Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices.

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Characterization and mutation anaylsis of a cold-active bacterial hormone-sensitive lipase from Salinisphaera sp. P7-4.

In mammals, hormone sensitive lipase (EC 3.1.1.79, HSL) catalyzes the hydrolysis of triacylglycerols as well as the modifications of a broad range of hydrophobic substrates containing ester linkages. HSLs are composed of an N-terminal ligand-binding domain and a C-terminal catalytic domain. Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the C-terminal domain of mammalian HSLs, have a catalytic triad composed of Ser, His, and Asp. Here, a novel cold-active hormone-sensitive lipase (SaHSL) from Salinisphaera sp. P7-4 was identified, functionally characterized, and subjected to site-directed mutations. The enzymatic properties of SaHSL were investigated using several biochemical and biophysical methods. Interestingly, SaHSL exhibited the ability to act on a broad range of substrates including glyceryl tributyrate and glucose pentaacetate. Homology modeling and site-directed mutagenesis indicated that hydrophobic residues (Leu156, Phe164, and Val204) around the substrate-binding pocket were involved in substrate recognition. In addition, highly conserved amino acids (Glu201, Arg207, Leu208, and Asp227) in the regulatory regions were found to be responsible for substrate specificity, thermostability, and enantioselectivity. In summary, this work provides new insights into the understanding of the C-terminal domain of HSL family and evidence that SaHSL can be used in a wide range of industrial applications.

The first description of a hormone-sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealed Bjerkandera adusta BaEstB as a novel esterase.

The heterologous expression and characterization of a Hormone-Sensitive Lipases (HSL) esterase (BaEstB) from the Basidiomycete fungus Bjerkandera adusta is reported for the first time. According to structural analysis, amino acid similarities and conservation of particular motifs, it was established that this enzyme belongs to the (HSL) family. The cDNA sequence consisted of 969 nucleotides, while the gene comprised 1133, including three introns of 57, 50, and 57 nucleotides. Through three-dimensional modeling and phylogenetic analysis, we conclude that BaEstB is an ortholog of the previously described RmEstB-HSL from the phylogenetically distant fungus Rhizomucor miehei. The purified BaEstB was characterized in terms of its specificity for the hydrolysis of different acyl substrates confirming its low lipolytic activity and a noticeable esterase activity. The biochemical characterization of BaEstB, the DLS analysis and the kinetic parameters determination revealed this enzyme as a true esterase, preferentially found in a dimeric state, displaying activity under alkaline conditions and relative low temperature (pH = 10, 20°C). Our data suggest that BaEstB is more active on substrates with short acyl chains and bulky aromatic moieties. Phylogenetic data allow us to suggest that a number of fungal hypothetical proteins could belong to the HSL family.

Mycobacterium tuberculosis rv1400c encodes functional lipase/esterase.

Lipases catalyze the hydrolysis of triglycerides (TAG). Open reading frames (ORF) predicted to encode enzymes involved in fatty acids breakdown are abundant in Mycobacterium tuberculosis genome. To define the function of M. tuberculosis rv1400c (LipI), a putative Hormone Sensitive Lipase (HSL) subfamily ORF, the rv1400c was cloned, expressed and purified in Escherichia coli as fusion protein. The purified LipI preferred short carbon chain substrates with an optimal activity at 37 °C/pH 8.0 and stable between pH 6.0 to 9.0. Its specific activity was calculated to 35.71 U/mg with pNP-butyrate as a preferred substrate. SDS, CTAB and Zn2+ can inhibit this enzyme. The conserved residues Ser165 and His291 were shown to be important for the catalysis activity of Rv1400c by site-directed mutagenesis. The biochemical and genetical data showed M. tuberculosis LipI might be a good candidate catalyst for polyunsaturated fatty acids.

Properties, structure, and applications of microbial sterol esterases.

According to their substrate preferences, carboxylic ester hydrolases are organized in smaller clusters. Among them, sterol esterases (EC 3.1.1.13), also known as cholesterol esterases, act on fatty acid esters of cholesterol and other sterols in aqueous media, and are also able to catalyze synthesis by esterification or transesterification in the presence of organic solvents. Mammalian cholesterol esterases are intracellular enzymes that have been extensively studied since they are essential in lipid metabolism and cholesterol absorption, and the natural role of some microbial sterol esterases is supposed to be similar. However, besides these intracellular enzymes, a number of microbes produce extracellular sterol esterases, which show broad stability, selectivity, or wide substrate specificity, making them interesting for the industry. In spite of this, there is little information about microbial sterol esterases, and only a small amount of them have been characterized. Some of the most commercially exploited cholesterol esterases are produced by Pseudomonas species and by Candida rugosa, although in the last case they are usually described and named as "high substrate versatility lipases." From a structural point of view, most of them belong to the α/ß-hydrolase superfamily and have a conserved "catalytic triad" formed by His, an acidic amino acid and a Ser residue that is located in a highly conserved GXSXG sequence. In this review, the information available on microbial sterol esterases has been gathered, taking into account their origin, production and purification, heterologous expression, structure, stability, or substrate specificity, which are the main properties that make them attractive for different applications. Moreover, a comprehensive phylogenetic analysis on available sequences of cholesterol esterases has been done, including putative sequences deduced from public genomes.

Expression and functional characterization of human lysosomal acid lipase gene (LIPA) mutation responsible for cholesteryl ester storage disease (CESD) phenotype.

Lysosomal acid lipase (LAL) is a serine hydrolase which hydrolyzes cholesteryl ester and triglycerides delivered to the lysosomes into free cholesterol and free fatty acids. Mutations in the LAL gene (LIPA) result in accumulation of triglycerides and cholesterol esters in various tissues of the body, leading to pathological conditions such as Wolman's disease (WD) and cholesteryl ester storage disease (CESD). CESD patients homozygous for His295Tyr (H295Y) mutation have less than 5% of normal LAL activity. To shed light on the molecular basis for this loss-of-function phenotype, we have generated the recombinant H295Y enzyme and studied its biophysical and biochemical properties. No significant differences were observed in the expression levels or glycosylation patterns between the mutant and the wild type LAL. However, the H295Y mutant displayed only residual enzymatic activity (<5%) compared to the wild type. While wild type LAL is mostly a monomer at pH 5.0, the vast majority H295Y exists as a high molecular soluble aggregate. Besides, the H295Y mutant has a 20°C lower melting temperature compared to the wild type. Transient expression studies in WD fibroblasts showed that mutation of His295 to other amino acids resulted in a significant loss of enzymatic activity. A homology model of LAL revealed that His295 is located on an α-helix of the cap domain and could be important for tethering it to its core domain. The observed loss-of-function phenotype in CESD patients might arise from a combination of protein destabilization and the shift to a non-functional soluble aggregate.

The Chlamydia trachomatis CT149 protein exhibits esterase activity in vitro and catalyzes cholesteryl ester hydrolysis when expressed in HeLa cells.

Chlamydia, like other intracellular bacteria, are auxotrophic for a variety of essential metabolites and obtain cholesterol and fatty acids from their eukaryotic host cell, however not many Chlamydia-specific enzymes have been identified that are involved in lipid metabolism. In silico analysis of one candidate Chlamydia trachomatis enzyme, annotated as a conserved putative hydrolase (CT149), identified two lipase/esterase GXSXG motifs, and a potential cholesterol recognition/interaction amino acid consensus (CRAC) sequence. His-tag purified recombinant CT149 exhibited ester hydrolysis activity in a nitrophenyl acetate-based cell-free assay system. When cholesteryl linoleate was used as substrate, ester hydrolysis occurred and production of cholesterol was detected by high performance liquid chromatography. Exogenous expression of transfected CT149 in HeLa cells resulted in a significant decrease of cytoplasmic cholesteryl esters within 48 h. These results demonstrate that CT149 has cholesterol esterase activity and is likely to contribute to the hydrolysis of eukaryotic cholesteryl esters during intracellular chlamydial growth.

Thermal stabilization of psychrophilic enzymes: a case study of the cold-active hormone-sensitive lipase from Psychrobacter sp. TA144.

Cold-adapted enzymes possess high specific activity at low and moderate temperatures with respect to their mesophilic and thermophilic homologs; it is accepted that they have a less rigid and more flexible structure in the region surrounding the active site. However, the low stability of such molecules could represent the main barrier for their application in some industrial bioprocesses. The aim of this article was to investigate the ability of the naturally occurring osmolytes to increase the thermal stability and the specific activity of the cold-active lipase from Psychrobacter sp. TA144 (PsyHSL), which belongs to the hormone-sensitive lipase group. The effect of trimethylamine N-oxide (TMAO), betaine, and L-proline addition on the activity and thermal stability of PsyHSL was investigated by means of biochemical and biophysical techniques. It turned out that in the presence of 3 M TMAO, the enzyme specific activity enhanced up to 250% at 50°C, while the addition of 1 M TMAO increased the thermostability fivefold at 45°C. Our experiments demonstrated that, even in the case of a psychrophilic enzyme, osmoprotectants, particularly TMAO, addition may be considered an efficient strategy to improve the protein thermal stability and specific activity at higher temperatures.

Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity.

Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes.

Rainbow trout (Oncorhynchus mykiss) possess two hormone-sensitive lipase-encoding mRNAs that are differentially expressed and independently regulated by nutritional state.

Teleost fish store lipids among several tissues primarily as triacylglycerol (TG). Upon metabolic demand, stored TGs are hydrolyzed by hormone-sensitive lipase (HSL). In this study, two distinct cDNAs encoding HSL were isolated, cloned, and sequenced from adipose tissue of rainbow trout. The full-length cDNAs, designated HSL1 and HSL2, were 2562-bp and 2887-bp in length, respectively, and share 82% nucleotide identity. Phylogenetic analysis suggests that the two HSLs derive from paralogous genes that may have arisen during a teleost-specific genome duplication event. Quantitative real-time PCR revealed that HSL1 and HSL2 were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. HSL1 and HSL2 mRNAs were detected in the brain, spleen, pancreas, kidney, gill, intestine, heart, and white muscle, but were most abundant in the red muscle, liver, and adipose tissue. HSL1 mRNA was more abundant than HSL2 mRNA in the adipose tissue, whereas HSL2 mRNA was more abundant than HSL1 mRNA in the liver. Short term fasting (4 weeks) increased HSL1 and HSL2 mRNA expression in the adipose tissue, but only HSL1 mRNA levels increased in the liver and the red muscle. During a prolonged fast (6 weeks), there was continued elevation of HSL1 and HSL2 mRNA levels in the liver and muscle; HSL mRNA expression in mesenteric fat declined, coincident with depletion of mesenteric fat mass. Refeeding fish reduced HSL expression to levels seen in continuously fed fish. These findings indicate that the pattern of HSL expression is consistent with the diverse lipid storage pattern of fish and suggest that distinct mechanisms serve to regulate differential expression of the two HSLs in tissues and during a progressive fast.

Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area.

Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL. In this study, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than unphosphorylated HSL. Taken together, our results show that HSL increases its hydrophobic nature upon phosphorylation by PKA. This suggests that PKA phosphorylation induces a conformational change that increases the exposed hydrophobic surface and thereby facilitates binding of HSL to the lipid substrate.

Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase.

Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 A using synchrotron radiation. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 A, beta = 97.1 degrees.

Novel family of cholesterol esterases produced by actinomycetes bacteria.

Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly-Xaa-Ser-Xaa-Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.

Novel cholesterol esterase secreted by Streptomyces persists during aqueous long-term storage.

We isolated a moderate thermophilic actinomycete, Streptomyces sp. X9, from soil and purified cholesterol esterase (CHE) from the culture medium to homogeneity. The molecular masses of the purified CHE estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration chromatography were 23.6 and 163 kDa, respectively, indicating that the enzyme assumes an oligomeric form. Heavy metals such as Hg2+ and Ag+ similarly inhibited the activity of the CHE in the same manner as those of other bacterial CHEs. The activity of Streptomyces sp. X9 CHE was susceptible to dithiothreitol, beta-mercaptoethanol and p-chloromercuribenzoate, but resistant to phenylmethylsulfonyl fluoride, unlike those of other bacterial CHEs. The purified CHE could utilize both cholesteryl and p-nitrophenyl (pNP) esters of fatty acids as substrates. Steady-state kinetics revealed respective Km values for cholesteryl myristate and pNP-myristate of 0.34 and 1.1 mM, indicating that the cholesteryl residue is important for catalysis. We also found that the Km for the pNP esters are dependent on the chain length of the substrate fatty acid residues. These results indicate that the novel CHE specifically hydrolyzes substrates by recognizing both cholesteryl and fatty acid moieties. The enzyme was stable during long-term aqueous storage at room temperature, indicating its potential application as a diagnostic reagent.

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200.

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5-12 and active at pH 5.5-6, showing optimal activity at pH 7.0 at 45 degrees C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5-20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.

Characterization of the C-terminal region of molecular forms of human milk bile salt-stimulated lipase.

UNLABELLED: Bile salt-stimulated lipase (BSSL) in human milk exists in multiple molecular forms and it has been shown that approximately one-third of lactating mothers secrete two forms. AIM: to determine the structural features of BSSL that may give rise to this heterogeneity. METHODS: Oligosaccharides present in the proline-rich region in the C-terminus of BSSL were investigated using deglycosylating enzymes and lectin affinity probing to determine the origin of the multiple molecular forms. RESULTS: It was found that the variability in the molecular mass of BSSL is due predominantly to glycosylation. The molecular forms contain similar sugar chains; all forms possess the core disaccharide Galbeta1-3GalNAc and beta-D-galactose, fucose linked at alpha1-6 and sialic acid linkage alpha2-3 to galactose. CONCLUSION: The molecular mass difference in the BSSL molecular forms cannot be attributed to the type of carbohydrate moiety in the sugar chains of the N- and O-linked sites suggesting that the differences arise from the extent or quantity of glycosylation. The oligosaccharides in the C-terminal region contain Lewis x and b and, less prominently, Lewis a antigenic structures. Owing to the presence of these blood-group-related antigenic determinants, the C-terminal region of BSSL may have an adhesive function in cell-cell interactions.

Production, isolation and characterization of a sterol esterase from Ophiostoma piceae.

We studied extracellular sterol esterase production by the ascomycete Ophiostoma piceae in liquid culture. Esterase activity was found in low levels in glucose medium but it was strongly induced by olive oil. An esterase was purified from the 0.5% olive oil-supplemented cultures using ultrafiltration followed by a single chromatographic step on a hydrophobic interaction column. The enzyme was a glycoprotein with 8% N-linked carbohydrate content, a molecular mass by SDS/PAGE around 56.5 kDa and an isoelectric point of 3.3. Its N-terminal sequence was TTVNVKYPEGEVV. Substrate specificity studies showed that the O. piceae esterase hydrolyzes p-nitrophenol esters, tributyrin, triolein and different cholesterol esters. Both affinity (Km) and catalytic constant (k(cat)) were positively affected by the length of the fatty acid esterifying glycerol and cholesterol. The presence of double bonds in the acyl chain increased the enzyme efficiency, although it affected the k(cat) values rather than the Km on the cholesterol esters. The O. piceae enzyme showed no interfacial activation. This enzyme could have biotechnological applications in paper manufacturing since it efficiently hydrolyzes both triglycerides and sterol esters, which form pitch deposits during manufacturing of softwood and hardwood paper pulps, respectively.

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa, with its application to cleaning lipid-stained contact lenses.

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.