Rhodococcus sp. strain CR-53 LipR, the first member of a new bacterial lipase family (family X) displaying an unusual Y-type oxyanion hole, similar to the Candida antarctica lipase clan

Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.     

Variations among four commercial lipases from Chromobacterium viscosum in ester synthesis

The properties of four commercial lipases from Chromobacterium viscosum, CvL 1–4, in ester synthesis were investigated. Three lipases showed a high synthetic activity in esterification, with conversions of oleic acid as high as 86–95% in 24 h, whereas one (CvL 1) gave a poor result of only 11% with the same quantity of 9 mg crude lipase preparation. The elution profiles of the four lipases from Sephacryl S-100 HR differed and SDS-PAGE suggested that while CvL 1 lipase had two equivalent protein bands of molecular size 33 and 27 kDa, respectively, the other three lipases showed only one main protein band of 33 kDa. Isoelectric focusing revealed that all of the lipases contained several isoforms, but the proportions of the isoforms varied. Furthermore, both aggregated and single lipase forms obtained after gel filtration were able to catalyse ester synthesis, but the two lipases from CvL 1 showed lower synthetic activities than the others.     

cDNA cloning and characterization of Geotrichum candidum lipase II

Geotrichum candidum produces two extracellular lipases, I and II. A lipase II cDNA clone was isolated from a cDNA library by colony hybridization using the 32P-labeled fragment of lipase I cDNA isolated previously. The nucleotide sequence of lipase II cDNA determined by the dideoxy chain terminating method includes the N- and C-terminal amino acid sequences of lipase II, and the overall amino acid composition deduced from the cDNA coincides with that deduced on amino acid analysis of this protein. The cloned lipase II cDNA codes a protein of 544 amino acids and a part of the signal sequence of 13 amino acids. The peptide chain lengths of lipases I and II are the same, their overall identity being 84%. Furthermore, four Cys residues are completely conserved, which may participate in the formation of disulfide bridges. A homology search indicated that the G. candidum lipases and Candida cyclindracea lipase are homologous enzymes and that they are members of the cholinesterase family.     

In silico characterization of thermostable lipases

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.     

Highly selective synthesis of 1,3-oleoyl-2-palmitoylglycerol by lipase catalysis.

1,3-Oleoyl-2-palmitoylglycerol (OPO), an important structured triglyceride in infant nutrition, was synthesized by a two-step process in high yields and purity using sn1,3-regiospecific lipases. In the first step, tripalmitin (TP) was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn1,3-regiospecific lipases yielding the corresponding 2-monopalmitin (2-MP). The 2-MP was isolated in up to 85% yield and >95% purity by crystallization and esterified in the second step with oleic acid using the same lipases to form the structured triglyceride OPO in up to 78% yield containing 96% palmitic acid in the sn2-position. Water activity, solvent, as well as carrier for lipase immobilization strongly influenced the yield and purity of products in both steps. The best results were achieved with lipases from Rhizomucor miehei and Rhizopus delemar immobilized on EP 100 and equilibrated to a water activity of 0.43. Special emphasis was given to develop this process in solvents that are allowed to be used in foodstuffs and to perform the second step in a solvent-free system.     

Comparative and functional genomics of lipases in holometabolous insects

Lipases have key roles in insect lipid acquisition, storage and mobilisation and are also fundamental to many physiological processes underpinning insect reproduction, development, defence from pathogens and oxidative stress, and pheromone signalling. We have screened the recently sequenced genomes of five species from four orders of holometabolous insects, the dipterans Drosophila melanogaster and Anopheles gambiae, the hymenopteran Apis mellifera, the moth Bombyx mori and the beetle Tribolium castaneum, for the six major lipase families that are also found in other organisms. The two most numerous families in the insects, the neutral and acid lipases, are also the main families in mammals, albeit not in Caenorhabditis elegans, plants or microbes. Total numbers of the lipases vary two-fold across the five insect species, from numbers similar to those in mammals up to numbers comparable to those seen in C. elegans. Whilst there is a high degree of orthology with mammalian lipases in the other four families, the great majority of the insect neutral and acid lipases have arisen since the insect orders themselves diverged. Intriguingly, about 10% of the insect neutral and acid lipases have lost motifs critical for catalytic function. Examination of the length of lid and loop regions of the neutral lipase sequences suggest that most of the insect lipases lack triacylglycerol (TAG) hydrolysis activity, although the acid lipases all have intact cap domains required for TAG hydrolysis. We have also reviewed the sequence databases and scientific literature for insights into the expression profiles and functions of the insect neutral and acid lipases and the orthologues of the mammalian adipose triglyceride lipase which has a pivotal role in lipid mobilisation. These data suggest that some of the acid and neutral lipase diversity may be due to a requirement for rapid accumulation of dietary lipids. The different roles required of lipases at the four discrete life stages of holometabolous insects may also contribute to the diversity of lipases required by insects. In addition, insects use lipases to perform roles for which there are no correlates in mammals, including as yolk and male accessory gland proteins.     

Enantioselective recognition mechanism of secondary alcohol by surfactant-coated lipases in nonaqueous media.

The enantioselective recognition mechanism of secondary alcohol by lipases originated from Candida rugosa and Pseudomonas cepacia was elucidated on the basis of the kinetic study of the esterification of alcohol with lauric acid in isooctane. To obtain inherent kinetic parameters, we utilized a surfactant-coated lipase whose conformation is considered to be an "open" form in a homogeneous organic solvent. Based on the experimental results, the enantioselectivity of lipases was found to be derived from the difference in the V(max) values between the two enantiomers. The same result was observed when lipases of different origin and substrates with different molecular structures were applied. © 1999 John Wiley & Sons, Inc.     

基于不同标度伪氨基酸组成预测脂肪酶的类型

从序列出发预测某蛋白质是否为脂肪酶以及属于哪种脂肪酶具有重要的理论和应用价值.提出了基于Z标度和T标度的伪氨基酸组成方法提取序列特征值,采用了k-近邻算法回答上述问题.经参数选择后,三种方法在各自最优运行参数下,其1倍交叉验证的结果为:对脂肪酶和非脂肪酶预测精度分别为92.8%、91.4%和91.3%;对脂肪酶类型预测的精度分别为92.3%、90.3%和89.7%.其中基于Z标度伪氨基酸组成效果最佳.基于T标度的次之,但均明显优于其他6种常见的特征值提取方法,并对其可能的原因进行了探讨.     

Generation of lipase-containing static emulsions in silicone spheres for synthesis in organic media

Because of the broad versatility of lipases as biocatalysts, interest has for some years been focused on the improvement of the economy of processes using these enzymes, especially by appropriate immobilisation. In this study, a method was developed to emulsify aqueous solutions of lipase A of Candida antarctica (CALA) and lipase of Thermomyces lanuginosa (TLL) in silicone elastomers yielding elastic beads. The persistent water-organic interface created by this static emulsion enabled an improved performance of the immobilised lipases due to the well known fact that from a kinetic point of view these enzymes show a higher efficiency in biphasic than in monophasic systems. The entrapped lipases catalysed the esterification of octanol and caprylic acid in hexane with an activity that, related to the free enzyme, was enhanced about 31-fold for CALA and 250-fold for TLL. Comparison to the activity of the same enzymes in sol–gels revealed that for CALA immobilisation in static emulsion was the only method yielding active biocatalysts, whereas activation of TLL was in the same range in static emulsion and sol–gels. However, apparent activity of TLL in static emulsion was considerably higher than in sol–gels due to the feasible high enzyme loading. The results indicate that immobilising lipases as static emulsion is a technique suitable for biotechnological application. Moreover, a transfer to enzymes of other classes seems possible.     

Can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride?

To address the question can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride, we have characterised the selectivity of lipases from a wide range of oilseeds with diverse fatty acid compositions. For this study, a novel hydrolysis assay using a fully randomised oil, was developed. From some seed sources (e.g. Cinnamomum camphora), lipases show high preference for particular fatty acids, whilst from others (e.g. Brassica napus, Theobroma cacao80% saturated or 'unusual' fatty acids may contain lipases which exhibit selectivity. It therefore follows that since the majority of seeds are composed of unsaturated fatty acids, that highly selective lipases will be unusual in nature. However lipases from some species of the Cuphea genera show exceptionally high preference for particular fatty acids. For example, lipase from seeds of Cuphea procumbans has over 20-fold selectivity for C10:0.     

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.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

Acid Lipase Inhibitor in Chicken Plasma Identified as Apolipoprotein A-I

We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895–898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken.

The purified inhibitor migrated with the same mobility on SDS–PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.     

Lipoprotein lipases and vitellogenins in relation to the known three-dimensional structure of pancreatic lipase

A 106-residue region of high similarity between lipoprotein/pancreatic/hepatic lipases and Drosophila vitellogenins encompasses four beta-strands with all residues but one strictly conserved or conservatively replaced between the structures, and enclosing the putative active site Ser-152. The properties suggest a common folding pattern but the region probably does not function as an 'interface recognition site' in the lipases, although it might well bind fatty acid esters of ecdysteroids or single lipid molecules in the vitellogenins. C-terminally of this 106-residue region, a surface loop ('flap') covers the active site. No residue within this loop is conserved through all lipases, but adjacent segments exhibit 60-70% residue identity. Hepatic and lipoprotein lipases probably hydrolyze both soluble and emulsified substrates at the same site. They lack residues corresponding to a second active site postulated in pancreatic lipase to account for hydrolysis of soluble substrates. In addition, due to structural differences the flap could prevent entry of soluble substrate molecules into the active site of pancreatic lipase.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a Proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5 kDa) and 347 aa (38.6 kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

Lipase-catalyzed ethanolysis of borage oil: a kinetic study

Ethanolysis of borage oil catalyzed by two commercial lipases (from Pseudomonas cepacia and Candida antarctica) was studied using two different methodologies. Multiresponse models derived from a generalized Michaelis-Menten mechanism were utilized to describe the rates of formation of ethyl esters of the primary fatty acids present in the precursor oil. The relative rate constants determined for each of the fatty acid residues indicated that both lipases discriminate against release of gamma-linolenic acid residues under the reaction conditions studied. However, both lipases also released some of the residues located at the sn-2 position, indicating that for the experimental conditions studied, both lipases are nonspecific. Moreover, inactivation of Novozym 435 was rapid. Because the half-life of this enzyme (ca. 2.2 h) is comparable to the half-life of the reaction, the intrinsic reaction rate and enzyme deactivation must both be considered in modeling the kinetics.     

Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli

The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.     

Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.     

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family.

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three-dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10-residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.     

Lipase specificity towards eicosapentaenoic acid and docosahexaenoic acid depends on substrate structure

The fatty acid specificity of five lipases towards eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was evaluated in the hydrolysis of fish oil, squid oil and a model system. The model system contained methyl esters of EPA, DHA and palmitic acid. All the investigated lipases discriminated against both EPA and DHA more in the model system than in the natural oils. Thus both EPA and DHA were more easily hydrolysed from a glyceride than from a methyl ester. In the model system, the lipase from Candida rugosa showed the highest discrimination against DHA, while the lipases from Pseudomonas fluorescens and Pseudomonas cepacia discriminated against EPA the most. In a glyceride, the fatty acid specificity of lipases towards EPA and DHA was affected by the positional distribution of the fatty acids and the glyceride structure due to the regiospecificity and triglyceride specificity of the lipase. In the oils, the Pseudomonas lipases also discriminated against EPA the most, while DHA was initially discriminated the most by the lipase from Thermomyces lanuginosus. However, after longer reaction times the enrichment of DHA in the glyceride fraction of the oils was greatest for the lipase from C. rugosa.