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Metalotionein (MT) berpotensi besar dalam pemuliharaan alam sekitar disebabkan peranannya yang penting dalam metabolisme dan penyahtoksikan ion logam melalui proses pengkelatan ion logam. cDNA MT1 daripada Eleusine indica L. (eiMT1) telah diamplifikasi dan diekspreskan dalam Escherichia coli. Pengekspresan protein rekombinan diaruh menggunakan 1 mM IPTG pada 37°C selama 4 jam. Protein rekombinan dengan berat molekul 12 kDa telah diperoleh dan ditulenkan menggunakan dua kaedah penulenan iaitu kromatografi penukaran ion dan kromatografi penurasan gel. Analisis jujukan protein rekombinan telah mengesahkan bahawa protein eiMT yang diekspreskan mengandungi jujukan asid amino sebagaimana dijangkakan bagi protein yang dikodkan oleh cDNA eiMT1. Untuk menilai keupayaan protein eiMT ini memberikan toleransi terhadap logam berat bagi E. coli, sel bakteria yang ditransformasikan dengan plasmid yang membawa cDNA eiMT1 telah dikulturkan dalam media yang mengandungi ion tembaga (Cu), zink (Zn), besi (Fe), kromium (Cr) dan kadmium (Cd). Sel yang mengekspres eiMT didapati menunjukkan tahap pertumbuhan yang lebih tinggi berbanding sel jenis liar di dalam media yang mengandungi kesemua ion logam serta mampu bermandiri di dalam Cu, Zn, Fe dan Cr sehingga kepekatan 500 μM manakala bagi Cd sehingga kepekatan logam 400 μM. Hasil kajian ini menunjukkan bahawa eiMT membolehkan peningkatan toleransi sel E. coli terhadap logam berat dan memainkan peranan penting dalam menyingkirkan ion logam berlebihan. Hal ini mencadangkan bahawa eiMT juga kemungkinan mampu melakukan penyahtoksikan ion logam berlebihan dalam E. indica. Oleh itu, sel E. coli yang membawa cDNA eiMT di samping tumbuhan E. indica sendiri boleh diaplikasikan dalam proses remediasi dengan menyerap logam berat dalam persekitaran yang tercemar.

The Rhodophyta, Kappaphycus alvarezii, is widely cultivated for its highly valuable kappa carrageenan. K.alvarezii has also been known for its capacity to absorb and accumulate excess heavy metal from itssurrounding. The study was undertaken to unravel the effect of different cadmium treatments towards K.alvarezii at different exposure times by determining the biomass change percentage, algal cell viability andthe changes in chlorophyll and protein content at Cd concentrations ranging from 50 to 500 μM for 5, 10and 15 days. While there was no significant change in the percentage of the biomass change, the viabilityof the algal cells increased significantly with increasing Cd concentration and exposure time. This wasaccompanied by significant increase in protein content following similar pattern as shown by the viabilityof the cells. In contrast, chlorophylls a and b content decreased significantly with increasing Cdconcentration. The ability of the alga to uptake Cd was also increased with increasing Cd concentrationspossibly indicating that the ability of K. alvarezii to survive the high level of Cd might be due to its abilityto accumulate heavy metal. The results from this research revealed the unique manner in which K. alvareziiresponds to Cd stress which is unlike any other algae. Thus the findings obtained from this study maycontribute important information on how macroalgae adapt to heavy metal pollution.

The chemical formation of terpenes in nature is carried out by terpene synthases as the main biocatalysts to guide the carbocation intermediate to form structurally diverse compounds including acyclic, mono- and multiple cyclic products. Despite intensive study of the enzyme active site, the mechanism of specific terpene biosynthesis remains unclear. Here we demonstrate that a single mutation of the amino acid L454G or L454A in the active site of Persicaria minor β-sesquiphellandrene synthase leads to a more promiscuous enzyme that is capable of producing additional hydroxylated sesquiterpenes such as sesquicineole, sesquisabinene hydrate and α-bisabolol. Furthermore, the same L454 residue mutation (L454G or L454A) in the active site also improves the protein homogeneity compared to the wild type protein. Taken together, our results demonstrate that residue Leucine 454 in the active site of β-sesquiphellandrene synthase is important for sesquiterpene product diversity as well as the protein homogeneity in solution.

In this paper, the performance of ring cavity Brillouin Erbium fiber laser (BEFL) within L-band wavelength region are discussed. Introducing Erbium doped fiber as a secondary gain medium into conventional Brillouin fiber laser configuration is the best alternative to maximize the Brillouin Stokes power (output power) and to sustain the gain flatness. Few parameters comprising length of fiber, pump power and tunable laser source power are varied to obtain the optimum output power. From the simulation process, a maximum Brillouin Stokes power of 54.75 dBm is produced as 16 dBm Brillouin pump power and 1480 nm pump laser are injected to the configuration.

Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large-scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs) which were deposited in dbEST in the National Center of Biotechnology Information (NCBI). From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304), flavonol synthase, FLS (JG705819) and leucoanthocyanidin dioxygenase, LDOX (JG745247) were selected for further examination by quantitative RT-PCR (qRT-PCR) in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.

β-Galactosidase (EC 3.2.1.23) is a hydrolase which plays an important role in cell wall modification and fruit softening during ripening. In this study, three full-length β-galactosidase cDNA clones were successfully obtained from papaya mesocarp using different approaches. pPGBII which is 2,771 bp in size, was isolated from a papaya ripe mesocarp cDNA library using a heterologous probe. The other two cDNA clones, pBG(a) and pBG(b), which are 3,168 and 2,580 bp in size, respectively, were amplified from ripe papaya fruit using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) approaches. The pPBGII, pBG(a) and pBG(b) cDNAs, respectively, were expected to yield a putative mature polypeptide of 79, 91 and 56 kDa with isoelectric points (pI) of 8.12, 8.75 and 8.26. The genomic DNA gel blot analysis indicated that all of the β-galactosidase genes exist as multiple copies in the papaya genome hence they belong to a multigene family. All three cDNA clones were expressed in fruits during ripening with varying patterns. In mesocarp, pPBGII mRNA was only expressed during ripening and peaked at the half-ripe stage when the fruits undergo dramatic softening. Meanwhile, pBG(a) mRNA expression increased from the immature green stage to the half-ripe stage where it reached maximum level before declining. pBG(b) mRNA level accumulated abundantly at the mature green stage and decreased thereafter. Therefore, we suggest that pPBGII and pBG(a) cDNA clones characterized in this work may be involved in fruit softening during papaya ripening while the fruit-specific pBG(b) may be related to early ripening stage.

Elicitation, the plant-based biotechnology approach that utilizes the ability of plant roots to absorb and secrete a vast variety of bioactive compounds, was studied on Polygonum minus using jasmonic acid (JA) as an elicitor. To understand the overall molecular responses of P. minus roots to JA induction, a subtracted cDNA library was constructed using the suppression subtractive hybridization (SSH) method. From a total of 1,344 randomly selected colonies, 190 clones were shown to be differentially expressed using Reverse Northern hybridization. BLAST analysis revealed that clones were similar to genes associated with the biosynthesis of aromatic compounds through the oxylipin pathway, such as alcohol dehydrogenase and lipoxygenase. Putative clones involved in the shikimate pathway, including S-adenosyl-l-methionine synthetase and S-adenosyl-l-homocysteine hydrolase, were identified with predicted roles in phenylpropanoids’ biosynthesis. Genes responding to abiotic stress unique to JA elicitation, such as ELI3-1, glutathione S-transferase and peroxidase 1, were also identified. The kelch-repeat containing F-box family protein, a possible transcription factor in response to JA elicitation was also found. The results of the RT-PCR showed that the eight selected clones were strongly up-regulated, except for lipoxygenase, which showed a slightly higher expression of the transcript levels in response to the JA elicitation.

Burkholderia pseudomallei, a tropical pathogen and the causative agent of melioidosis, is known to secrete a serine metalloprotease (MprA) into the internal milieu of the infectious host. This protein has been shown to cause extensive damage to mammalian physiological proteins and its role in the pathogenesis of melioidosis is still under investigation. Previously, we have reported on the epitope mapping of B. pseudomallei protease that revealed a consensus peptide sequence of serine–methionine–alanine (SMA). The serine within this motif is involved in the serine protease catalytic triad and the SMA domain of the B. pseudomallei serine protease could be a major immunodominant domain. We undertook to further characterize the mprA gene and protein to gain deeper insight into the role and mechanism of this protein. Preliminary analysis showed that the crude lysate of expressed recombinant protease was able to hydrolyze gelatin, azocasein and skimmed milk. Further biochemical characterization demonstrated that the expressed protein maintained good proteolytic activity over a wide pH range of 5–11, is stable from 4 °C up to 68 °C and partially digested immunoglobulins A and G, transferrin and myosin. The proteolytic activity was strongly inhibited by phenylmethylsulfonyl fluoride. From our in vitro experimental evidence, we propose a proenzyme processing mechanism similar to that of subtilisin to produce the mature active protease.

Aquilaria species are very important economic plant for production of resin locally known as gaharu in Malaysia. There are five species that can be found in Malaysia and the most important Aquilaria species for gaharu production is A. malaccensis. Molecular markers for Aquilaria species are still insufficient and require more efficient, robust and reproducible molecular marker. Inter-simple sequence repeat (ISSR) markers are highly polymorphic and have high reproducibility which will be useful in areas of genetic diversity, phylogenetic studies, gene tagging, genome mapping and evolutionary biology in a wide range of crop species. Five selected ISSR primers were used to identify four Aquilaria species commonly found in Malaysia namely A. malaccensis, A. sub-integra, A. crassna and A. hirta. All the primers showed sufficient polymorphism to distinguish between the four species. Hence, the markers derived from ISSR can be used for molecular identification of Aquilaria spp. in ensuring homogenous species for plantation which may improve the quality of resin derived from known and certified materials.

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.

Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, addition of 15% (v/v) glycerol to the protein purification buffer and removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

The purpose of this research is to demonstrate surface roughness and chip formation by the machining of Aluminium silicon alloy (AlSic) matrix composite, reinforced with aluminium nitride (AlN), with three types of carbide inserts present. Experiments were conducted at various cutting speeds, feed rates, and depths of cut, according to the Taguchi method, using a standard orthogonal array L9 (34). The effects of cutting speeds, feed rates, depths of cut, and types of tool on surface roughness during the milling operation were evaluated using Taguchi optimization methodology, using the signal-to-noise (S/N) ratio. The surface finish produced is very important in determining whether the quality of the machined part is within specification and permissible tolerance limits. It is understood that chip formation is a fundamental element that influences tool performance. The analysis of chip formation was done using a Sometech SV-35 video microscope. The analysis of results, using the S/N ratio, concluded that a combination of low feed rate, low depth of cut, medium cutting speed, and an uncoated tool, gave a remarkable surface finish. The chips formed from the experiment varied from semi–continuous to discontinuous.

β-Galactosidases (EC 3.2.1.23) from ripe papaya (Carica papaya L. cv. Eksotika) fruits having galactanase activities were fractionated by a combination of cation exchange and gel-filtration chromatography into three isoforms, viz., β-galactosidase I, II and III. The native proteins of the respective isoforms have apparent molecular masses of 67, 67 and 55 kDa, each showing one predominant polypeptide upon SDS-PAGE of about 31 and 33 kDa for β-galactosidases I and III, respectively, and of 67 kDa for β-galactosidase II. The β-galactosidase I protein, which was undetectable in immature fruits, appeared to be specifically accumulated during ripening. The β-galactosidase II protein was present in developing fruits, but its level seemed to decrease with ripening. β-Galactosidase I seemed to be an important softening enzyme: its activity increased dramatically (4- to 8-fold) to a peak early during ripening and correlated closely with differential softening as related to position in the fruit tissue. The inner mesocarp tissue was softer, and its wall pectins were modified earlier and firmness decreased more rapidly during ripening compared to the outer mesocarp tissue. β-Galactosidase II also may contribute significantly to softening because of its ability to catalyse increased solubility and depolymerization of pectins as well as through its ability to modify the alkali-soluble hemicellulose fraction of the cell wall. The physiological significance of both β-galactosidase isoforms may partly be attributed to their functional capacity as β-(1,4)-galactanases.

Human secretory group IIa phospholipase A2 (hIIa-PLA2) contains a large number of prominent cationic patches on its molecular surface and has exceptionally high affinity for anionic surfaces, including anionic membranes. To identify the cationic amino acid residues that support binding of hIIa-PLA2 to anionic membranes, we have performed extensive site-directed mutagenesis of this protein and measured vesicle binding and interfacial kinetic properties of the mutants using polymerized liposomes and nonpolymerized anionic vesicles. Unlike other secretory PLA2s, which have a few cationic residues that support binding of enzyme to anionic membranes, interfacial binding of hIIa-PLA2 is driven in part by electrostatic interactions involving a number of cationic residues forming patches on the putative interfacial binding surface. Among these residues, the amino-terminal patch composed of Arg-7, Lys-10, and Lys-16 makes the most significant contribution to interfacial adsorption, and this is supplemented by contributions from other patches, most notably Lys-74/Lys-87/Arg-92 and Lys-124/Arg-127. For these mutants, complete vesicle binding occurs in the presence of high vesicle concentrations, and under these conditions the mutants display specific activities comparable to that of wild-type enzyme. These studies indicate that electrostatic interactions between surface lysine and arginine residues and the interface contribute to interfacial binding of hIIa-PLA2 to anionic vesicles and that cationic residues closest to the opening of the active-site slot make the most important interactions with the membrane. However, because the wild type binds extremely tightly to anionic vesicles, it was not possible to exactly determine what fraction of the total interfacial binding energy is due to electrostatics.

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).

The relationship between a putative metallothionein gene (MT) and exposure to cadmium (Cd) in blood cockles (Anadara granosa) is reported. In a 96-h dose-response experiment, mortality of cockles was found to proportionately increase in the range of 0.2-5.0 mg/l Cd with a calculated LC(50) of 2.94 mg/l. Exposure to 0.25 mg/l Cd for 16 days caused significant increases (P

Human nonpancreatic (group IIa) secreted phospholipase A2 (human sPLA2) is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated. It is probable that the enzyme normally acts as an acute-phase protein whose function is to facilitate the removal of infectious organisms or damaged host cells as part of the normal inflammatory response. The enzyme shows negligible activity with phosphatidylcholine (PC) vesicles and cell membranes, presumably reflecting the enzyme's lack of ability to bind productively to such condensed neutral interfaces. Mammalian pancreatic enzymes show modest activity with such interfaces and contain a unique tryptophan at position 3, which is part of the presumptive interfacial binding surface of these enzymes. Human sPLA2 does not contain tryptophan. The amphiphilic indole side chain of tryptophan is noted for its ability to penetrate the lipid interface of membranes, and tryptophan residues appear to be associated with the ability of lipases and phospholipases A2 to bind to and hydrolyze such interfaces. We have investigated in detail the properties of a V3W mutant of human sPLA2, which has a unique tryptophan on the interfacial binding surface of this enzyme. Although this enzyme shows a modest ( approximately 50%) reduction in activity when anionic substrates are used under standard assay conditions, the activity of the enzyme on phosphatidylcholine vesicles and cell membranes is dramatically increased compared with human sPLA2. This is particularly the case with small unilamellar vesicles of PC, where activity is enhanced over 250-fold compared to the almost zero activity expressed by human sPLA2. This enhanced activity is best explained by increased interfacial binding and activation of the V3W mutant and is not due to enhanced active-site binding and hydrolysis. The results highlight the important role that tryptophan residues can play in interfacial binding, particularly to condensed zwitterionic interfaces. The interfacial characteristics of the mutant human enzyme now resemble more closely the mammalian pancreatic enzymes that already have a tryptophan at position 3.

Phosphatidyl-choline (PC) vesicles and normal cell membranes are resistant to hydrolysis by human group II secreted PLA2, an enzyme that can attain high concentrations in extracellular fluids during many inflammatory processes. This highly cationic enzyme (pI>10.5) has a marked preference for anionic phospholipid interfaces, normally present within the cell. Therefore, the ability of one such anionic phospholipid, phosphatidic acid (PA), to enhance the activity of this enzyme has been investigated in detail. Results using model membrane vesicles and a continuous fluorescence assay highlight the ability of low molar proportions of PA to stimulate vesicle hydrolysis and this stimulation with increasing PA was parallelled by enhanced interfacial binding. In contrast, no productive binding of this enzyme could be detected to the surface of pure PC vesicles. The enhancement of hydrolysis in the presence of PA could also be achieved by prior treatment of pure PC vesicles with PLD, an effect that was dependent on the concentration of PLD and the duration of exposure to this enzyme. The fluorescence assay also allowed cell membranes and whole cells to be used as substrates and whereas such membrane presentations were refractory to hydrolysis by the human enzyme, prior treatment with PLD allowed hydrolysis using concentrations of this PLA2 that would be found extracellularly under inflammatory conditions. These results highlight the potential for PA, generated at the surface of the cell membrane, to be hydrolysed by extracellular human sPLA2 with the generation of lysophosphatidic acid and other lipid mediators and provides one possible mechanism whereby this human sPLA2 could become pro-inflammatory.

A gene coding for human non-pancreatic (group II) secreted phospholipase A 2 (hnpsPLA 2 ) has been constructed by the single-step ligation of twelve synthetic oligonucleotides. The gene has been cloned into a modification of the bacterial expression vector pET 11 which allows protein over-expression as inclusion bodies and enables about 3 mg/litre of pure refolded fully active enzyme to be obtained. The protein was expressed as a 1-Ala mutant (N1A) to allow removal of the initiator methionine by the Escherichia coli amino-peptidase. This mutant had very similar properties to the wild-type enzyme. A double mutant, N1A, V3W was also constructed and expressed in high yield. This tryptophan-containing mutant showed similar properties to the wild-type and N1A mutant but had about 40% of the activity under the assay conditions used. This tryptophan was used as a reporter group for interfacial binding and its properties were compared to those of the corresponding tryptophan in PLA 2 from porcine pancreas. Expression of the wild-type gene sequence for hnpsPLA 2 in E. coli gave the expected mutant protein still with the initiator methionine and with much reduced activity. Interfacial binding of all hnpsPLA2 mutants to anionic phospholipids was very similar when assessed by fluorescence methods. Comparisons of these mutants with the pancreatic enzyme revealed significant differences in terms of the effect of calcium on interfacial binding. The ability to express reasonably large amounts of the N1A mutant in E. coli will provide a basis for future site directed mutagenesis studies of this important human enzyme.

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain's endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25-1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.