Now showing items 21-40 of 4590

    • A NOVEL CHOLESTEROL ESTER HYDROLASE FROM PSEUDOMONASSPEC

      Hanke, Christian; Schumacher, Günther; Boehringer Mannheim GmbH, Research Center Penzberg, Nonnenwald 2, D-8122 Penzberg, F.R.G. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      The genefor a cholesterol ester hydrolase (CE) [E.C.3.1.1.13] of a Pseudomonas spec. strain was cloned in pBR322 by screening a plasmid library with synthetic oligonucleotides derived from tryptic fragments ofthe purified enzyme. Subsequent sequence analysis of two overlapping clones showed a large open reading frame of 0.9 kb predicting a protein with a molecular weight of 30.700 kD. It contains a consensus-like signal peptide (SP) of 24 amino acids and a conserved sequence around the putative active center which is homologousto that of some other prokaryotic as well as eukaryotic esterases. No enzymatic activity as well as no immunological reaction could be detected in E. coli cells harbouring the CE-gene with 1.5 kb of its own upstream sequence on high copy plasmids. In order to improve gene expression the CE-gene was cloned behind strong E. coli promoters, the translation initiation region was optimized and several modifications of the SP-sequence were tested. The amount of CE after induction was 5 to 10 % oftotal protein but all of the enzyme was present in inclusion bodies. Again no enzymatic activity could be detected neither inside the E. coli cells nor in the culture medium. As the production of an enzymatically active CE in E. coli was not feasible, the whole expression cassette including promoter, SP and CE-gene was cloned into mobilizing vector pBT306-1, a vector system compatible for gram-negative organisms. Transconjugants with this vector in a CEnegative Pseudomonas strain showed fivefold higher CE-activity than the original strain. Using the same plasmid no activity could be observed in Pseudomonas putida strain 2440. As CE is a lipoprotein we assumethat specific lipids are necessary for enzymatic activity.
    • PRODUCTION OF LIPASE BY SPOROTRICHUM (CHRYSOSPORIUM) THERMOPHILE APINIS IMMOBILIZED IN ALGINATE AND POROUS GLASS BEADS

      Johri, B. N.; Alurralde, J. L.; Kressdorf, B.; Klein, J. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Spores and mycelia of Sporotrichum (Chrysosporium) thermophile Apinis were immobilized in alginate and porous glass beads for lipase production in batch replacement culture and a column reactor. Young mycelia immobilized in alginate produced higher levels of lipase than free cells or spores immobilized in alginate or glass beads. Alginate beads were successfully used in batch culture for more than 300 h without any signs of breakage and little cell washout. Porous glass beads proved a better carrier for spore derived inocula but activity levels were low. Crude lipase of S. thermophile was stable upto 90°C both in free and immobilized state and thus requires further characterization.
    • COMPARATIVE ANALYSIS OF LIPASES IN VIEW OF PROTEIN DESIGN

      Kordel, Marianne; Menge, Ulrich; Morelle, Gilles; Erdmann, Helmut; Schmid, Rolf D.; GBF, Gesellschaft fiir Biotechnologische Forschung mbH, Dept. of Enzyme Technology, Mascheroder Weg 1, D-3300 Braunschweig (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      The consensus sequences containing the active serine residue of 21 lipases were examined for structural properties by secondary structure prediction and hydrophobicity plots. Mostof the G-X-S-X-G peptides were found to form a turn structure andto be buried,i.e. inaccessible to water. The structural characters of a second serine containing consensus peptide describedin literature were compared to those of the G-X-S-X-G sequences. In addition, we investigated,if a correlation of the structural features of these peptides to the substrate specificity (regio specificity and fatty acid specificity) can be found.
    • CLONING, SEQUENCING AND REGULATION OF THE LIPASE GENE FROM PSEUDOMONASSP. M-12-33

      Nakanishi, Yuji; Watanabe, Hidemi; Washizu, Kinya; Narahashi, Yoshiko; Kurono, Yoshiaki; Amano Pharmaceutical Co., Ltd., Kunotsubo, Nishiharu-cho, Nishikasugai-gun, Aichi, Japan; The Institute of Physical and Chemical Research, Hirosawa, Wako-shi, Saitama, Japan (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Thelipase gene from Pseudomonas sp. M-12-33 was cloned. The cloned DNA was 2.9 Kb in length, which was essential for production of the lipase and contained two open reading frames, lipA and lipX. The lipA gene was supposed to comprise 1092 nucleotides and give a preproprotein of 364 aminoacids which was then processed to a mature lipase protein of 320 amino acids. On the other hand, the lipX gene was assumedto code a protein of 344 amino acids concerned with someregulation of the enzyme production.
    • CHARACTERIZATION AND OVER-EXPRESSION OF A CLONED PSEUDOMONAS LIPASE GENE

      Hom, S. S. M.; Scott, E. M.; Atchison, R. E.; Picataggio, S.; Mielenz, J. R.; Henkel Research Corporation. Santa Rosa, California, U.S.A. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Triacylglycerollipases (E.C.3.1.1.3) are ubiquitous amongst microbes, plants, and animals and catalyse the hydrolysis of ester bonds of triglycerides to form glycerol and fatty acids. On the other hand, lipases catalyse the reverse esterification reaction in low water conditions, forming glycerides from glycerol and free fatty acids. Moreover, some lipases catalyse transesterification through the exchange ofesterified fatty acids with free fatty acids. These reactions can either be selective toward a specific ester bond in triglycerides or completely non-specific. Besides positional specificity, some lipases demonstrate selectivity toward a particular fatty acid substrate. Since lipases have wide versatility, considerable interest in the industrial uses of lipases has recently developed. Industrial applications of lipases include enzymatic fat splitting, accelerated cheese ripening, production of cocoa butter substitutes, and as a detergent additive. Also, the enanatioselectivity of certain lipases offers an attractive opportunity for the preparation of chiral intermediates for pharmaceutical syntheses. Thelipase from Pseudomonassp. ATCC 21808is of particular interest for potential industrial applications becauseofits high temperature optimum for enzymatic activity (65 deg. C), its thermostability (no activity loss after one week at 50 deg C in a phosphate buffer), and activity over a broad pH range (4-9).
    • CHARACTERISTICS OF A NEW LIPASE FROM A Thermus sp BACTERIUM

      Silva, A. M. G. M.; Cabral, J. M.; Costa, M. S.; Garcia, F. A. P.; Chemical Engineering Department, Univ. of Coimbra, P-3049 Coimbra Codex; Biochemical Engineering Laboratory, I.S.T., P-1096 Lisboa Codex; Department of Zoology, University of Coimbra, P-3049 Coimbra Codex (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Thirty strains of the genus Thermus, isolated from hot sprins in Portugal, were screened for the secrection of lipases. In the end, the strain LFF1 received our attention for further characterization. We report here some kinetic properties of the crude extracellular extract when used in a reversed micellar system of AOT in isooctane for the hydrolysis of triolein. In common with other lipases, this extract showed maxima at pH 7 and temperatures in the range 40-50 °C, but significant residual activities were also observed at higher (up to 80 °C) and lower (downto -3.5 °C) temperatures. The hydrolysis oftriolein in the micellar system followed an apparent Michaelis- Menten kinetic mechanism with K,,(app)=7.1%(v/v) and V_.„(app)=55.5 mole/(ml.h.mg protein). The specific activity of the extract decreased continuously with increasing concentrations of protein encapsulated in the reversed micelles. The aqueousextract lost less than 9% of its activity when stored at 4 °C for almost 3 months.
    • USTILAGO MAYDIS LIPOLYTIC ENZYMES: CHARACTERIZATION AND PARTIAL PURIFICATION

      Lang, S.; Katsiwela, E.; Kleppe, F.; Wagner, Fritz; Institute of Biochemistry and Biotechnology, Technical University, D-3300 Braunschweig, West-Germany (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      The crude lipase preparation of Ustilago maydis ATCC 14826 (after growth on coconut oil) was studied with respect to hydrolysis and esterification potential, resp., as well as to purification of lipolytic enzymes. Concerning substrate specifity (hydrolysis) among triglycerides short chain substances were cleaved to an higher extent than long chain or unsaturated compounds. Obvious inhibition of lipase activity was observed when additional amounts of linoleic or linolenic acid were used during hydrolysis of coconut oil. After Amberlite XAD-2 immobilization and transfer into n-hexane the wax ester synthesis potential was confirmed. The purification of crude lipase preparation by chromatographic methods led to two lipolytic enzymes.
    • MEDIA ENGINEERING IN THE CATALYSIS BY LIPASES

      Otero, Cristina; Pastor, Eitel; Rua, Maria L.; Ballesteros, Antonio; Unidad de Biocatalisis, Instituto de Catdalisis, c.S.1I.C., 28006 Madrid, Spain (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Two reactions catalyzed by lipases have been studied, namely i) Transesterification between ethyl butyrate and glycerol in a two-phase system. The optimal water content in the mixture was 5%. Only monobutyrin was obtained. ii) Hydrolysis of p-nitrophenyl esters in microemulsions of sodium bis-2-ethylhexyl in heptane; its specificity constant was an order of magnitude lower than in aqueous mediun.
    • LIPASE FROM PSEUDOMONAS SP.: REACTIONS, CLONING, AND AMINO ACID SEQUENCE ANALYSIS

      Nishioka, Takaaki; Chihara-Shiomi, Mikiko; Yoshikawa, Kazuhiro; Inagaki, Minoru; Yamamoto, Yukio; Hiratake, Jun; Baba, Naomichi; Oda, Jun'ichi (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Lipases catalyze reactions not only in water but also in organic solvents. We have developed several asymmetric organic syntheses using lipase from Pseudomonas fluorescens in organic solvents. However, all attempts to acylate [1,1’-binaphthyl]-2,2’-diol with the lipase using enol esters failed. Among the lipases we screened for this substrate, lipase from Pseudomonas sp. is the only enzyme that catalyzes both stereoselective acylation of the diol and deacylation of its esters in organic solvents. The lipase shows higher activity in the hydrolysis of triglyceride into glycerol when compared with other lipases. Because of these characteristic reactivities of the lipase from Pseudomonas sp., we were interested in the cloning and sequencing of the gene of the enzyme and amino acid sequence analysis. The gene cloned from Pseudomonas sp. genome DNAwas 933 base pairs and inserted in plasmid pKK233-2. E. ‘coli JM109 transformed with the recombinant plasmid showed lipolysis asa zone of clearing around a colony on agar plate containing tributyrin. Amino acid sequence deduced from the DNA sequence is similar to that of lipase from Pseudomonas fragi (47 % identical). Content of serine residue is unusually high as 12 %. Sequence alignment with other 19 amino acid sequences of lipases suggested that Ser82, His251, and Asp209 are the catalytic residues.
    • THE LIPASE GENE OF BACILLUS SUBTILIS 168

      Colson, C.; Dartois, V.; Schanck, K.; Baulard, A.; Laboratoire de Génétique Microbienne (GENE), Université Catholique de Louvain, Place Croix du Sud, 4 (bte 3), B-1348 Louvainla- Neuve, Belgium (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Among Gram-positive bacteria, strain 168 of Bacillus subtilis constitutes a tool of choice for basic genetic and molecular studies. We madethe observation that this strain exhibits an extracellular lipolytic activity. We have undertaken to characterize the corresponding gene(s). Shotgun cloning of Bacillus subtilis 168 DNAin E. coli yielded two types of lipasepositive clones designated lipA and lipB. However, the lipB enzyme wasrather an esterase, on the basis of the preferential cleavage of esters of short chain fatty acids and of the absence of fluorescent reaction on triolein/rhodamin G medium. By multiple Tn5 transposon inactivations, gene lipA was estimated to be about 700 basepairs long. Both genes were inactivated in B. subtilis by reciprocal recombination with the homologous gene disrupted in vitro by a DNA segmentcontaining an antibiotic resistance (lipA::Km; lipB::Cm). The resulting strain expressed very little - if any - residual extracellular lipase-esterase activity. Mapping experiments indicated that lipA is a new locus at about 22° whereaslipB, at about 306° could correspondto an esterase gene (estB) previously described. Sequencing of genelipA is in progress. Besides, we are seeking conditions to | optimize the yield of extracellular lipase (culture conditions, genetic background, transcription signals) as a prerequisite for its large scale purification.
    • STAPHYLOCOCCALLIPASES AND PHOSPHOLIPASES

      Götz, Friedrich; Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 28, D-7400 Tübingen, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      For many years it has been known that certain Staphylococci produce and release lipolytic enzymesinto the culture medium. In order to obtain more information about the genetic and biochemical properties, the lipase genes of Staphylococcus hyicus and Staphylococcus aureus were cloned and sequenced. The S. hyicus lipase gene was cloned and expressed in Staphylococcus carnosus. From this organism, the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kD. This protein was purified and the N-terminal sequence revealed that the primary gene product was processed at the proposed signal peptide cleavagesite. In S. hyicus, the DNA-donorstrain, the lipase was further processed by an extracellular protease to a 46 kD protein, which exhibited a 3-fold higher specific activity as compared to the 86 kD protein. The 46 kD protein waspurified to homogeneity. The lipolytic activity was dependent upon the presence of Ca2*. The purified lipase revealed an extremely broad specificity. The enzyme hydrolysed not only triglycerides, but also naturally occurring phosphatidylcholines and lysophospholipidsto free fatty acids and water-soluble products. The 641 aminoacid residue S. hyicus lipase is organized as a pre-pro-lipase, consisting of a 38 aminoacid signal peptide, a 207 aminoacid pro-peptide, and a 396 amino acid mature lipase. Secondary structure predictions revealed that the pro-peptideis characterized by hydrophilic protein domains, while in the mature lipase, hydrophobic protein domains are predominate. It was demonstrated by gene fusionstudies that the pro-peptide is necessary for efficient lipase secretion.
    • A MOLECULAR VIEW OF LIPOPROTEIN LIPASE AND HEPATIC LIPASE STRUCTURE AND FUNCTION

      Ameis, Detlev; Kobayashi, Junji; Davis, Richard C.; Stahnke, Gisela; Ben-Zeev, Osnat; Wong, Howard; Doolittle, Mark H.; Will, Hans; Greten, Heiner; Schotz, Michael C.; et al. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Lipolytic enzymes play a pivotal role in the metabolism of triglyceride-rich lipoproteins circulating in plasma. The primary structures of many lipases have now been elucidated by molecular cloning of their cDNAs. To gain further insights into the molecular biology of lipoprotein lipase (LPL) and hepatic lipase (HL), we have determined their genomic organization. Both enzymes are members of a lipase gene family. Based on information derived from the genomic structures it is now possible to directly assess the molecular basis of familial LPLdefiency, a rare disorder of lipid metabolism involving the massive accumulation of chylomicrons in the plasma. Employing gene amplification techniques with LPL exon-specific oligonucleotide primers and direct DNA sequence determination, we have characterized a kindred with classical familial LPL-deficiency. Loss of LPL enzymatic activity was found to be caused by an amino acid substitution close to the putative active site.
    • CLONING, EXPRESSION AND CHARACTERIZATION OF CUTINASE, A FUNGAL LIPOLYTIC ENZYME

      Lauwereys, Mark; de Geus, Pieter; De Meutter, J.; Stanssens, P.; Matthyssens, G.; Plant Genetic Systems N.V., J. Plateaustraat 22, B-9000 Gent, Belgium (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      A cutinase from the fungus Fusarium solani pisi has been overproduced in E. coli by placing a phoA-signal/cutinase hybrid gene under the control of the tac promoter. Due to its periplasmic location the recombinant enzyme can be easily purified in large quantities. Assays using p-nitrophenylbutyrate suggest that the overproduced and authentic enzyme are catalytically equivalent. The specific activities on tributyrin (4000u/mg) and triolein (800u/mg) demonstrate the lipolytic nature of the enzyme. The cutinase, however, differs from classical lipases in that no measurable activation around the CMC of the tributyrin substrate is observed. We also provide evidence that the recombinant enzyme is quite thermostable.
    • COMPARATIVE STUDY ON PRIMARY STRUCTURES OF TWO LIPASES FROM GEOTRICHUM CANDIDUM

      Shimada, Yuji; Sugihara, Akio; Tominaga, Yoshio; Osaka Municipal Technical Research Institute, Morinomiya, Joto-ku, Osaka 536, JAPAN (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Geotrichum(G.) candidum produces two extracellular lipases I and II, the lipase I being the predominant component. The lipase I and II cDNAs were cloned, and their nucleotide sequences were determined. The nucleotide sequences included the N- and Cterminal amino acid sequences and the partial amino acid sequences of the lipases. The mature lipases I and II were of the same length, their overall identity being 84%, Furthermore, four Cys residues were completely conserved, which may participate in the formation of disulfide bridges. A homology search indicated that the G. candidum lipases and Candida cylindracea lipase are homologous enzymes, and that they are members of the cholinesterase family.
    • PANCREATIC COLIPASE:STRUCTURAL AND IMMUNOLOGICAL STUDIES

      Sarda, L.; Institut de Chimie Biologique,Faculté St-Charles,F-13331 Marseille 3,France (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Lipases hydrolyze triacylglycerols in the form of micellar aggregates or emulsions. Adsorption of the enzyme to lipid particles is a key step in interfacial catalysis. Pancreatic lipase is inhibited by bile salt and phospholipid which prevent enzyme binding to interface.Inhibition is specifically reversed by colipase,a protein of 10.5 KD found with lipase in the pancreatic secretion.Human,porcine and equine colipases have been sequenced.The amino acid sequence of the human and rat proteins has been deduced from the nucleotide sequence of cloned cDNA.Comparison of the primary structures reveals extensive homology.Colipase is secreted aS a precursor form (procolipase) and activated by tryptic cleavage at one single bond (Arg,-Gly,). Limited proteolysis increases the lipid binding capacity of colipase.Kinetic studies of the activation of bile salt inhibited pancreatic lipase by colipase have provided evidence that the cofactor forms a stoechiometric active complex with enzyme at interface.Results of binding studies with model substrates are consistent with the view that colipase binds to interface coated with bile salt and further anchors lipase to its substrate.It has been postulated that colipase possesses two specific surface domains for binding to lipid and lipase.Attempts to identify the lipid and lipase binding sites were made using a physico-chemical approach.Spectroscopic studies have shown that the region containing the three tyrosine residues is involved in the binding to lipid-water interface.Chemical modification of the free carboxyl groups of residues Glu), and/or Asp, which has no effect on lipid binding prevents interaction with lipase.Polyclonal and monoclonal (MAb) antibodies have been raised against porcine colipase and used for the characterization of the binding sites in spatial relationship with antigenic regions.Results of studies carried out with eight MAb and fractions of polyclonal antibodies separated on immobilized synthetic peptides bring evidence that the tyrosine containing region and the N-terminal fragment are involved in lipid binding and that Glu, is implicated in interaction with lipase.
    • HUMAN GASTRIC LIPASE

      Carriere, F.; Moreau, H.; Gargouri, Y.; Cudrey, C.; Ferrato, F.; Bernadac, A.; Benicourt, C.; Junien, J. L.; Verger, R.; Centre de Biochimie et de Biologie Moléculaire du C.N.R.S., 31, chemin Joseph Aiguier, 13402 Marseille cedex 9 (France); Institut de Recherche Jouveinal, 1, Rue des Moissons, B.P. 100, 94265 Fresnes.(France) (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Ninety percentofthe dietary lipids in humans are triglycerides which constitute the essential part of the 100g to 150g daily fat intake in industrialized countries. It was thoughtuntil recently that the hydrolysis of dietary triglycerides began in the intestinal lumen and was catalysed exclusively by pancreatic lipase. Studies on gastrointestinal lipolysis have underestimated several importantpoints, particularly the role of gastric lipolysis. It is now well established that Human Gastric Lipase (HGL), is the first lipolytic enzyme involved in dietary fat digestion. HGL originates entirely from the fundic mucosa. Nolipolytic activity was detected in the lingual, pharyngeal or oesophagus areas. Using immunocytolocalization techniques, cells producing HGLwereidentified as the chief cells of gastric fundic glands already known to biosynthesize pepsin. HGL was purified to electrophoretic homogeneity (MW = 50 kDa)from gastric juice. It is a glycoprotein with a glycan moiety amounting about 15 to 20 % ofthe total protein weight. The complete amino acid sequence of HGL,derived from cDNA sequence, shows 80 % homology with rat lingual lipase. No structural homology exists between human gastric lipase and pancreatic lipase, except the G-X-S-X-G sequence found in otherlipases andserine esterases. This sequence containsa serine analogousto the essential Ser- 152 in human pancreatic lipase. HGL contains one free sulfhydryl group whichis essential to the expressionoflipaseactivity. HGL hydrolyses short chain (tributyrin) and long chain (Intralipide) triacylglycerols at similar rates. HGL activity is very dependentupon the interfacial tension between triacylglycerol and water. In the presence of amphiphiles such as bile salts or alimentary proteins, the tributyrin-waterinterfacial tension decreases and HGLis activated. Thus HGLis capable of hydrolyzing triglyceride emulsionsin the presence ofbile salts concentration prevailing in the upper small intestine and in the presence of alimentary proteins. These observations could explain the high dietary lipid absorption observed underpancreatic lipase deficiency. In vitro studies showed that prehydrolysis by HGLofIntralipide emulsion enable it to be subsequently hydrolyzed by humanpancreatic lipase. Fatty acid liberated by HGL probablytriggerthe later action of pancreatic lipase by changing the interfacial tension.
    • STEREOSELECTIVITY OF LIPASES : Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases

      Rogalska, E.; Ransac, S.; Verger, R.; Centre de Biochimie et de Biologie Moléculaire du C.N.R.S., 31, chemin Joseph Aiguier, 13402 Marseille cedex 9 (France) (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      In the present study, porcine pancreatic lipase (PPL), rabbit gastric lipase (RGL) and human gastric lipase (HGL) stereospecificity towards chemically like, but sterically non equivalent ester groups within onesingle triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin ortriolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by HPLC. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of HGL and RGL,expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% fortriolein, respectively. The corresponding values with PPL were 3% in the caseof trioctanoin and 8% in that of triolein. It is worth noting that RGL, unlike HGL, became morestereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the moststriking catalytic differences observed between these twogastric lipases.
    • Identification of the THL binding site on human pancreatic lipase

      Peng, Q.; Hadvary, P.; Maerki, H. P.; Pharmaceutical Research Department, F. Hoffmann-La Roche Ltd. CH-4002 Basel, Switzerland (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Pancreatic lipase is considered as a serine hydrolase that plays a key function in dietary fat absorption by hydrolysing triglycerides into diglycerides and subsequently into monoglycerides and free fatty acids. Although it has been assumed [1,2] that porcine pancreatic lipase has two functionally important sites: a catalytic site involving Serj109, and a topographically distinct interfacial "recognition site" or " substrate binding site" controlled by Serı52, the catalytic mechanism has not been demonstrated experimentally thus far. On the other hand, the X-ray structure of human pancreatic lipase [3] shows clearly that Serj52 forms a triad with His263 and Asp 176 which givesa spatial superposition with the catalytic triad of the serine protease trypsin (Fig.1). \ 1102 esp Recently Tetrahyrolipstatin exe (THL), a_ selective and irreversible inhibitor of er pancreatic lipase has been used to identify the THL binding site on porcine pancreatic lipase. The result showed that THL binds to Serı52 of the lipase covalently [4]. The same attempt was also made for human pancreatic lipase, the data indicate strongly that ee human and porcine pancreatic see lipases share high : similarity/identity not only in their primary structure and enzymatic characteristics, but also in their catalytic mechanism.
    • LIPASES IN REVERSE MICELLES

      Walde, Peter; Luisi, Pier Luigi; Institut für Polymere, Eidgenössische Technische Hochschule, Zürich (Switzerland) (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Two independent spectroscopic methods have been developed to assay lipases continuously with triacylglycerol substrates in a reverse micellar solution. With the two methods, a simple and unique possibility is offered to study the kinetics and the specificity of lipases, embedded in a system which possibly mimics the biologically relevant environment of lipolytic enzymes. Preliminary activity data are presented for the colipase dependent human pancreatic lipase in reverse micelles, and we have started to investigate the conformational behavior of this enzyme by means of circular dichroism and fluorescence spectroscopy.
    • CLONING AND EXPRESSION OF INDUSTRIALLY IMPORTANT FUNGALLIPASES

      Boel, Esper; Huge-Jensen, Birgitte; Wöldike, Helle F.; Gormsen, Erik; Christensen, Mogens; Andreasen, Frank; Thim, Lars; Novo Nordisk A/S, Novo Allé, DK-2880 Bagsveerd, Denmark (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Triglyceride lipases from various organisms have a numberof potential industrial applications exemplified by their use in flavour enhancement, in production of esters and specialty fats, and in household detergents. We have cloned and expressed two fungallipases: the 1,3-positional specific lipase from Rhizomucor miehei (1,2), commercialized as Lipozyme™, and lipase from Humicola lanuginosa commercialized for use in household detergents (Lipolase™). Production of these lipases and other enzymesforindustrial application has necessitated the developmentof an efficient recombinant expression system. We have focusedour efforts on the development of Aspergillus as a recombinant host system for this purpose. Aspergillus has the capacity to secrete large amounts of active hydrolytic enzymes such as for example glucoamylases and amylases. Cloning of strong promoters from the corresponding genes (3,4) and development of a transformation- and selection system (4) has allowed expression from heterologous cDNA genesin Aspergillus oryzae. As first attempt to obtain heterologous expression we transferred an aspartic proteinase cDNA (5) from Rhizomucor miehei into A. oryzae and obtained high levels of secreted, active and correctly processed enzyme (4). These experiments have later been extended to fungal triglyceride lipases (6,7).