Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

Various lipases for producing products enriched with polyenic acids in fish fat hydrolysis]

The enzymic hydrolysis of fish with lipases from various sources was studied. The lipase from the fungus Rhizopus microsporus preferentially removes saturated fatty acids, while lipase from the pyloric caeca of salmon unsaturated fatty acids upon hydrolysis of fish fats. The enzymes can be used to obtain fatty products enriched with eicosanopentaenoic acid, mono- and diacylglycerols by enzymic hydrolysis of the ivasi fat.     

Comparative study of thermostabilty and ester synthesis ability of free and immobilized lipases on cross linked silica gel

A novel support has been utilized for immobilization of lipase, which was prepared by amination of silica with ethanolamine followed by cross linking with glutaraldehyde. Lipases from Rhizopus oryzae 3562 and Enterobacter aerogenes were immobilized on activated silica gel, where they retained 60 and 50% of respective original activity. The thermal stability of the immobilized lipases was significantly improved in comparison to the free forms while the pH stability remained unchanged. E. aerogenes and R. oryzae 3562 lipases retained 75 and 97% of respective initial activity on incubation at 90 degrees C, whereas both the free forms became inactive at this temperature. The conversion yield of isoamyl acetate was found to be higher with the immobilized fungal (90 vs. 21%) and bacterial lipases (64 vs. 18%) than the respective free forms. Immobilized R. oryzae 3562 lipases retained 50% activity for isoamyl acetate synthesis up to ten cycles whereas it was eight cycles for E. aerogenes.     

Esterification reactions catalyzed by lipases in microemulsions: the role of enzyme localization in relation to its selectivity

The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis-(2-ethylhexyl)sulfo-succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies. (c) 1993 John Wiley & Sons, Inc.     

Glyceride synthesis by four kinds of microbial lipase

Apart from their usual mechanism of action, lipases from Aspergillus niger and Rhizopus delemar also catalyzed the synthesis of glycerides from oleic acid and glycerol. Lipases from Geotrichum candidum and Penicillium cyclopium were inactivated by oleic acid, but were stable in the presence of casein, albumin or buffer of appropriate pH. Lipases from Aspergillus niger and Rhizopus delemar synthesized glycerides from, not only fatty acid, but dibasic acids and aromatic acids, making ester bonds only at position 1 and 3 of glycerol. In contrast, lipases from Geotricum candidum and Penicillium cyclopium synthesized glycerides only from long chain fatty acids, and made ester bonds at all three available positions of the glycerol molecule.     

Synthesis of various kinds of esters by four microbial lipases

Ester synthesis by microbial lipases, using homogeneous enzyme preparations, were investigated. The amount of synthesized ester was estimated by alkalimetry, and products were identified by thin-layer chromatography and infrared spectroscopy. Lipases from Aspergillus niger, Rhizopus delemar, Geotrichum candidum and Penicillium cyclopium synthesized esters from oleic acid and various primary alcohols. Only Geotrichum candidum lipase synthesized esters of secondary alcohols. Esters of tertiary alcohols, phenols or sugar alcohols were not synthesized by any lipase. Rather high concentrations of alcohol were required to synthesize the esters of ethylene glycol, propylene glycol or trimethylene glycol. Lipases from Aspergillus niger and Rhizopus delemar synthesized oleyl esters of various fatty acids and some dibasic acids. In contrast, lipases from Geotrichum candidum and Penicillium cyclopium synthesized oleyl esters only from medium or long chain fatty acids.     

胶陀螺发酵液的研究

对胶陀螺自然发酵后的发酵液的发酵菌种和化学成分进行了研究。结果表明 ,发酵液中富含组氨酸、赖氨酸、酪氨酸等氨基酸和钙、镁、磷、铁、锌等无机元素 ,同时含有柠檬酸、酒石酸、乙醇、乙甲醚基环氧乙烷、丁内酯及 3- (3-羰基 - 4 -羟基苯基 ) -D -丙氨酸等物质 ,发酵菌种为黑根霉。     

Specificity and glyceride synthesis by mycelial lipases of Rhizopus delemar

Mycelial lipase activity of the mould Rhizopus delemar was purified by gel filtration chromatography to three distinct proteins of notable lipase activity. The three enzymes were designated A′, B′ and C′, according to elution volumes from a Sephadex G150 column. The capacity of the three lipases to catalyse glyceride synthesis from free fatty acids and glycerol indicated a tendency towards short-chain and unsaturated fatty acids in preference to long-chain saturated fatty acids. The postional specificity of all lipases involved in such synthetic reactions indicated the formation of ester bonds at positions 1 and 3 of glycerol.     

Computer-Aided Analysis of Spatial Structure of Some Hydrolytic Enzymes

Using the MolScript version 2.1 computer program for protein molecule modeling and X-ray structure analysis data the spatial structures of several hydrolytic enzymes have been compared. These include glucoamylase from Aspergillus awamori and Saccharomycopsis fibuligera and lipases from Rhizopus japonicus. Results on homology of amino acid sequences and topology of secondary structure elements were obtained. 3D models of these enzymes with positioning of functionally important groups in the active site cavity were built.     

Staphylococcal lipases: biochemical and molecular characterization

To date, the nucleotide sequences of nine different lipase genes from six Staphylococcus species, three from S. epidermidis, two from S. aureus, and one each from S. haemolyticus, S. hyicus, S. warneri, and S. xylosus, have been determined. All deduced lipase proteins are similarly organized as pre-pro-proteins, with pre-regions corresponding to a signal peptide of 35 to 38 amino acids, a pro-peptide of 207 to 321 amino acids with an overall hydrophilic character, and a mature peptide comprising 383 to 396 amino acids. The lipases are secreted in the pro-form and are afterwards processed to the mature form by specific proteases. The pro-peptide of the S. hyicus lipase is necessary for efficient translocation and for protection against proteolytic degradation. Despite being very similar in their primary structures the staphylococcal lipases show significant differences in their biochemical and catalytic properties, such as substrate selectivity, pH optimum and interfacial activation. The lipase from S. hyicus is unique among the staphylococcal and bacterial lipases in that it has not only lipase activity, but also a high phospho-lipase activity. All staphylococcal lipases are dependent on Ca(2+), which is thought to have a function in stabilizing the tertiary structure of the lipases. Evidence exists that staphylococcal lipases like other bacterial lipases, possess a lid-like domain that might be involved in the interfacial activation of these enzymes.     

Cloning,characterization, and expression of cDNA encoding a lipase from Kurtzmanomyces sp. I-11

A cDNA clone of the lipase secreted by Kurtzmanomyces sp. I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase. The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively. The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74% identity) and weakly to other lipases. The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved. A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.     

Multiple crystal forms of lipases from Geotrichum candidum

Multiple stable crystal forms of two lipases from the fungus Geotrichum candidum have been obtained. The diffraction pattern extends to beyond 2.0 A resolution. Similarity of the cell dimensions of various forms suggested similar packing of molecules in different crystals. This was confirmed by rotation function results. Four heavy-atoms derivatives have been identified.     

Lipase-catalyzed hydrolysis of Crambe oil in AOT-isooctane reversed micelles

Summary The hydrolysis by 1,3-specific lipases (Humicola lanuginosa, Mucor miehei, Rhizopus delemar andRhizopus javanicus) of the highly symmetric, high molecular weight triglycerides fromCrambe abyssinica (Crambe) seed oil is studied in an AOT-stabilized microemulsion system. Enzyme kinetic data shows that, of the lipases studied,Rhizopus javanicus lipases exerts the highest hydrolytic activity towards this new seed oil.     

Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.     

根霉脂肪酶的研究进展

根霉属微生物是脂肪酶的一个重要来源,多种根霉脂肪酶已被分离并鉴定。本文将对根霉脂肪酶的菌种筛选、发酵、纯化、结构、分子生物学研究及应用进行概述。     

华根霉脂肪酶有机相合成酶活的研究

通过比较7种微生物脂肪酶的有机相合成酶活、水相水解酶活及在正庚烷中催化己酸乙酯合成的能力,证明了合成酶活与水解酶活相关性不高,合成酶活比水解酶活更能反映脂肪酶的合成能力。通过比较两株华根霉(Rhizopus chinensis)脂肪酶酶活,发现合成酶活相差较大,表明相同种属微生物的脂肪酶合成酶活存在不同。对．Rhizopus chinensis-2液态发酵产脂肪酶进程研究发现,水解酶活高峰先于合成酶活高峰大约12h。将不同培养时间的Rhizopus chinensis-2全细胞脂肪酶用于催化己酸乙酯合成,具有高合成酶活的全细胞脂肪酶催化己酸乙酯合成反应较快。因此,全细胞脂肪酶用于催化有机相酯合成反应时,具有高脂肪酶合成酶活的菌体具有较好的催化酯合成能力。     

Inhibition of lipases from Chromobacterium viscosum and Rhizopus oryzae by tetrahydrolipstatin

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.     

Towards novel biocatalysts via protein design: the case of lipases

Abstract: During the past 3 years, the tertiary structures of several lipases have been solved by X-ray analysis. The structures revealed unique features such as hydrophobic 'patches' on the surface, presumably involved in lipid supersubstrate binding, and a lid structure which covers the active site in the absence of substrate. Only very recently the first X-ray structure of a bacterial lipase has been solved, and further structural features different from lipases of eukaryotic origin became apparent. Many lipase genes have been cloned and sequenced recently, and expression systems for the preparation of recombinant enzymes in good yields are available. As an example, the lipase from Rhizopus oryzae has been successfully expressed by us in Escherichia coli , and the resulting inclusion bodies were renatured in high yields. Consequently, the mechanism of action of lipases is now being studied via site-directed mutagenesis, and the rational design of lipases for the selective transformation of substrates is presently addressed in several laboratories.     

Inhibition of Incorporation of l-Alanine into Uridine-diphospho N-acetylmuramic Acid by Glycine

Lipases from Aspergillus niger and Rhizopus delemar hydrolyzed triolein and produced l,2 (2,3)-diolein and 2-monoolein. These two lipases appears to have strong specificity towards the outer chains of the triglyceride. Comparing the proportions of fatty acids in position 1 (3) of cocoa butter with proportions of fatty acids liberated after limited hydrolysis of cocoa butter, it becomes clear that these two lipases do not hydrolyze the ester bond in position 2 of the triglyceride.

On the other hand, lipases from Geotrichum candidum Link and Penicillium cyclopium Westring attacked the fatty acid chains regardless of their positions. Geotrichum candidum lipase liberated oleic acid and palmitic acid in preference to stearic acid from cocoa butter.