Rhizopus Oryzae Lipase Research Articles

Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties

The mature lipase of the fungus Rhizopus oryzae (ROL) was functionally expressed and secreted in the methylotrophic yeast Pichia pastoris. In a batch cultivation, where methanol feeding was linked to the dissolved oxygen content in the cultivation solution, a lipase activity of 500 000 units per liter (60 mg active lipase per liter) of culture was achieved after initial glycerol feeding of the culture. Recombinant ROL lipase was purified to homogeneity by a simple two-step purification procedure and had a specific activity of 8571 U mg −1 (triolein, 30°C, pH 8.1) which is comparable with the purified native enzyme. The properties of the recombinant lipase were similar to those reported both for the native lipase and for the enzyme expressed in Escherichia coli and refolded from inactive inclusion bodies.

Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols.

The binding site of sn-1(3)-regioselective Rhizopus oryzae lipase (ROL) has been engineered to change the stereoselectivity of hydrolysis of triacylglycerol substrates and analogs. Two types of prochiral triradylglycerols were considered: 'flexible' substrates with ether, benzylether or ester groups, and 'rigid' substrates with amide or phenyl groups, respectively, in the sn-2 position. The molecular basis of sn-1(3) stereoselectivity of ROL was investigated by modeling the interactions between substrates and ROL, and the model was confirmed by experimental determination of the stereoselectivity of wild-type and mutated ROL. For the substrates, the following rules were derived: (i) stereopreference of ROL toward triradylglycerols depends on the substrate structure. Substrates with 'flexible' sn-2 substituents are preferably hydrolyzed at sn-1, 'rigid' substrates at sn-3. (ii) Stereopreference of ROL toward triradylglycerols can be predicted by analyzing the geometry of the substrate docked to ROL: if the torsion angle phiO3-C3 of glycerol is more than 150 degrees, the substrate will preferably be hydrolyzed in sn-1, otherwise in sn-3. For ROL, the following rules were derived: (i) residue 258 affects stereoselectivity by steric interactions with the sn-2 substituent rather than polar interactions. To a lower extent, stereoselectivity is influenced by mutations further apart (L254) from residue 258. (ii) With 'rigid' substrates, increasing the size of the binding site (mutations L258A and L258S) shifts stereoselectivity of hydrolysis toward sn-1, decreasing its size (L258F and L258F/L254F) toward sn-3.

Extracellular production of active Rhizopus oryzae lipase by Saccharomyces cerevisiae

The gene encoding Rhizopus oryzae lipase (ROL) was successfully expressed, and extracellularly active ROL was produced by Saccharomyces cerevisiae. The genes encoding the mature region (mROL) and the mROL having the pro sequence (ProROL) were fused to the gene encoding the pre- or prepro-α-factor leader region, and expressed under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL), which is strongly functional in S. cerevisiae. Efficient lipase activity was observed in the culture medium when the gene encoding ProROL was expressed. The recombinant lipase (rROL) produced by S. cerevisiae was purified by ethanol precipitation, butyl-Toyopearl 650M chromatography, and Sephacryl S-100 HR gel filtration to a single band by native-PAGE. However, the single band was found to consist of two proteins with different molecular masses (35 kDa and 46 kDa) on SDS-PAGE. The N-terminal analysis showed that 35 kDa protein was processed in the prosequence, and attached 28 amino acids of the C-terminal part of the prosequence, named rPro28ROL, while the 46 kDa protein had the entire pro-region, named rProROL. The preference of rROL for a series of triglycerides was determined using the titrimetric assay. rROL showed higher activity on saturated substrates having C 8-, C 10-, C 12-, and C 14- fatty acid chains in triglycerides. Extracellular lipase activity reached 2880 U/ l at 120 h of cultivation in YPD medium.

Computer-aided modelling of stereoselective triglyceride hydrolysis catalyzed by Rhizopus oryzae lipase

Lipase from Rhizopus oryzae catalyzes the stereoselective hydrolysis of triglycerides and analogues. Stereopreference and degree of enantiomeric excess of the product varies with the structure of the substrate: trioctanoylglycerol (‘ester’) and the sn-2 analogues 2-X-1,3-dioctanoylpropandiol, where 2-X = 2- O-octyl (‘ether’) and 2-hexyl (‘alkane’), are preferentially hydrolyzed at sn-1, substitution by 2-phenyl (‘phenyl’) reverses the Stereopreference to sn-3. We have modelled the stereoselectivity of Rhizopus oryzae lipase by docking the tetrahedral intermediates of these substrates in two orientations, appropriate to hydrolysis at sn-1 or sn-3, respectively. The initial complexes were further relaxed by molecular dynamics simulations. The favoured orientation of a substrate is characterized by three factors: (1) The substrate fits well into the binding site. The glycerol backbone is relaxed and the sn-2 chain points to a well defined hydrophobic binding site. (2) The oxyanion is stabilized by an extra hydrogen bond from the side chain of Thr 83. (3) The substrate lacks repulsive interactions with protein side chains, especially of Leu 258. Our model is consistent with experimental data and explains qualitatively the ranking of four different substrates with regard to stereoselectivity. It can be used to design lipase mutants with modified stereoselectivity.

The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence.

The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position - 68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.

Stereospecific synthesis of selected triglycerides: comments on acyl migration and analysis of configuration

Symmetrical and stereospecifically defined triglycerides have been prepared from racemic and ( R)- and ( S)-glycidol, respectively. The synthetic sequence allows the incorporation of sensitive fatty acids such as linolenic acid. Justification for retention of configuration and absence of acyl migrations during the synthesis is provided by the 1H-NMR spectra of triglycerides that are replaced at the sn-1 and sn-3 positions with enantiomers of α-methoxytrifluoromethylphenylacetic acid ( bis-MTPA esters). Partial hydrolysis of synthetic symmetrical triglycerides catalyzed by the lipase of Rhizopus oryzae was used to demonstrate the homogeneity of these preparations.

Interesterification of Milk Fat with Oleic Acid Catalyzed by Immobilized Rhizopus oryzae Lipase

Milk fat was interesterified with oleic acid by catalysis of an immobilized lipase in a microaqueous two-phase system. A commercial lipase from Rhizopus oryzae and a controlled pore glass carrier were selected for preparation of an immobilized lipase. The prepared immobilized lipase showed a Michaelis constant of 77mM and a maximum velocity of 40 U/g of carrier on the hydrolysis of triolein. Conditions for interesterification catalyzed by the immobilized lipase were optimized by the reaction between trimyristin and oleic acid under various conditions. The interesterification of milk fat with oleic acid was performed in isooctane with .3% (vol/vol) water content. The fatty acid composition and thermal characteristics of the triglycerides of the interesterified milk fat were investigated. The interesterified milk fat had about 50% more oleic acid and a significantly lower palmitic acid content than those of the original milk fat. The crystallization and melting curves obtained by differential scanning calorimetry analysis showed that the transition temperature of the major milk fat peaks decreased by 7.6 and 5.4°C, respectively. The results suggest that the prepared immobilized lipase can induce rather specific interesterification between oleic acid and palmitic acid in the milk fat triglycerides, which produces a lower melting milk fat, without losses of the short-chain fatty acid composition.