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  1. Rahman RN, Tejo BA, Basri M, Rahman MB, Khan F, Zain SM, et al.
    Appl Biochem Biotechnol, 2004 8 12;118(1-3):11-20.
    PMID: 15304735
    Candida rugosa lipase was modified via reductive alkylation to increase its hydrophobicity to work better in organic solvents. The free amino group of lysines was alkylated using propionaldehyde with different degrees of modification obtained (49 and 86%). Far-ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent showed that such chemical modifications at the enzyme surface caused a loss in secondary and tertiary structure that is attributed to the enzyme unfolding. Using molecular modeling, we propose that in an aqueous environment the loss in protein structure of the modified lipase is owing to disruption of stabilizing salt bridges, particularly of surface lysines. Indeed, molecular modeling and simulation of a salt bridge formed by Lys-75 to Asp-79, in a nonpolar environment, suggests the adoption of a more flexible alkylated lysine that may explain higher lipase activity in organic solvents on alkylation.
    Matched MeSH terms: Lipase/isolation & purification
  2. Tan CH, Show PL, Ooi CW, Ng EP, Lan JC, Ling TC
    Biotechnol J, 2015 Jan;10(1):31-44.
    PMID: 25273633 DOI: 10.1002/biot.201400301
    Microbial lipases are popular biocatalysts due to their ability to catalyse diverse reactions such as hydrolysis, esterification, and acidolysis. Lipases function efficiently on various substrates in aqueous and non-aqueous media. Lipases are chemo-, regio-, and enantio-specific, and are useful in various industries, including those manufacturing food, detergents, and pharmaceuticals. A large number of lipases from fungal and bacterial sources have been isolated and purified to homogeneity. This success is attributed to the development of both conventional and novel purification techniques. This review highlights the use of these techniques in lipase purification, including conventional techniques such as: (i) ammonium sulphate fractionation; (ii) ion-exchange; (iii) gel filtration and affinity chromatography; as well as novel techniques such as (iv) reverse micellar system; (v) membrane processes; (vi) immunopurification; (vi) aqueous two-phase system; and (vii) aqueous two-phase floatation. A summary of the purification schemes for various bacterial and fungal lipases are also provided.
    Matched MeSH terms: Lipase/isolation & purification*
  3. Loo JL, Khoramnia A, Lai OM, Long K, Ghazali HM
    Molecules, 2014 Jun 23;19(6):8556-70.
    PMID: 24959682 DOI: 10.3390/molecules19068556
    Mycelium-bound lipase (MBL), from a locally isolated Geotrichum candidum strain, was produced and characterized as a natural immobilized lipase. A time course study of its lipolytic activity in 1 L liquid broth revealed the maximum MBL activity at 4 h for mycelium cells harvested after 54 h. The yield and specific activity of MBL were 3.87 g/L dry weight and 508.33 U/g protein, respectively, while less than 0.2 U/mL lipase activity was detected in the culture supernatant. Prolonged incubation caused release of the bound lipase into the growth medium. The growth pattern of G. candidum, and production and properties of MBL were not affected by the scale. The stability of mycelia harboring lipase (MBL), harvested and lyophilized after 54 h, studied at 4 °C depicted a loss of 4.3% and 30% in MBL activity after 1 and 8 months, while the activity of free lipase was totally lost after 14 days of storage. The MBL from G. candidum displayed high substrate selectivity for unsaturated fatty acids containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.
    Matched MeSH terms: Lipase/isolation & purification
  4. Shariff FM, Rahman RN, Basri M, Salleh AB
    Int J Mol Sci, 2011;12(5):2917-34.
    PMID: 21686158 DOI: 10.3390/ijms12052917
    A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5-99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55-80 °C and at a pH of 6-10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ß-strands, 23.6% turns and 35.6% random conformations.
    Matched MeSH terms: Lipase/isolation & purification
  5. Leow TC, Rahman RN, Basri M, Salleh AB
    Extremophiles, 2007 May;11(3):527-35.
    PMID: 17426920
    A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra.
    Matched MeSH terms: Lipase/isolation & purification
  6. Rahman RN, Baharum SN, Basri M, Salleh AB
    Anal Biochem, 2005 Jun 15;341(2):267-74.
    PMID: 15907872
    An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees C and pH 6-9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.
    Matched MeSH terms: Lipase/isolation & purification*
  7. Amid M, Manap MY, Hussin M, Mustafa S
    Molecules, 2015 Jun 17;20(6):11184-201.
    PMID: 26091076 DOI: 10.3390/molecules200611184
    Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.
    Matched MeSH terms: Lipase/isolation & purification*
  8. Salwoom L, Raja Abd Rahman RNZ, Salleh AB, Mohd Shariff F, Convey P, Pearce D, et al.
    Molecules, 2019 Feb 16;24(4).
    PMID: 30781467 DOI: 10.3390/molecules24040715
    In recent years, studies on psychrophilic lipases have been an emerging area of research in the field of enzymology. This study focuses on bacterial strains isolated from anthropogenically-influenced soil samples collected around Signy Island Research Station (South Orkney Islands, maritime Antarctic). Limited information on lipase activities from bacteria isolated from Signy station is currently available. The presence of lipase genes was determined using real time quantification PCR (qPCR) in samples obtained from three different locations on Signy Island. Twenty strains from the location with highest lipase gene detection were screened for lipolytic activities at a temperature of 4 °C, and from this one strain was selected for further examination based on the highest enzymatic activities obtained. Analysis of 16S rRNA sequence data of this strain showed the highest level of sequence similarity (98%) to a Pseudomonas sp. strain also isolated from Antarctica. In order to increase lipase production of this psychrophilic strain, optimisation of different parameters of physical and nutritional factors were investigated. Optimal production was obtained at 10 °C and pH 7.0, at 150 rev/min shaking rate over 36 h incubation.
    Matched MeSH terms: Lipase/isolation & purification*
  9. Shariff FM, Leow TC, Mukred AD, Salleh AB, Basri M, Rahman RN
    J Basic Microbiol, 2007 Oct;47(5):406-12.
    PMID: 17910105
    A thermophilic bacterium, Bacillus sp. strain L2 was isolated from a hot spring in Perak, Malaysia. An extracellular lipase activity was detected through plate and broth assays at 70 degrees C after 28 h of incubation. The L2 lipase production was growth dependent as revealed by a number of factors affecting the secretion of extracelullar lipase. As for nutritional factors, casamino acids, trehalose, Ca(2+) and Tween 60 were found to be more effective for lipase production. The optimum physical condition for L2 lipase production was obtained at 70 degrees C after 28 h of cultivation time, at pH 7.0, 150 rpm of agitation rate and 1% of starting inoculum size. The activity staining of crude L2 lipase revealed a clearing zone at 39 kDa.
    Matched MeSH terms: Lipase/isolation & purification
  10. Abd Rahman RN, Leow TC, Salleh AB, Basri M
    BMC Microbiol, 2007;7:77.
    PMID: 17692114
    Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78 degrees C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5-99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.
    Matched MeSH terms: Lipase/isolation & purification*
  11. Kam YC, Woo KK, Ong LGA
    Molecules, 2017 Dec 08;22(12).
    PMID: 29292721 DOI: 10.3390/molecules22122106
    Lipases with unique characteristics are of value in industrial applications, especially those targeting cost-effectiveness and less downstream processes. The aims of this research were to: (i) optimize the fermentation parameters via solid state fermentation (SSF); and (ii) study the performance in hydrolysis and esterification processes of the one-step partially purified Schizophyllum commune UTARA1 lipases. Lipase was produced by cultivating S. commune UTARA1 on sugarcane bagasse (SB) with used cooking oil (UCO) via SSF and its production was optimized using Design-Expert® 7.0.0. Fractions 30% (ScLipA) and 70% (ScLipB) which contained high lipase activity were obtained by stepwise (NH₄)₂SO₄ precipitation. Crude fish oil, coconut oil and butter were used to investigate the lipase hydrolysis capabilities by a free glycerol assay. Results showed that ScLipA has affinities for long, medium and short chain triglycerides, as all the oils investigated were degraded, whereas ScLipB has affinities for long chain triglycerides as it only degrades crude fish oil. During esterification, ScLipA was able to synthesize trilaurin and triacetin. Conversely, ScLipB was specific towards the formation of 2-mono-olein and triacetin. From the results obtained, it was determined that ScLipA and ScLipB are sn-2 regioselective lipases. Hence, the one-step partial purification strategy proved to be feasible for partial purification of S. commune UTARA1 lipases that has potential use in industrial applications.
    Matched MeSH terms: Lipase/isolation & purification
  12. Alhelli AM, Abdul Manap MY, Mohammed AS, Mirhosseini H, Suliman E, Shad Z, et al.
    PMID: 27836491 DOI: 10.1016/j.jchromb.2016.10.037
    This report shows the partitioning and purification of alkaline extracellular lipase from Penicillium candidum (PCA 1/TT031) by solid-state fermentation (SSF). In the present analysis, some of the important parameters such as PEG concentration, PEG molecular mass, salt concentration and buffer concentration were optimised through the response surface methodology (RSM). The optimum aqueous two-phase systems (ATPS) environment consisted of 13.8% (w/w) phosphate buffer, 9.2% (w/w) PEG-3000 and 3.3% (w/w) NaCl at 25°C. The RSM approach was proved to be the most suitable methodology for the recovery of desired enzymes. In this method, the enzyme partitioned into the top phase of the PEG-buffer-NaCl ATPS. Under this experimental environment, the purification factor was found to be 33.9, the partition coefficient was 4.0 and the yield was found to be 84.0% of lipase. Moreover, the experimental and predicted results were in considerable agreement, which established the reliability and validity of the proposed model. The ATPS methodology is proven to be effective for the primary recovery of lipase at a low cost with a large loading capacity and possibility of linear scale up. In addition to using the existing methodologies for improving enzyme production, the use of statistical optimisation of the constituents of phases through RSM continues to be the basic and practical method.
    Matched MeSH terms: Lipase/isolation & purification*
  13. Olusesan AT, Azura LK, Forghani B, Bakar FA, Mohamed AK, Radu S, et al.
    N Biotechnol, 2011 Oct;28(6):738-45.
    PMID: 21238617 DOI: 10.1016/j.nbt.2011.01.002
    Thermostable lipase produced by a genotypically identified extremophilic Bacillus subtilis NS 8 was purified 500-fold to homogeneity with a recovery of 16% by ultrafiltration, DEAE-Toyopearl 650M and Sephadex G-75 column. The purified enzyme showed a prominent single band with a molecular weight of 45 kDa. The optimum pH and temperature for activity of lipase were 7.0 and 60°C, respectively. The enzyme was stable in the pH range between 7.0 and 9.0 and temperature range between 40 and 70°C. It showed high stability with half-lives of 273.38 min at 60°C, 51.04 min at 70°C and 41.58 min at 80°C. The D-values at 60, 70 and 80°C were 788.70, 169.59 and 138.15 min, respectively. The enzyme's enthalpy, entropy and Gibb's free energy were in the range of 70.07-70.40 kJ mol(-1), -83.58 to -77.32 kJ mol(-1)K(-1) and 95.60-98.96 kJ mol(-1), respectively. Lipase activity was slightly enhanced when treated with Mg(2+) but there was no significant enhancement or inhibition of the activity with Ca(2+). However, other metal ions markedly inhibited its activity. Of all the natural vegetable oils tested, it had slightly higher hydrolytic activity on soybean oil compared to other oils. On TLC plate, the enzyme showed non-regioselective activity for triolein hydrolysis.
    Matched MeSH terms: Lipase/isolation & purification*
  14. Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB
    Protein J, 2014 Jun;33(3):296-307.
    PMID: 24777627 DOI: 10.1007/s10930-014-9560-3
    The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
    Matched MeSH terms: Lipase/isolation & purification
  15. Show PL, Tan CP, Shamsul Anuar M, Ariff A, Yusof YA, Chen SK, et al.
    Bioresour Technol, 2012 Jul;116:226-33.
    PMID: 22061444 DOI: 10.1016/j.biortech.2011.09.131
    An extractive fermentation technique was developed using a thermoseparating reagent to form a two-phase system for simultaneous cell cultivation and downstream processing of extracellular Burkholderia cepacia lipase. A 10% (w/w) solution of ethylene oxide-propylene oxide (EOPO) with a molecular mass of 3900 g/mol and pH 8.5, a 200 rpm speed, and 30 °C were selected as the optimal conditions for lipase production (55 U/ml). Repetitive batch fermentation was performed by continuous replacement of the top phase every 24h, which resulted in an average cell growth mass of 4.7 g/L for 10 extractive batches over 240 h. In scaling-up the process, a bench-scale bioreactor was tested under the conditions that had been optimized in flasks. The production rate and recovery yield were higher in the bioreactor compared to fermentation performed in flasks.
    Matched MeSH terms: Lipase/isolation & purification
  16. Ali MS, Yun CC, Chor AL, Rahman RN, Basri M, Salleh AB
    Protein J, 2012 Mar;31(3):229-37.
    PMID: 22350313 DOI: 10.1007/s10930-012-9395-8
    A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
    Matched MeSH terms: Lipase/isolation & purification*
  17. Eko Sukohidayat NH, Zarei M, Baharin BS, Manap MY
    Molecules, 2018 Jul 20;23(7).
    PMID: 30037038 DOI: 10.3390/molecules23071800
    Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30⁻60 °C and pH range of 6⁻11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na⁺, Mg2+, K⁺ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.
    Matched MeSH terms: Lipase/isolation & purification*
  18. Ganasen M, Yaacob N, Rahman RN, Leow AT, Basri M, Salleh AB, et al.
    Int J Biol Macromol, 2016 Nov;92:1266-1276.
    PMID: 27506122 DOI: 10.1016/j.ijbiomac.2016.06.095
    Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.
    Matched MeSH terms: Lipase/isolation & purification
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