Investigation of Human Cyclooxygenase-2 Glycosylation Heterogeneity and Protein Expression in Insect and Mammalian Cell Expression Systems

Human cyclooxygenase-2 (hCox-2) is a key enzyme in the biosynthesis of prostaglandins and the target of nonsteroidal anti-inflammatory drugs. Recombinant hCox-2 overexpressed in a vaccinia virus (VV)-COS-7 system comprises two glycoforms. Removal of the N-glycosylation consensus sequence at Asn⁵⁸⁰(N580Q and S582A mutants) resulted in the expression of protein comprising a single glycoform, consistent with the partial N-glycosylation at this site in the wild-type (WT) enzyme. The specific cyclooxygenase activities of the purified WT and N580Q mutant were equivalent (40 ± 3 μmol O₂/min/mg) and titrations with diclofenac showed no difference in inhibitor sensitivities of WT and both mutants. Results of the expression of WT and N580Q hCox-2 in aDrosophilaS2 cell system were also consistent with the N-glycosylation at this site, but low levels of activity were obtained. High levels of N-glycosylation heterogeneity are observed in hCox-2 expressed using recombinant baculovirus (BV) in Sf9 cells. Expression of a double N-glycosylation site mutant in Sf9 cells, N580Q/N592Q, resulted in a decrease in glycosylation but no clear decrease in heterogeneity, indicating that the high degree of N-glycosylation heterogeneity observed with the BV-Sf9 system is not due to partial glycosylation of both Asn⁵⁸⁰and Asn⁵⁹². N-linked oligosaccharide profiling of purified VV and BV WT and S582A mutant hCox-2 showed the presence of high mannose structures, (Man)_n(GlcNAc)₂,n= 9, 8, 7, 6. The S582A mutant was the most homogeneous with (Man)₉(GlcNAc)₂comprising greater than 50% of oligosaccharides present. Analysis of purified VV WT and S582A mutant hCox-2 by liquid chromatography–electrospray ionization–mass spectrometry showed an envelope of peaks separated by approximately 160 Da, corresponding to differences of a single monosaccharide. The difference between the highest mass peaks of the two envelopes, of approximately 1500 Da, is consistent with the wild-type enzyme containing an additional high mannose oligosaccharide.     

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX₁ promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X₃₃ and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X₃₃ cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His₆-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His₆-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His₆-ROLw) and the mutant (His₆-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.     

Site-specific N-glycosylation of human chorionic gonadotrophin--structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy.

Glycopeptides representing individual N-glycosylation sites of the heterodimeric glycoprotein hormone human chorionic gonadotrophin (hCG) were obtained from subunits hCG alpha (N-glycosylated at Asn-52 and Asn-78) and hCG beta (N-glycosylated at Asn-13 and Asn-30) by digestion with trypsin and chymotrypsin, respectively. Following purification by reverse-phase HPLC and identification by amino acid sequencing, the glycopeptides were analysed by one- and two-dimensional 1H NMR spectroscopy. The results are summarized as follows: (i) oligosaccharides attached to Asn-52 of hCG alpha comprised monosialylated 'monoantenary' NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-4'), disialylated diantennary NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N2), and the monosialylated hybrid-type structures NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-A) and NeuAc alpha 2-3Gal-beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-AB) in a ratio approaching 5:2:2:1; (ii) Asn-78 of hCG alpha carried N2 and N1-4' almost exclusively (ratio approximately 3:2); (iii) both N-glycosylation sites of hCG beta contained predominantly component N2, partially (approximately 25%) and completely alpha 1-6-fucosylated at the N-acetylglucosamine linked to Asn-13 and Asn-30, respectively. The distinct site-specific distribution of the oligosaccharide structures among individual N-glycosylation sites of hCG appears to reflect primarily the influence of the surrounding protein structure on the substrate accessibility of the Golgi processing enzymes alpha-mannosidase II, GlcNAc transferase II and alpha 1,6-fucosyltransferase.     

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.     

Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.     

Functional properties of glycosylated lysozyme secreted in Pichia pastoris

Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn(19)-Tyr(20)-Thr(21)), G49N (Asn(49)- Ser(50)-Thr(51)), R21T/G49N (Asn(19)-Tyr(20)-Thr(21)/Asn(49)-Ser(50)-Thr(51)), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc(2)Man(9-11), while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc(4)Man(27-32). The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.     

Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris

Escherichia coli pH 2.5 acid phosphatase gene (appA) and three mutants were expressed in Pichia pastoris to assess the effect of strategic mutations or deletion on the enzyme (EcAP) biochemical properties. Mutants A131N/ V134N/D207N/S211N, C200N/D207N/S211N, and A131N/ V134N/C200N/D207N/S211N had four, two, and four additional potential N-glycosylation sites, respectively. Extracellular phytase and acid phosphatase activities were produced by these mutants and the intact enzyme r-AppA. The N-glycosylation level was higher in mutants A131N/V134N/D207N/S211N (48%) and A131N/V134N/ C200N/D207N/S211N (89%) than that in r-AppA (14%). Despite no enhancement of glycosylation, mutant C200N/ D207N/S211N was different from r-AppA in the following properties. First, it was more active at pH 3.5-5.5. Second, it retained more (P < 0.01) phytase activity than that of r-AppA. Third, its specific activity of phytase was 54% higher. Lastly, its apparent catalytic efficiency kcat/Km for either p-nitrophenyl phosphate (5.8 x 10(5) vs 2.0 x 10(5) min(-1) M(-1)) or sodium phytate (6.9 x 10(6) vs 1.1 x 10(6) min(-1) M(-1)) was improved by factors of 1.9- and 5.3-fold, respectively. Based on the recently published E. coli phytase crystal structure, substitution of C200N in mutant C200N/D207N/S211N seems to eliminate the disulfide bond between the G helix and the GH loop in the alpha-domain of the protein. This change may modulate the domain flexibility and thereby the catalytic efficiency and thermostability of the enzyme.     

Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man9GlcNAc2 from Man9GlcNAc2-PP-Dol while others receive Man5GlcNA(2 from Man5GlcNAc2-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the alpha-mannosyltransferase that converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man9GlcNAc2 and processed no further than Man5GlcNAc2 in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man5GlcNAc2-PP-Dol is the only route to complex N-glycans in T. brucei and that Man9GlcNAc2-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man5GlcNAc2 or Man9GlcNAc2 transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.     

Heterologous expression of an extra-cellular lipase from the basidiomycete Pleurotus sapidus

The 1641 bp cDNA encoding an extra-cellular lipase of the basidiomycete Pleurotus sapidus (Lip2) was cloned from a cDNA library. Expression of the cDNA in Escherichia coli, with and without signal sequence, led to the production of recombinant Lip2, mainly as inclusion bodies with low catalytic activity. Refolding yielded catalytically active protein. A C-terminal His tag was used for purification and immunochemical detection. The recombinant lipase hydrolysed xanthophyll esters with high efficiency, and omitting the signal sequence did not alter the catalytic properties. The P. sapidus lipase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus characterised on the molecular level and expressed in a manageable host.     

Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V(HH) antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.     

On the contentious sequence and glycosylation motif of the ribosome inactivating plant protein gelonin

The amino acid sequence and the glycosylation motif of the ribosome inactivating protein (RIP) gelonin are identified by Fourier transform ion cyclotron resonance mass spectrometry. Intact gelonin as isolated from the seeds of Gelonium multiflorum consists of at least three different post-translational modified forms: analysis of gelonin peptides as obtained by proteolytic digestion is consistent with the amino acid sequence published by Nolan et al. High resolution mass determination established a glycosylation pattern of GlcNAc2Man(3-5)Xyl. N189 was identified as glycosylation site. The proposed glycan structure is consistent with a standard plant N-glycosylation pattern as found in other RIP. Based on these results we suggest that gelonin is located in the vacuole of Gelonium multiflorum seeds.     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

Enantioselectivity of Candida rugosa lipases (Lip1, Lip3, and Lip4) towards 2-bromo phenylacetic acid octyl esters controlled by a single amino acid

Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phenomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R-enantiomer (E-value = 15), while Lip1 and Lip3 showed an S-enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S- or R-enantiomer preference of C. rugosa lipases. This led to the construction by site-directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2-bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)-2-bromo-phenylacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination.     

Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.     

Proteomic analysis of N-glycosylation in mosquito dopachrome conversion enzyme

A novel dopachrome conversion enzyme (DCE) is present in insects and involved in their melanization pathway. DCE shares no sequence homology with any noninsect species from bacteria to humans. Several DCE sequences have been available, but enzyme structure and catalytic mechanism are unclear. This study concerns DCE PTMs, especially glycosylation. A mosquito DCE was purified and its monosaccharide composition, N-glycosylation site, and oligosaccharide structures were determined. Results showed that N-acetyl D-glucosamine and D-mannose are the major monosaccharides and L-fucose, D-xylose, and D-arabinose are the minor ones in mosquito DCE. Glycosylation site and oligosaccharide structures were elucidated from MS and MS/MS spectra of trypsin-digested DCE glycopeptides. A single N-glycosylation site (Asn285 -Glu-Thr) was identified in DCE and was proven to be fully glycosylated. Man3GlcNAc2, Man3(Fuc)1-2GlcNAc2, and their truncated structures were the dominant oligosaccharides. In addition, high mannose-type structures (Man4-7(Fuc)GlcNAc2) were also identified. Removal of DCE N-oligosaccharides with peptide N-glycosidase (PNGase F) decreased its activity and thermal stability. However, partial DCE deglycosylation with alpha-mannosidase or alpha-fucosidase somewhat stimulated its activity and improved its thermal stability. During mass spectrometric analysis of DCE glycopeptides, their CID patterns were highly intriguing, in that some glycopeptides underwent both C-terminal rearrangement and formation of dimeric structures during CID. Results of this study provide an interesting example in terms of potential complexity of the glycopeptide CID fragmentation pattern.     

Site-directed amino acid substitutions in the hydroxylase alpha subunit of butane monooxygenase from Pseudomonas butanovora: Implications for substrates knocking at the gate

Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase alpha subunit of butane monooxygenase (BMOH-alpha) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-alpha were altered to the corresponding residues from the alpha subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 microM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 microM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-alpha.     

High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris

The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532Da which was 2451Da greater than the mass calculated from the sequence of the protein (50,081Da) and 1536Da greater than the mass of the native human protein (50,996Da). In vitro deglycosylation experiments by peptide:N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.     

Systematic mutagenesis of potential glycosylation sites of lysosomal acid lipase

Lysosomal acid lipase (LAL; EC 3.1.1.13) is a key enzyme in the intracellular lipid metabolism. It hydrolyzes exogenous triglycerides and cholesterol esters taken up by various cell types. LAL has six potential N-glycosylation sites and one potential O-glycosylation site. Elimination of each of the six Asn-(X)-Ser/Thr sites by site-directed mutagenesis and expression in baculovirus-infected Spodoptera frugiperda cells resulted in two single-mutant enzymes without lipolytic activities (N134Q and N246Q) and four mutants with preserved activities. The two inactive mutants were not detectable on immunoblot analysis, indicating that they were not secreted. Six double mutants in all possible combinations except for the two inactive single mutants were produced and expressed. Double mutants in combination with the N9 glycosylation site showed reduced activities as compared to the other mutants or the wild-type enzyme. Kinetic data of LAL glycosylation mutants indicate that substrate affinity of N9Q was not changed, but k (cat) of N9 mutants was reduced distinctly compared to the wild-type enzyme. Peanut agglutinin lectin did not recognize LAL, demonstrating that the protein has no core1 structure (Galbeta 1-3 GalNAc) of O-glycosylation. These data indicate that at least two of the six N-glycosylation sites are used in native lipase. N134 and N246 were found to be essential for LAL activity. We conclude that glycosylation plays an important role in the formation of functional LAL.     

Expression, purification, and characterization of His-tagged Staphylococcus xylosus lipase wild-type and its mutant Asp 290 Ala

The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.     

Engineering of Yarrowia lipolytica lipase Lip8p by circular permutation to alter substrate and temperature characteristics

Applications of lipases are mainly based on their catalytic efficiency and substrate specificity. In this study, circular permutation (CP), an unconventional protein engineering technique, was employed to acquire active mutants of Yarrowia lipolytica lipase Lip8p. A total of 21 mutant lipases exhibited significant shifts in substrate specificity. Cp128, the most active enzyme mutant, showed higher catalytic activity (14.5-fold) and higher affinity (4.6-fold) (decreased K _m) to p-nitrophenyl-myristate (pNP-C₁₄) than wild type (WT). Based on the three-dimensional (3D) structure model of the Lip8p, we found that most of the functional mutation occurred in the surface-exposed loop region in close proximity to the lid domain (S112–F122), which implies the steric effect of the lid on lipase activity and substrate specificity. The temperature properties of Cp128 were also investigated. In contrast to the optimal temperature of 45 °C for the WT enzyme, Cp128 exhibited the maximal activity at 37 °C. But it is noteworthy that there is no change in thermostability.