Use of specific polyclonal antibodies to detect heterogeneous lipases from Geotrichum candidum.

Geotrichum candidum CMICC 335426 was previously shown to produce two lipases termed lipase A and lipase B, lipase B being highly specific for hydrolysis of esters of cis-delta 9 fatty acids. We now describe the isolation of polyclonal antibodies specific for lipase A and lipase B. These antibodies were used in Western blotting techniques to detect the appearance of the lipases during the course of the fermentation of G. candidum CMICC 335426. A and B were found to be produced simultaneously in the extracellular medium at the start of the growth phase. The two lipases were always present at similar levels in the medium. The specific antibodies were then used to detect the presence of A- and B-like lipases in crude lipase samples from other strains of G. candidum. The lipases were found at different levels in all these samples, and the specificities of the crude lipases varied significantly from one strain to another. Differences in specificity could therefore be explained by different levels of specific (B-type) and non-specific (A-type) lipases in the medium. This was verified by purifying A- and B-type lipases from the G. candidum strain ATCC 34614.     

Overexpression of lipase A and B of Geotrichum candidum in Pichia pastoris: High-level production and some properties of functional expressed lipase B

Lipase A and B from Geotrichum candidum CMICC 335426 were functionally expressed and secreted in Pichia pastoris. Lipase B exhibits a unique substrate specificity for long-chain cis-9 unsaturated triacylglycerols. Thus, using different cultivation conditions, the production of recombinant lipase B was optimised, yielding 200 000 Units per liter culture. After overexpression lipase B was purified by diafiltration (372 U/mg). The investigated enzyme properties were comparable to those reported for the native enzyme.     

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.     

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.     

Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity

Summary Three strains of Geotrichum candidum (ATCC 34614, NRRL Y-552 and NRRL Y-553) were examined for lipase production and activity. Variables including medium, pH, temperature, agitation rate and incubation time were examined to define the optimal culture conditions. Growth on oil in complex medium at 30°C, 300 rpm, and pH 7 produced maximal lipase activity. Fatty acid specificity of these strains and of two crude G. candidum enzyme preparations (lipase 26557 RP, Rhône Poulenc and lipase GC-4, Amano) was measured using equimolar mixtures of methyl or butyl esters of palmitic and oleic acids. The lipase from NRRL Y-553 and lipase 26557 RP displayed preferential specificity for hydrolyzing oleic acid esters, while the lipases from ATCC 34614, NRRL Y-552 and lipase GC-4 failed to discriminate between plamitic and oleic acids.     

The midgut trypsins of shrimp (Penaeus monodon). High efficiency toward native protein substrates including collagens

Two shrimp trypsins have been purified from the midguts of Penaeid shrimps by various chromatographies and HPLC. The molecular masses of them are 27 and 29 kDa, respectively. They show the typical specificity of trypsin for substrates and inhibitors, and their N-terminal amino-acid sequences are homologous to those of other trypsins. The shrimp enzymes are very acidic (pI less than or equal to 2.4), and show distinctively low Km for the synthetic amide substrates. They also hydrolyse various native proteins more efficiently than bovine trypsin in vitro. However, the anionic shrimp trypsins do not have special preference for basic protein substrates over the acidic one. Collagenolytic activity of the midgut extract was mainly due to serine proteases. The collagenolytic activity of the purified shrimp trypsin was confirmed by assays with either soluble or insoluble native type I collagens. In comparison with the other trypsins from the Crustacean decapods, the shrimp enzymes have four pairs of disulfide bonds, intermediary between the crayfish trypsin (three pairs) and the crab trypsin (five pairs), and are immunochemically different from them.     

Purification and characterization of extracellular lipases from Ophiostoma piliferum

Interest in lipases from microorganisms, animals, and plants has greatly increased in the past decade due to their applications in biotransformations and organic syntheses. We are reporting the purification and characterization of two lipases from the fungus, Ophiostoma piliferum, a saprophytic organism commonly found on wood. A major and a minor lipase have been co-purified by hydrophobic interaction chromatography on octyl sepharose FF, followed by ion exchange chromatography on Q sepharose FF. The lipases bound very tightly to octyl sepharose resulting in greater than 100-fold purification in this one step. The major lipase has a molecular weight of approximately 60 kDa, a pI of 3.79, and is glycosylated as determined by PAS staining. The minor lipase, which composes 10% of the total protein, has a pI of 3.6, and molecular weight of approximately 52 kDa and did not stain with the PAS reagent. Deglycosylation of the major lipase produced two proteins of lower molecular weight, a 55 kDa protein and a 52 kDa protein. The deglycosylated protein at 52 kDa co-migrates with the minor lipase on SDS-PAGE gels. N-terminal amino acid sequencing of the major and minor lipases indicated both lipases have the same N-termini and MALDI-TOF mass spectral analysis showed similar peptide patterns. Available data indicate that the lipases are derived from the same protein and appear to differ in their post-translational modification as evidenced by their pIs and molecular weight difference. The pH rate profile and thermal stability were determined for the purified O. piliferum lipase and were consistent with a mesophilic lipase. In aqueous solution, the lipases exhibited a higher rate of hydrolysis for p-nitrophenylbutyrate (C4) than for p-nitrophenylstearate (C18), which is an unexpected result.     

Esterase and lipase in camel tick Hyalomma dromedarii (Acari: Ixodidae) during embryogenesis

Esterase and lipase activity showed significant changes during embryogenesis of camel tick Hyalomma dromedarii. From the elution profile of chromatography on DEAE-cellulose, six forms of H. dromedarii esterase (El to EVI) can be distinguished. Esterase EIII was purified to homogeneity after chromatography on Sepharose 6B. The molecular mass of esterase EIII was 45 kDa for the native enzyme and represented a monomer of 45 kDa by SDS-PAGE. Esterase EIII had an acidic pI at 5.3. Lipase activity was detected in the same DEAE-cellulose peaks (LI to LVI) of H. dromedarii esterases. The highest lipase activity was exhibited by lipase LIII. Esterase EIII and lipase LIII were compared with respect to Michaelis constant, substrate specificity, temperature optimum, heat stability, pH optimum, effect of metal ions and inhibitors. This study suggests that H. dromedarii lipolytic enzymes may play a central role in the interconversion of lipovitellins during embryogenesis.     

Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.     

Separation and characterization of 61- and 57-kDa lipases from Geotrichum candidum ATCC 66592.

Two lipolytic proteins (61 and 57 kDa) present in a Sephadex G-100 fraction of extracellular lipase from Geotrichum candidum ATCC 66592 were separated using high-performance liquid chromatography. Crossed electrofocusing immunoelectrophoresis was used to demonstrate that the 61-kDa lipase fraction contained two forms of lipase with pI 4.5 and 4.7. However, when deglycosylated with endoglycosidase H, the two forms gained an identical pI, 4.6. The 57-kDa lipase fraction contained one form of lipase with pI close to 4.5. Although the 61- and 57-kDa lipases were immunologically identical, the substrate specificity differed. Thus, the 61-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was 60% of the initial velocity of hydrolysis of oleic acid methyl ester, whereas the 57-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was only 7% of the initial velocity of hydrolysis of oleic acid methyl ester.     

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.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

Purification and characterization of two distinct lipases from Geotrichum candidum

Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidum were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35-fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56,000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.     

Cellulases of Penicillium verruculosum

Nine major cellulolytic enzymes were isolated from a culture broth of a mutant strain of the fungus Penicillium verruculosum: five endo-1, 4-β-glucanases (EGs) having molecular masses 25, 33, 39, 52, and 70 kDa, and four cellobiohydrolases (CBHs: 50, 55, 60, and 66 kDa). Based on amino acid similarities of short sequenced fragments and peptide mass fingerprinting, the isolated enzymes were preliminary classified into different families of glycoside hydrolases: Cel5A (EG IIa, 39 kDa), Cel5B (EG IIb, 33 kDa), Cel6A (CBH II, two forms: 50 and 60 kDa), Cel7A (CBH I: 55 and 66 kDa), Cel7B (EG I: 52 and 70 kDa). The 25 kDa enzyme was identical to the previously isolated Cel12A (EG III). The family assignment was further confirmed by the studies of the substrate specificity of the purified enzymes. High-molecular-weight forms of the Cel6A, Cel7A, and Cel7B were found to possess a cellulose-binding module (CBM), while the catalytically active low-molecular-weight forms of the enzymes, as well as other cellulases, lacked the CBM. Properties of the isolated enzymes, such as substrate specificity toward different polysaccharides and synthetic glycosides, effect of pH and temperature on the enzyme activity and stability, adsorption on Avicel cellulose and kinetics of its hydrolysis, were investigated.     

Purification and characterization of isoforms of beta-galactosidases in mung bean seedlings

Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.     

Purification and properties of the lipases from Rhodotorula pilimanae Hedrick and Burke

Summary The Rhodotorula pilimanae CBS 5804 strain secretes into the culture medium two lipases: their pH optima are 4 and 7. The two lipases were purified by precipitation with acetone followed by chromatography on SP-Sephadex C50 and Sephadex G200. The purification factors achieved in comparison with the supernatant culture were x74 for lipase I and x90 for lipase II. The molecular weights were estimated at 172,800 and 21,400 for lipase I and lipase II, respectively. Their activities are optimal between 45°C and 55°C. The activation energies were 5.9 kcal·mole^-1 for lipase I and 12.4 kcal·mole^-1 for lipase II. The inactivation energies were about 21.9 and 17.7 kcal·mole^-1 for lipase I and lipase II, respectively. The enzymes are slightly inhibited by Cu²⁺, Co²⁺, Hg²⁺, Mn²⁺, N-acetylacetone, acetic acid and sodium lauryl sulphate. EDTA did not affect their enzymatic activity. These two lipases are secreted in the culture media in the absence of inducer; their biosynthesis is not inhibited by glucose. These lipases hydrolyse primarily the 1-(or 3-)position of all triglycerides tested.     

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.     

Detection and partial purification of two lipases fromCandida rugosa

Summary Two distinct lipases produced byCanadida rugosa were identified and separated by a high resolution anion-exchange column (Mono Q) after an ethanol extraction of the crude lipase. From this Mono Q column, lipase I eluted at 0.05 M NaCl whereas lipase II eluted at 0.15 M NaCl. The less anionic nature of lipase I was also confirmed by native polyacrylamide gel electrophoresis as well as isoelectrophoresis. Both proteins have an apparent molecular weight of 58,000 by SDS-PAGE. The isoelectric points of lipase I and II are 5.6 and 5.8 respectively.     

Properties of hemicellulases of the enzyme complex from Trichoderma longibrachiatum

Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained previously based on the strain Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pi 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, beta-xylosidase (beta-XYL; 80 kDa, pI 4.5) and alpha-L-arabinofuranosidase I (alpha-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of the preparations Celloviridin G20x and Xy-beten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (alpha-L-AF I or beta-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with alpha-L-AF I.