Differential behaviour of Pseudomonas sp. 42A2 LipC,a lipase showing greater versatility than its counterpart LipA

Growth of Pseudomonas sp. 42A2 on oleic acid releases polymerized hydroxy-fatty acids as a result of several enzymatic conversions that could involve one or more lipases. To test this hypothesis, the lipolytic system of strain Pseudomonas sp. 42A2 was analyzed, revealing the presence of at least an intracellular carboxylesterase and a secreted lipase. Consensus primers derived from a conserved region of bacterial lipase subfamilies I.1 and I.2 allowed isolation of two secreted lipase genes, lipA and lipC, highly homologous to those of Pseudomonas aeruginosa PAO1. Homologous cloning of the isolated lipA and lipC genes was performed in Pseudomonas sp. 42A2 for LipA and LipC over-expression. The overproduced lipases were further purified and characterized, both showing preference for medium fatty acid chain-length substrates. However, significant differences could be detected between LipA and LipC in terms of enzyme kinetics and behaviour pattern. Accordingly, LipA showed maximum activity at moderate temperatures, and displayed a typical Michaelis–Menten kinetics. On the contrary, LipC was more active at low temperatures and displayed partial interfacial activation, showing a shift in substrate specificity when assayed at different temperatures, and displaying increased activity in the presence of certain heavy metal ions. The versatile properties shown by LipC suggest that this lipase could be expressed in response to variable environmental conditions.     

Optical Resolution of (±)-Metyrosine

A lipase gene (lip) and its activator gene (act) on a 2.9 kb BglII-EcoRI fragment from Pseudomonas sp. KWI-56 were cloned in Escherichia coli using pUC19 as a vector plasmid. From the sequencing results, the open reading frames of the lip and the act were found to contain 1092 and 1032 nucleotides, respectively. The act existed downstream of the lip with the same orientation. When the lip was expressed in E. coli using the lac promoter on the pUC plasmid vector, the lipase activity of E. coli carrying both the lip and the act was 200-fold greater than that carrying only the lip. This result suggested the act was important in the expression of the lip in E. coli.     

A new alkaline lipase obtained from the metagenome of marine sponge <Emphasis Type="Italic">Ircinia</Emphasis> sp.

Microorganisms associated with marine sponges are potential resources for marine enzymes. In this study, culture-independent metagenomic approach was used to isolate lipases from the complex microbiome of the sponge Ircinia sp. obtained from the South China Sea. A metagenomic library was constructed, containing 6568 clones, and functional screening on 1 % tributyrin agar resulted in the identification of a positive lipase clone (35F4). Following sequence analysis 35F4 clone was found to contain a putative lipase gene lipA. Sequence analysis of the predicted amino acid sequence of LipA revealed that it is a member of subfamily I.1 of lipases, with 63 % amino acid similarity to the lactonizing lipase from Aeromonas veronii (WP_021231793). Based on the predicted secondary structure, LipA was predicted to be an alkaline enzyme by sequence/structure analysis. Heterologous expression of lipA in E. coli BL21 (DE3) was performed and the characterization of the recombinant enzyme LipA showed that it is an alkaline enzyme with high tolerance to organic solvents. The isolated lipase LipA was active in the broad alkaline range, with the highest activity at pH 9.0, and had a high level of stability over a pH range of 7.0–12.0. The activity of LipA was increased in the presence of 5 mM Ca²⁺ and some organic solvents, e.g. methanol, acetone and isopropanol. The optimum temperature for the activity of LipA is 40 °C and the molecular weight of LipA was determined to be ~30 kDa by SDS-PAGE. LipA is an alkaline lipase and shows good tolerance to some organic solvents, which make it of potential utility in the detergent industry and enzyme mediated organic synthesis. The result of this study has broadened the diversity of known lipolytic genes and demonstrated that marine sponges are an important source for new enzymes.     

lip2, a novel lipase gene cloned from Aspergillus niger exhibits enzymatic characteristics distinct from its previously identified family member

We have cloned a novel lipase gene, lip2, from Aspergillus niger and expressed it in Escherichia coli. Upon purification of the recombinant Lip2 protein, its properties were characterized. In comparison with a previously identified lipase Lip1, both enzymes are acid lipases (optimal pH <6.5), Ca²⁺-dependent and PMSF-sensitive, but have different molecular weights (35 and 43 kDa), optimal substrate spectra (C10 and C8), optimal reaction temperatures (45 and 50°C) and thermal stability. Circular dichroism spectroscopy revealed that Lip2 contains a typical Ca²⁺-active site. This first report on the cloning of the Lip2 gene and its enzymatic characteristics may greatly facilitate its potential industrial application.     

A Bacillus subtilis locus encoding several gene products affecting transport of cations

A 3.6-kb DNA fragment from Bacillus subtilis was found to complement the K⁺ uptake-deficient Escherichia coli strain TK2420. Transformation with a pKLO61 plasmid harboring this fragment conferred the capacity to grow on a minimal medium containing only 10 mM K⁺. Insertional mutagenesis and subcloning identified a single gene responsible for the complementation. This gene coded for an apparent homolog of E. coli TrkA. Sequence analysis of the cloned region also revealed three additional open reading frames. These included: a gene encoding a homolog to the czcD gene product of Alcaligenes eutrophus, a lysR-type regulatory gene which was found to enhance Na⁺ resistance in E. coli NM81 (ΔnhaA) in a separate complementation test, and an orfD with no significant similarity to sequences deposited in Genbank.     

Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml^?1) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.     

Cloning,expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963

A novel lipase gene from an organic solvent degradable strain Pseudomonas fluorescens JCM5963 was cloned, sequenced, and overexpressed as an N-terminus His-tag fusion protein in E. coli. The alignment of amino acid sequences revealed that the protein contained a lipase motif and shared a medium or high similarity with lipases from other Pseudomonas strains. It could be defined as a member of subfamily I.1 lipase. Most of the recombinant proteins expressed as enzymatically active aggregates soluble in 20 mM Tris–HCl buffer (pH 8.0) containing sodium deoxycholate are remarkably different from most subfamily I.1 and I.2 members of Pseudomonas lipases expressed as inactive inclusion body formerly described in E. coli. The recombinant lipase (rPFL) was purified to homogeneity by Ni-NTA affinity chromatography and Sephacryl S-200 gel filtration chromatography. The purified lipase was stable in broad ranges of temperatures and pH values, with the optimal temperature and pH value being 55 °C and 9.0, respectively. Its activity was found to increase in the presence of metal ions such as Ca²⁺, Sn²⁺ and some non-ionic surfactants. In addition, rPFL was activated by and remained stable in a series of water-miscible organic solvents solutions and highly tolerant to some water-immiscible organic solvents. These features render this novel lipase attraction for biotechnological applications in the field of organic synthesis and detergent additives.     

Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris

Aspergillus niger lipases are important biocatalysts for a broad range of industrial applications. To enhance the expression level of a newly cloned lipase gene lip2 of A. niger in Pichia pastoris, we applied codon optimization and synthesized the full length codon-optimized gene by a two-step gene synthesis strategy. This strategy consists of an assembly PCR for several small DNA fragments and enzymatic digestion and ligation steps to ligate these fragments into the full-length gene. First, the full-length lip2 gene was divided into three fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) with the additions of proper restriction sites, and separately amplified by assembly PCR reactions. Second, three PCR amplified fragments were digested and ligated into the full-length lip2 gene. In the two-step gene synthesis, synthesis of smaller DNA fragments resulted in a significant lower level of nonspecific mismatching among oligonucleotides and a very low mutational rate of the PCR products, demonstrating the superiority of the method. When compared with the originally cloned lip2 gene of A. niger, the new codon optimized lip2 gene expressed at a significantly higher level in yeasts after methanol induction for 72 h, and both the enzyme activity and protein content reached maximal levels of 191 U/ml and 154 mg/1, with 11.6- and 5.3-fold increases, respectively.     

The XmnI restriction-modification system: Cloning,expression, sequence organization and similarity between the R and M genes

The xmnIRM genes from Xanthomonas manihotis 7AS1 have been cloned and expressed in Escherichia coli. The nucleotide (nt) sequences of both genes were determined. The XmnI methyltransferase (MTase)-encoding gene is 1861 by in length and codes for 620 amino acids (aa) (68660 Da). The restriction endonuclease (ENase)-encoding gene is 959 by long and therefore codes for a 319-aa protein (35275 Da). The two genes are aligned tail to tail and they overlap at their respective stop codons. About 4 × 10⁴ units/g wet cell paste of R·XmnI was obtained following IPTG induction in a suitable E. coli host. The xmnIR gene is expressed from the T7 promoter. M·XmnI probably modifies the first A in the sequence, GAA(N)₄TTC. The xmnIR and M genes contain regions of conserved similarity and probably evolved from a common ancestor. M·XmnI is loosely related to M·EcoRI. The XmnI R-M system and the type-I R-M systems probably derived from a common ancestor.     

Effects of carbon sources on extracellular lipase production and lipA transcription in Acinetobacter calcoaceticus

  The effects of lactic acid, oleic acid, gum arabic and their mutual interactions, on the production of extracellular lipase and the regulation of the expression of the lipase encoding gene (lipA) in Acinetobacter calcoaceticus were investigated. Formation of extracellular lipase was measured in culture supernatants of wild-type strain BD413 and expression of the lipA gene was monitored in vivo with a chromosomal fusion of lipA to lacZ. At the level of lipA expression only oleic acid had a significant effect; it lowered expression. Neither gum arabic nor lactic acid had any effect on lipA expression. On the other hand, the yield of extracellular lipase increased 2–5 times by the addition of gum arabic, possibly due to the release of cell surface-bound lipase. An interaction between oleic acid and lactic acid was also detected. Journal of Industrial Microbiology & Biotechnology (2000) 24, 25–30. Received 03 May 1999/ Accepted in revised form 04 September 1999     

Engineering of <Emphasis Type="Italic">Bacillus</Emphasis> lipase by directed evolution for enhanced thermal stability: effect of isoleucine to threonine mutation at protein surface

A lip gene from a Bacillus isolate was cloned and expressed in E. coli. By thermal denaturation analysis, T_1/2 of lipase was observed to be 7 min at 50°C with less than 10% activity after 1 h incubation at 50°C. To expand the functionality of cloned lipase, attempts have been made to create thermostable variants of lip gene. A lipase variant with an isoleucine to threonine amino acid substitution at the protein surface was isolated that demonstrated higher thermostability than its wild type predecessor. To explore the structure–function relationship, the lip gene product of wild type (WT) and mutant was characterized in detail. The mutation enhanced the specific activity of enzyme by 2-folds when compared with WT. The mutant enzyme showed enhanced T_1/2 of 21 min at 50°C. The kinetic parameters of the mutant enzyme were significantly altered. The mutant enzyme displayed higher affinity for substrate (decreased K _m ) in comparison to the wild type. The k _cat and catalytic efficiency (k _cat/K _m ) of mutant were also enhanced by two and five times, respectively, as compared with the WT. The mutation resides on the part of helix which is exposed to the solvent and away from the catalytic triad. The replacement of a solvent exposed hydrophobic residue (Ile) in WT with a hydrophilic residue (Thr) in mutant might impart thermostability to the protein structure.     

Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: Expression of a cold-active lipase with high specific activity

Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4 °C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5′ and 3′ regions of the coding sequence of the related protein.This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25 °C.     

Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli

To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml⁻¹ was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l⁻¹. A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml⁻¹ and GL-7-ACA acylase activity of 950 U l⁻¹ were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly.     

Evidence for intrachromosomal gene conversion in cultured mouse cells

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK^? cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK⁺ was applied to detect recombinational events between different TK^? genes. TK⁺ segregants were observed at a frequency of 10^?4–10^?5 in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK⁺ cells at frequencies of 10^?7 or less. Physical analysis of the TK⁺ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho I endonuclease digestion in each TK⁺ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.     

Cloning the trpR gene.

Summary In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage . From one such secondary site lysogen, strain SP265, one may isolate deletions that remove all or part of the trpR gene and other genes in the deo-thr sector of the E. coli chromosome. Specialized transducing phages harboring serB ⁺ and trpR ⁺ were liberated following induction of SP265. All such phages were N-defective, bio-type pseudolysogens whose DNA persisted in the form of plasmids. A collection of transducing phages, differing in their complement of bacterial DNA, was used to locate cleavage sites for bamHI, SalI, and PvuI within the deoD-trpR region of the E. coli genome. The trpR gene lies within a specific 950 base pair BamHI-PvuI segment.A 1250 base pair BamHI fragment carrying a functional trpR gene was cloned into the amplifiable plasmid pBR322. A single SalI site in this fragment was shown to lie within the trpR gene.In two situations where increased gene dosage might generate elevated amounts of Trp repressor (N-defective trpR ⁺ pseudolysogens and strains harboring pBR322 trpR ⁺ plasmids) neither tryptophan auxotrophy, enhanced sensitivity to DL-5-methyltryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.Journal Paper No. 7426 of the Purdue University Agricultural Experiment Station     

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.     

Studies on lipase directed export of Escherichia coli beta-lactamase in Staphylococcus carnosus

Summary The lipase (lip) gene of Staphylococcus hyicus was used to study the expression of the Escherichia coli -lactamase (bla) gene in S. carnosus. The bla gene, devoid of its promotor and most of the signal sequence, was fused to the lip structural gene at various positions. A set of 11 secretion vectors (pLL1 to pLL11) was isolated and analysed. All secretion vectors caused -lactamase production and activity in S. carnosus. However, the amount of hybrid proteins secreted was influenced by the length of the NH₂-terminal lipase portion. An increased concentration, comparable to that of the native lipase, of secreted lipase/-lactamase hybrid proteins was only found when the lipase portion of the construct comprised more than 101 amino acids of the NH₂-terminal region of the lipase preprotein; the proposed lipase signal peptide is 36 amino acids long. If the hybrid proteins constructed contained 101 or less amino acids of the NH₂-terminal lipase preprotein, only low amounts of secreted hybrid proteins were detectable and a significant portion of the hybrid proteins and -lactamase activity was found in the cellular fraction. The results indicate that the lipase possesses adjacent to the signal peptide a peptide domain that is essential for the secretion of the lipase/-lactamase hybrid proteins.Abbreviations Cm chloramphenicol - bla gene beta lactamase coding gene of Escherichia coli - lip gene lipase-coding gene of Staphylococcus hyicus - PA polyacrylamide - PAGE PA gel electrophoresis - SDS sodium dodecyl sulphate - [] indicates plasmid-carrier state     

Cloning and expression in Escherichia coli of the BanI and BanIII restriction-modification systems from Bacillus aneurinolyticus

The genes coding for the GGPyPuCC-specific (BanI) and ATCGAT-specific (BanIII) restriction-modification systems of Bacillus aneurinolyticus IAM1077 were cloned and expressed in Escherichia coli using pBR322 as a vector. The plasmids carrying the BanI and BanIII restriction-modification genes were designated pBanIRM8 and pBanIIIRM12, respectively. The restriction maps of these recombinant plasmids were constructed. These two plasmids were stably maintained in E. coli HB101. However, when E. coli JM109 was used as a host, pBanIIIRM12 was efficiently propagated but pBanIRM8 was not. The HB101 cells carrying only the restriction gene of BanIII were viable, but the BanI restriction gene carrier could not form colonies on agar plates. The growth of bacteriophage λ was strongly restricted only in the F. coli HB101 cells harboring pBanIRM8. These facts indicate that the BanI restriction enzyme is expressed and functions more efficiently than BanI modification enzyme in E. coli.