Pichia pastoris X33. A lipase activity of 85.2 U/mg dry cell weight was obtained from recombinant P. pastoris. The copy numbers of inserted lipase gene were determined 2.09 ± 0.06 by real time PCR absolute quantification method. The enzyme showed the best stability in pH 7.0–10.0 and at temperature 37 °C–40 °C and was also stable in hydrophilic organic solvents. Ca2+, Mg2+, Mn2+ and Cu2+ ions enhanced enzyme activity, whereas Fe2+ and Zn2+ ions and some detergents like SDS, CTAB, Tween 20 and 80 dramatically decreased the activity of the enzyme. The results demonstrate that our whole cell biocatalyst exhibited a good potential for biodiesel production from microalgae oil in 10 repeated batch cycles.

Preparation of ion-exchange supports via activation of epoxy-SiO2 with glycine to immobilize microbial lipase – Use of biocatalysts in hydrolysis and esterification reactions

Ion-exchange supports have been prepared via sequential functionalization of silica-based materials with (3‑Glycidyloxypropyl)trimethoxysilane (GPTMS) (Epx-SiO2) and activation with glycine (Gly-Epx-SiO2) in order to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. Rice husk silica (RHS) was selected as support with the aim of comparing its performance with commercial silica (Immobead S60S). Sequential functionalization/activation of SiO2-based supports has been confirmed by AFM, SEM and N2 adsorption-desorption analyses. Maximum TLL adsorption capacities of 14.8 ± 0.1 mg/g and 16.1 ± 0.6 mg/g using RHS and Immobead S60S as supports, respectively, have been reached. The Sips isotherm model has been used which was well fitted to experimental data on TLL adsorption. Catalytic activities of immobilized TLL were assayed by olive oil emulsion hydrolysis and butyl stearate synthesis via an esterification reaction. Hydrolytic activity of the biocatalyst prepared with a commercial support (357.6 ± 11.2 U/g) was slightly higher than that of Gly-Epx-SiO2 prepared with RHS (307.4 ± 7.2 U/g). On the other hand, both biocatalysts presented similar activity (around 90% conversion within 9–10 h of reaction) and reusability after 6 consecutive cycles of butyl stearate synthesis in batch systems.

Effect of Surfactant Structure on the Superactivity of Candida rugosa Lipase.

In this work, we present the effects of ionic and zwitterionic surfactants on the hydrolytic activity of Candida rugosa lipase (CRL), one of the most important and widely used microbial lipases. A series of amine N-oxide surfactants was studied to explore the relationship between their molecular structures and their effect on catalytic properties of CRL. These zwitterionic amphiphiles are known for their ability to form aggregates that can increase their size, thanks to a sphere-rod transition, without any additive. Enzyme activity seemed to be improved by morphological changes of micelles from spherical to rod-like, and the structure of the monomers played a crucial role in this transition. In fact, all the amine oxides investigated provoked superactivation, but the CRL activity increased by lengthening the alkyl chain of N-oxide surfactants, whereas it decreased in the presence of bulky head groups. Superactivity was mainly because of an increase in kcat (0.57 s-1 in buffer, 0.80-1.99 s-1 in surfactant solutions) and, in some cases, a decrease in KM (2 × 10-3 M in buffer, 1.08-4.28 × 10-3 M in surfactant solutions). Micelles seemed to play a dual role: superactivity occurred at surfactant concentrations higher than their critical micelle concentration, but, on the other hand, micelles subtracted the substrate from the bulk, making it unavailable for the catalysis.

REVIEW: Novel sources and functions of microbial lipases and their role in the infection mechanisms

Lipases belong to the family of serine hydrolases, which in turn include various esterase enzymes (E.C.3.1.1.1). They are involved in the cleavage of triacylglycerols to free fatty acids and glycerol in many important biological processes, as for instance routine metabolism of dietary triglycerides to cell signalling and inflammation. Lipases constitute a ubiquitous group of enzymes able to catalyse a number of different reactions, many of them of industrial interest. Particularly, microbial lipases exhibit a wide range of industrial applications, namely in pharmaceutical, food and detergents industry. The aim of this review is to summarize the recent achievements illustrating the importance and the versatility of microbial lipases, including their involvement in infection mechanisms.

Fatty Acids Effect on Lipase and Biosurfactant Induction in Rhodotorula Glutinis CMGB-RG5

Abstract Fatty acids represent important substrates for obtaining microbial lipases and biosurfactants. The yeast strain CMGBRG5 was identified as Rhodotorula glutinis using the BIOLOG MicroLog System. The opacity screening tests showed that R. glutinis CMGB-RG5 was able to produce lipases in presence of 1% Tween 80 after three days of incubation. Lipase induction was estimated as cell growth in presence of Tween 80, Tween 20, olive oil and tributyrin. After 48 hours, best results were obtained in presence of butyric acid, respectively, oleic acid: 2.2 × 107 cells/ml on tributyrin and 1.0 × 107 cells/ml on olive oil. Biosurfactant production was evaluated as emulsification index (E24%). After one week, high E24 values were obtained on fried sunflower oil (53%) and olive oil (35%). Crude and concentrated biosurfactants were tested against nine Candida strains. Best antimicrobial activity was observed for [20X] biosurfactants against C. tropicalis, C. guilliermondii and C. krusei. In conclusion, R. glutinis CGB-RG5 shows high potential for using fatty acids from various sources as unique carbon substrates for synthesis of biocompounds with high economic and biotechnological value.

Microbial Lipase Mediated by Health Beneficial Modification of Cholesterol and Flavors in Food Products: A Review.

The tremendous need of lipase for varied applications in biotechnology increases its economical value in food and allied industries. Patents suggest that lipase has an impressive number of applications viz. enhancements of flavor in food products (Cheese, butter, alcoholic beverages, milk chocolate and diet control food stuffs), detergent industry in removing oil, grease stain, organic chemical processing, textile industry, oleochemical industry, cosmetic industry and also as therapeutic agents in pharmaceutical industries. This communication extends the frontier of lipase catalyzed benefits to human body by lowering serum cholesterol and enhancement of flavor in different food products. Among all, multiple innovations going on in the field of lipase applications are widening its scope in food industries consistently. Therefore, in the present work an effort has been made to explore the utilization of lipase in the field of food product enhancement. Supplementation of food products with lipase results in modification of its physical, chemical and biochemical properties by enhancing its therapeutic activity. Lipases are the most important enzymes used in food industries. They are utilized as industrial catalysts for lipid hydrolysis. Because of lipases hydrolysis nature it is widely exploited to catalyze lipids or fats in different food products and enhancement of food flavors.

Novel lipases discovery specifically from marine organisms for industrial production and practical applications

Abstract Lipases are acknowledged as relevant biocatalysts for numerous important applications in food, detergents and pharmaceutical processing, ester and peptide synthesis, transesterifications, biosurfactant production, and in the resolution of racemic mixtures to produce optically active compounds. Lipases are produced by organisms of microbial, plant, and animal origin. Microbial lipases have attracted far more interest from researchers and industries than lipases from other sources, due both to their specific features and ease of production on large scale. Notwithstanding current achievements, there is still a quest for lipases with improved/novel catalytic features and improved stability, namely in harsh environments. Marine organisms can be an adequate source for such lipases as marine enzymes have proved useful for both process improvement and for the development of new manufacturing procedures/new products. The production of lipases is influenced by the composition of the culture medium, e.g. carbon and nitrogen sources, as well as by physical chemical parameters such as temperature and pH. In this review relevant, types of lipases from marine organisms are identified, their role is described and the novel features of these enzymes are discussed.

Synthetic and Natural Lipase Inhibitors.

Lipases are enzymes that catalyse the hydrolysis of ester bonds of triglycerides ranging among biocatalysts of considerable physiological significance and industrial potential. Better understanding of the catalytic functions and achieving the possibility to control the biocatalysis process, in particular exploring some activators and inhibitors of lipases, seems to be crucial in the context of novel applications. The lipase activity is a function of interfacial composition: the enzyme can be there activated as well as denaturated or deactivated and the interface is an appropriate site for modulating lipolysis. Lipase inhibitor, interacts directly with the enzyme and inhibits lipase action. Alternatively, some compounds can postpone the lipolytic reaction via adsorption to the interphase or to the substrate molecules. The aim of this review is to summarise the current knowledge concerning human, animal and microbial lipase inhibitors, which were grouped into two categories: synthetic lipase inhibitors (including phosphonates, boronic acids and fats analogues) and natural compounds (including β-lactones and some botanical foodstuffs - plant extracts and plant metabolites, mainly polyphenols and saponins as well as peptides and some dietary fibers). The topics discussed include also inhibition issues from the viewpoint of obesity treatment. Among natural compounds able to inhibit lipase activity are β- lactones including orlistat. Orlistat is the only registered drug for obesity treatment in many countries and lipases are essential enzymes for lipid absorption - thus fat absorption or obesity can be controlled by lipase inhibition, especially pancreatic lipase which is responsible for the hydrolysis of over 80% of total dietary fats. Its effectiveness in obesity treatment was also described.

Computational elucidation of phylogenetic, structural and functional characteristics of Pseudomonas Lipases

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) catalyzes tri-, di-, and monoacyl glycerol of fat into glycerol and fatty acids. It has important roles in the digestion of lipids in living organisms and industrially as laundry detergents along with proteases. The microbial lipases are more stable, active and economically feasible compared to plant and animal sources. Hence, much attention was given to the maximum production of the enzyme from the microbial sources. The phylogenetic analysis revealed that the amino acid sequence of lipase protein and their corresponding cDNA of Pseudomonas aeruginosa clustered with Pseudomonas stutzeri among different species of Pseudomonas, while P. aeruginosa PA1 clustered with P. aeruginosa SJTD-1 among different strains of P. aeruginosa. The lipase of P. aeruginosa PA1 was a monomeric, acidic and thermostable protein having a molecular weight ranging in between 32.72 to 34.89 kDa. The protein was abundant with random coils and alpha helices in its secondary structure. The tertiary model showed 96.310 score as an overall quality factor. Hence, this in silico study gives some useful information about the lipase protein without performing crystal structure assessment by X-ray Crystallography or NMR study in wet lab experiments which could be helpful for isolation and characterization of the enzyme in vitro.

Production of lipase from Streptomyces

Lipases constitute an important group of biocatalysts for biotechnological applications This enzyme can be produced by plants animals and microorganisms However microbial lipases are the most studied and occupy the center attraction because of their stability selectivity and broad substrate specificity Streptomyces are filamentous Gram positive bacteria found generally in the soil This microorganism is known mainly for their capacity to synthesize of numerous antibiotics and other secondary metabolites They also produce a large number of enzymes such as lipases which makes Streptomyces a genus with significant biotechnological potential

Screening and production of lipase from fungal organisms

Abstract Lipases (triacylglycerol acylhydrolases, (E.C. 3.1.1.3)) are a class of enzymes endowed with an ability to catalyze the hydrolysis of triglycerides to glycerol and free fatty acids. These are industrially significant, particularly microbial lipases. Among several diverse sources, lipases from fungal origin are much embraced owing to their high stability. These lipases are directly screened majorly by the plate assay technique involving various preferred and specific substrates and indirectly by appropriately growing the strains in liquid medium followed by analysing the activity of the filtrates. Production of fungal lipase is majorly by using solid state and submerged fermentation processes with considerable variation in their operational conditions, that are influenced by physico-chemical factors and which plays a major role in the optimum lipase production. Further, the immobilized fungal cells have also been employed for lipase production using various support materials. Keeping in view of all these aspects, this review focuses on the screening, production and utility of lipases obtained from prominent fungal organisms.

The performance of microbial lipase immobilized onto polyolefin supports for hydrolysis of high oleate sunflower oil

The application of immobilized microbial lipase for the catalytic hydrolysis of triglyceride esters is described. Sunflower oil was used as the primary substrate for the hydrolysis in the presence of microbial lipase extracted from the fungal yeast Candida rugosa OF360. Lipase was immobilized onto three different polyolefin supports: high density polypropylene (Accurel EG100 and EP100), and low density polyethylene (Accurel EP400). In the case of Accurel EP400 there was a strong positive linear relationship between enzyme concentration prior to immobilization and expressed enzyme activity. In contrast, Accurel EG100 exhibited no increase in expressed activity with increasing initial enzyme concentration. The presence of glutaraldehyde crosslinking agent resulted in enhanced expressed activity on EP100 and exhibited minimal change in EP400. Maximum immobilized efficiency was observed for the Accurel EP100 and EP400 at particle sizes in the 200 μm range. Overall, half-lives of immobilized enzyme were observed to be up to sevenfold that of enzyme in free solutions. Optimum pH values in the range 7–9 were confirmed. Optimum temperatures for expressed activity of lipase on the Accurel EP100 and EP400 were determined to be 30 °C and 45 °C respectively.

Temporal microbiota and biochemical profiles during production and ripening of Divle Cave cheese

The aim of this study was to evaluate the proteolysis and lipolysis profiles of a raw ewes' milk cheese during production and ripening. The microbial community of this cheese was quantified, and their roles during proteolysis and lipolysis were studied. We observed an accumulation of substantial amounts of peptides during ripening, which might be attributed to the abundant presence of lactic acid bacteria, as well as to secondary microbiota including fungi, yeasts and actinobacteria. Similarly, high lipolysis levels were obtained, which can be explained by the indigenous lipase in milk and microbial lipases. These results should be of great interest to industrial manufacturers of this cheese.

Microbial lipases and their applications – a review

This review focuses on the key aspects of lipases. Lipases (EC 3.1.1.3) are triacylglycerol acylhydrolases that act on carboxylic ester bonds. They breakdown triacylglycerides into glycerides (diglycerides or monoglycerides), fatty acids and glycerol. Their mass ranges from 19 kDa for B. stratosphericus to 92 kDa for P. gessardii. Their optimum temperature and pH ranges from 15 °C to 80 °C for Acinetobacter sp. and Janibacter sp. and 5 to 11 for P. gessardii and E. faecium respectively. Lipases chemo-, regio-, and enantio- specific features make them first choice of enzymes in research. Their kinetics for substrate hydrolysis depends on different esters. Mostly lipases are extracellular. Type 1 secretory system (T1SS) and Type 2 secretory system (T2SS) are involved in secreting lipases to external medium. They are found in eukaryotes and prokaryotes including animals, plants and microorganisms. Moreover, bacterial and fungal enzymes have diverse industrial applications in food, health, pharmaceutical, medical, textile, detergent, cosmetic and paper industries. Genetic engineering is employed to improve the properties of lipases. Their increasing demand in market has made them a hot topic in scientific research. Scientists are trying to discover novel lipase producing microorganisms due to their expanding commercial value. Keywords: Lipases, esterification, transesterification, biochemical and physicochemical properties, recombinant DNA technology

A Study on Marine Lipolytic Yeasts: Optimization and Characterization of Lipase Enzyme

Lipases are hydrolase enzymes which hydrolyze triglycerides into glycerol and free fatty acids. Microorganisms like bacteria, fungi and yeast are capable of producing lipases. These microbial lipases have wide range of industrial application. Lipases produce by the yeast are now gaining more attention with the help of new emerging enzyme technology. In this paper the lipolytic activity of marine yeasts were studied. Ten yeast strains were isolated from the marine samples collected from the coast of Port Blaire, Andaman and Nicobar Island, India. Primary screening was done in tributyrin agar medium and three isolates A, B and C showed positive activity by producing extracellular enzymes. The antioxidant activity of the isolates selected was done with DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, the isolates A,B,C showed 78%,77% and 66% respectively. Reducing power assay was also carried out. Secondary screening of the selected isolates was done to determine lipase activity using Tris-HCl buffer and phosphate buffer. The activity of A, B and C was found to be 48%, 63% and 60% respectively. The enzyme was extracted using different solvents as means of partial purification.

Realm of Thermoalkaline Lipases in Bioprocess Commodities.

For decades, microbial lipases are notably used as biocatalysts and efficiently catalyze various processes in many important industries. Biocatalysts are less corrosive to industrial equipment and due to their substrate specificity and regioselectivity they produced less harmful waste which promotes environmental sustainability. At present, thermostable and alkaline tolerant lipases have gained enormous interest as biocatalyst due to their stability and robustness under high temperature and alkaline environment operation. Several characteristics of the thermostable and alkaline tolerant lipases are discussed. Their molecular weight and resistance towards a range of temperature, pH, metal, and surfactants are compared. Their industrial applications in biodiesel, biodetergents, biodegreasing, and other types of bioconversions are also described. This review also discusses the advance of fermentation process for thermostable and alkaline tolerant lipases production focusing on the process development in microorganism selection and strain improvement, culture medium optimization via several optimization techniques (i.e., one-factor-at-a-time, surface response methodology, and artificial neural network), and other fermentation parameters (i.e., inoculums size, temperature, pH, agitation rate, dissolved oxygen tension (DOT), and aeration rate). Two common fermentation techniques for thermostable and alkaline tolerant lipases production which are solid-state and submerged fermentation methods are compared and discussed. Recent optimization approaches using evolutionary algorithms (i.e., Genetic Algorithm, Differential Evolution, and Particle Swarm Optimization) are also highlighted in this article.

Structural homogeneity in microbial lipases.

Enzyme lipase (triacylglycerol hydrolases E.C.3.1.1.3) catalyzes the hydrolysis of triacylglycerols to glycerol and free fatty acids. In recent years, progressively more consideration is being rewarded to lipases produced by microorganisms. In nature, some of the most important lipase-producing bacteria are Pseudomonas, Bacillus, Staphylococcus, etc., and fungi are Rhizopus sp., Aspergillus, Penicillium, Geotrichum, Mucor, Rhizomucor, etc. [1,2]. The bacterial lipases from Bacillus sp. exhibit interesting properties that make them potential candidates for biotechnological applications [1-3]. Bacillus alcalophilus are the most common lipase producing strains. Lipases are classified into different families based on the amino-acid sequences similarity and their biological properties [4]. Microbial lipases are relatively stable and are capable of catalyzing a variety of reactions; they hold immense potential for diverse industrial applications.

Optimized expression of the Starmerella bombicola lactone esterase in Pichia pastoris through temperature adaptation, codon-optimization and co-expression with HAC1

The Starmerella bombicola lactone esterase (SBLE) is a novel enzyme that, in vivo, catalyzes the intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment. In fact, this is an unusual reaction given the unfavorable conditions for dehydration. This characteristic strongly contributes to the potential of SBLE to become a 'green' tool in industrial applications. Indeed, lactonization occurs normally in organic solvents, an application for which microbial lipases are increasingly used as biocatalysts. Previously, we described the production of recombinant SBLE (rSBLE) in Pichia pastoris (syn. Komagataella phaffii). However, expression was not optimal to delve deeper into the enzyme's potential for industrial application. In the current study, we explored codon-optimization of the SBLE gene and we optimized the rSBLE expression protocol. Temperature reduction had the biggest impact followed by codon-optimization and co-expression of the HAC1 transcription factor. Combining these approaches, we achieved a 32-fold improvement of the yield during rSBLE production (from 0.75 mg/l to 24 mg/L culture) accompanied with a strong reduction of contaminants after affinity purification.

Biochemical properties of free and immobilized Candida viswanathii lipase on octyl-agarose support: Hydrolysis of triacylglycerol and soy lecithin

Abstract Microbial lipases are important enzymes in food and pharmaceutical industries. In this work, a Candida viswanathii lipase was purified by hydrophobic interaction chromatography on octyl Sepharose. The purification presented 78.4% yield and the enzyme was 8.7-fold purified, with specific activity 700.4 U/mg protein and 69 kDa molecular weight. Immobilization of the enzyme on the same support presented 72.5% yield and a derivative with 101% expressed activity (109.2 U/g support), indicating hyperactivation of the enzyme. Optimal activity for both free and immobilized lipase was observed at pH 4.0 and 45 °C. The free and immobilized lipase showed broad-range stability from acid to neutral pH, and apparent activation and stability on organic solvents. The derivative was 60-fold thermostabilized in relation to the free enzyme and fully retained its activity after four cycles of p-nitrophenyl palmitate hydrolysis. Slight activation was observed with dithiothreitol and β-mercaptoethanol. The free and immobilized lipase efficiently hydrolyzed monoesters, simple and mixed long chain triacylglycerols, as well as soy lecithin. The activity and stability in acid pH, the organic solvent tolerance and the lecithin hydrolysis indicate high potential application of the enzyme and its derivative in textile, food and pharma industries and for chemical synthesis.

Intact polar lipid export in the temperate western North Atlantic and Sargasso Sea

Intact polar diacylglycerolipids (IP-DAGs), abundant membrane components in phytoplankton and bacterioplankton, comprised 5.9±0.5% of suspended particulate organic carbon (POC) in the euphotic zone at two stations in the western North Atlantic Ocean. The IP-DAG assemblage included 9 phospholipid, glycolipid and betaine lipid classes that are routinely found in marine samples. We also report the pelagic occurrence of two recently discovered IP-DAGs, phosphatidyl-S,S-dimethylpropanethiol (PDPT) and a betaine-like lipid, both originally identified in Emiliania huxleyi. At the mesotrophic station north of the Gulf Stream, IP-DAGs comprised 0.62±0.38% of sinking POC collected in sediment traps, and at the oligotrophic station in the Sargasso Sea they comprised 0.31±0.05% of sinking POC.Phospholipids and glycolipids with polyunsaturated fatty acid (PUFA) moieties were relatively abundant in sinking particles at 150m compared with suspended particles in the euphotic zone at the mesotrophic station north of the Gulf Stream. The sinking particles were probably associated with the demise of a dinoflagellate or diatom bloom that resulted in a maximum total IP-DAG export flux of 1.8μmol/m2/day. At the oligotrophic station in the Sargasso Sea, sinking particles included microalgal glycolipids, phospholipids and betaine lipids with PUFAs; picocyanobacterial glycolipids with saturated and monounsaturated FAs; and bacterial phospholipids with saturated and monounsaturated FAs. The microalgal and bacterial IP-DAGs were relatively abundant in sinking particles compared with suspended particles. The IP-DAG export flux at the station in the Sargasso Sea was similar in three sediment traps deployed on consecutive days, with an average of 0.46±0.08μmol/m2/day.A diel cycle was observed in the IP-DAG euphotic zone inventory in the Sargasso Sea, with higher concentration in samples taken at dusk than at dawn, and we propose a model using this dynamic to calculate IP-DAG residence time. Export efficiency was calculated from the residence time and the ratio of IP-DAG export flux to euphotic zone inventory, with values ranging from 0.02% for PDPT to 6.9% for monogalactosyldiacylglycerol (MGDG) with PUFAs. The greater export flux and export efficiency of IP-DAGs with PUFAs contrasts with previous results for total FAs in sinking particles, which showed preferential export of saturated and monounsaturated FAs. The difference likely resulted from IP-DAGs with PUFAs being membrane components of rapidly sinking organic matter from microalgae. Alternatively, PUFA moieties cause steric hindrance of microbial lipase activity, potentially decreasing their hydrolysis rate and increasing the relative concentration of IP-DAGs with PUFAs in sinking particles.