Cloning and sequence analysis of cDNA encoding Rhizopus niveus lipase.

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.     

Cloning and Sequence Analysis of cDNA encoding Rhizopus niveus Lipase

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.     

Purification and substrate specificity of Staphylococcus hyicus lipase

The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.     

Human hepatoma (HepG2) cells secrete a single 65 K dalton triglyceride lipase immunologically identical to postheparin plasma hepatic lipase

A triacylglycerol lipase was isolated from the culture medium of HepG2 human hepatoma cells and its properties were compared to hepatic triglyceride lipase (H-TGL) from human postheparin plasma. The HepG2 cell enzyme bound to heparin-Sepharose, was eluted with 1 M NaCl and was not inhibited by 1 M salt. Western-blotting of the fractions from the heparin-Sepharose column with a monoclonal antibody prepared against postheparin plasma H-TGL and which binds to an epitope in the carboxyl-terminus of H-TGL gave a single immunoreactive protein band of 65 kDa. This finding of immunochemical identity was confirmed with polyclonal antibodies prepared against synthetic peptides of H-TGL corresponding to amino acid residues 82-94 near the amino-terminus and residues 468-477, the carboxyl-terminus of the enzyme. We conclude that HepG2 cells secrete a single triacylglycerol lipase with molecular weight properties and immunological characteristics identical to post-heparin plasma H-TGL.     

Characterization of turkey pancreatic lipase

Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 degrees C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 degrees C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 degrees C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (beta-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.     

Isolation and characterization of an extracellular lipase from Mucor sp strain isolated from palm fruit

During our screening of lipolytic fungus which may play a role in the acidification of palm oil, we have recently isolated a Mucor sp strain. Culture conditions were optimized and the highest lipase production amounting to 57 U/ml was achieved after 6 days of cultivation. The extracellular lipase was purified 1050-fold by ammonium sulfate precipitation, carboxymethyl–sephadex chromatography and Sephadex G75 gel filtration to a final specific activity of 6600 IU/mg. The molecular weight of the homogenous lipase was determined about 42 kDa by gel filtration and SDS–polyacrylamide gel electrophoresis. The purified lipase was determined as a glycoprotein with a pI of 6.2. The Nt sequence was determined as AspGluIleGluThrValGlyXPheThrMetAspLeuProProAsnProPro and showed no homology with the sequences of the known lipases suggesting that the enzyme may be a new lipase. The purified lipase hydrolyzed both synthetic and natural triglycerides with the optimal activity recorded on trioctanoin and sunflower oil, respectively. Its activity was strongly inhibited by Triton X-100 and SDS. Metal ions such as Fe³⁺, Fe²⁺ and Hg²⁺ also decreased the lipase activity.     

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

Abstract

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7?days at 25?°C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ～50?kDa and ～54?kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40?°C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25?°C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10?°C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation.     

Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells

The processing and secretion of newly synthesized hepatic lipase was characterized in FU5AH rat hepatoma cells. Pulse-chase experiments revealed two immunoreactive species with apparent molecular weights of 55,400 and 57,600. The 55.4 kDa species was detectable only in cell extracts, whereas the 57.6 kDa species was present in both cell extracts and media. Following a 5 min pulse with L-[35S]methionine and a 10 min chase, these two species represented only 0.003% of the total labelled protein. Quantitation of the 55.4 kDa and 57.6 kDa species in a chase time course taken together with their respective sensitivity and resistance to digestion with endo-beta-N-acetylglucosaminidase H indicates that the 55.4 kDa species is a high mannose precursor to the mature 57.6 kDa enzyme which contains only complex N-linked oligosaccharides. From a time course of endo-beta-N-acetylglucosaminidase H digestion, it was determined that hepatic lipase contains a minimum of two N-linked oligosaccharides. Treatment of the 55.4 kDa species with endo-beta-N-acetylglucosaminidase H yields a protein with a kDa value similar to that observed after treatment of the mature secreted enzyme with endo-beta-N-acetylglucosaminidase F or trifluoromethanesulfonic acid. Therefore, processing of N-linked oligosaccharides is probably the only post-translational modification responsible for the observed change in the apparent molecular weight of hepatic lipase. The half-residence times of hepatic lipase in the endoplasmic reticulum-cis Golgi region and in the cell were estimated at 34 min and 57 min, respectively. Newly synthesized hepatic lipase in Fu5AH cells is secreted constitutively and is not stored in an intracellular pool. Finally, little of the newly synthesized enzyme is degraded during the course of a 1 h chase.     

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.     

Molecular size of bovine lipoprotein lipase as determined by radiation inactivation

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.     

Purification and characterization of an intracellular lipase from pleopods of whiteleg shrimp (Litopenaeus vannamei)

An intracellular lipase present in the whiteleg shrimp Litopenaeus vannamei was detected in pleopods. The lipase from pleopods was purified and characterized by biochemical and kinetic parameters. Purified intracellular lipase has a molecular mass of 196kDa, the polypeptide is assembled by two monomers, 95.26 and 63.36kDa. The enzyme lacks glycosylation, and it has an isoelectric point of 5.0. The enzyme showed the highest activity at a temperature range of 30-40°C at pH 8.0-10.0. Activity was completely inhibited by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate, suggesting that the intracellular lipase is a serine lipase. The lipase hydrolyzes short and long-chain triacylglycerides, as well as naphthol derivatives at comparable rates in contrast to other sources of lipases. Specific activity of 930U mg(-1) and 416.56U mg(-1) was measured using triolein and tristearin at pH 8.0 at 30°C as substrates, respectively. The lipase showed a K(M,app) of 41.03mM and k(cat)/K(M,app) ratio of 4.88 using MUF-butyrate as the substrate. The intracellular lipase described for shrimp has a potential role in hydrolysis of triacylglycerides stored as fat body, as has been shown in humans.     

Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters

A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083 bp encoding a protein of 269 Aa with an estimated molecular mass of 30 kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.     

Variations among four commercial lipases from Chromobacterium viscosum in ester synthesis

The properties of four commercial lipases from Chromobacterium viscosum, CvL 1–4, in ester synthesis were investigated. Three lipases showed a high synthetic activity in esterification, with conversions of oleic acid as high as 86–95% in 24 h, whereas one (CvL 1) gave a poor result of only 11% with the same quantity of 9 mg crude lipase preparation. The elution profiles of the four lipases from Sephacryl S-100 HR differed and SDS-PAGE suggested that while CvL 1 lipase had two equivalent protein bands of molecular size 33 and 27 kDa, respectively, the other three lipases showed only one main protein band of 33 kDa. Isoelectric focusing revealed that all of the lipases contained several isoforms, but the proportions of the isoforms varied. Furthermore, both aggregated and single lipase forms obtained after gel filtration were able to catalyse ester synthesis, but the two lipases from CvL 1 showed lower synthetic activities than the others.     

Purification and characterization of an extracellular lipase of Galactomyces geotrichum

Summary Galactomyces geotrichum secretes an extracellular lipase with a relative molecular weight of 57±2 kDa in the unglycosylated state and 62±2 kDa when glycosylated. The enzyme has optimal activity at pH 7.75 and 30°C and has a pI of 5.2. The specificity of the lipase is similar to lipase A of Geotrichum candidum, but the amino acid composition of the lipases are different.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Human adipose tissue hormone-sensitive lipase: identification and comparison with other species

The mRNA for human hormone-sensitive lipase (HSL) was identified using Northern blot analysis and a cDNA-probe for rat HSL. As in the rat, human adipose tissue expresses a single mRNA species of 3.3 kb. Using Western blotting with a polyclonal rabbit antibody towards rat adipose tissue HSL, the corresponding enzyme in human adipose tissue was identified with an apparent 88 kDa polypeptide, thus slightly larger than the rat and bovine 84 kDa, and the mouse and guinea-pig 82 kDa species. Additional evidence for the identification was provided by the inhibition of HSL diacylglycerol lipase activity by the anti-rat HSL antibody, and by NaF, DFP and Hg2+, known inhibitors of HSL. The concentration of the enzyme, as reflected by its activity per g tissue and the specific activity was about two thirds of that in the rat adipose tissue (200 g rats). The identification of the human enzyme protein made it possible to directly demonstrate its phosphorylation by cAMP-dependent protein kinase, thus extending the previous report regarding activation of the lipase with this kinase and ATP-Mg2+ in human adipose tissue extracts (Khoo, J.C., Aquino, A.A. and Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131).     

High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM

The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0 mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg⁻¹), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65 °C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50 °C for more than 150 min.     

Structural modulation of salt-resistant rat-liver lipase alters the relative phospholipase and triacylglycerol hydrolase activities

This paper demonstrates that structural modification of the heparin-releasable salt-resistant lipase of rat liver (liver lipase) alters its relative capacity to hydrolyze phospholipid and triacylglycerol emulsions. Enzymatic activities were modified by immunoinhibition and proteolysis and by selective amino acid agents. Binding of three different monoclonal antibodies resulted in a lower extent of inhibition of phospholipase than of triacylglycerol hydrolase activity. Degradation of the enzyme by trypsin under mild conditions led to a decrease of both enzyme activities in a different way. Triacylglycerol hydrolase activity was less affected than the phospholipase activity. Visualization of the proteolysis of the purified enzyme by immunoblotting revealed the actual breakdown of a 58 kDa protein into a 53 kDa protein band and subsequently in a 48 kDa one. Incubation of the purified enzyme by N-tosyl-L-phenylethylchloromethyl ketone (acting on cysteine or histidine) or N-ethylmaleimide (a sulfhydryl reagent) did not influence either enzyme activity. On the other hand, after the selective modification of lysine residue(s) by phenylisothiocyanate, the phospholipase A1 activity was stimulated by 68%, whereas the triacylglycerol hydrolase activity was completely lost. The role of a lysine residue(s) in the activity of the enzyme towards phospholipid and triacylglycerol emulsions is discussed.     

Synthetic activity enhancement of membrane-bound lipase from <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> by pretreatment with isooctane

The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase™ firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4°C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.