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Food endogenous enzymes have impacts on color, texture and flavor of foods during food processing or preservation. Cold plasma is a novel non-thermal food processing technology, which has been extensively studied for contamination elimination and shelf life extension of foods. Particularly, much work has been reported about the effects of cold plasma on enzyme activities and alterations about enzymes conformational structures. It is thus necessary to understand the mechanisms of actions and applications of cold plasma technology in the conformation of food endogenous enzymes. This review focuses on the applications of cold plasma for the inactivation of various endogenous enzymes, including peroxidase, polyphenol oxidase, lysozyme, α-chymotrypsin, alkaline phosphatase, and pectin methylesterase. The activations of several enzymes, such as superoxide dismutase, catalase, and lipase, by cold plasma are also discussed. In addition, this review highlights the transformation of conformational structures including primary and spatial structures induced by chemical reactive species during cold plasma treatments, such as reactive oxygen species and reactive nitrogen species, especially, active sites consisting of prosthetic group and specific amino acids are demonstrated. Both extrinsic and intrinsic factors affecting cold plasma treatments are also described. In general, cold plasma exhibits the ability to activate or inactivate enzymes activities with affecting the conformational structures of enzyme. Further studies should be focused on exploration at molecular level for providing more insight on the interaction mechanism. In addition, equipment and process parameters of cold plasma operation for different fresh food products should be optimized for achieving appropriate control on enzyme variation and obtaining maximum efficiency.

Triacylglycerols (TAG) are the major form of energy storage in plants. TAG are primarily stored in seeds and fruits, but vegetative tissues also possess a high capacity for their synthesis and storage. These storage lipids are essential to plant development, being used in seedling growth during germination, pollen development, and sexual reproduction, for example. TAG are also an important source of edible oils for animal and human consumption, and are used for fuel and industrial feedstocks. The canonical pathway leading to TAG synthesis is the glycerol-3-phosphate, or Kennedy, pathway, which is an evolutionarily conserved process in most living organisms. The enzymatic machinery for synthesizing TAG is well known in several plant species, and the genes encoding these enzymes have been the focus of many studies. Here, we review recent progress on the understanding of evolutionary, functional and biotechnological aspects of the glycerol-3-phosphate pathway enzymes that produce TAG. We discuss current knowledge about their functional aspects, and summarize valuable insights into genetically engineered plants for enhancing TAG accumulation. Also, we highlight the evolutionary history of these genes and present a meta-analysis linking positive selection to gene family and plant diversification, and also to the domestication processes in oilseed crops., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Terpene synthase (TPS) genes were isolated and functionally characterized from three traditional edible plants, Acanthopanax sciadophylloides ("Koshiabura") and Acanthopanax sieboldianus ("Himeukogi"), belonging to the family Araliaceae, and Curcuma zedoaria (zedoary, "Gajutsu"), belonging to the family Zingiberaceae. These plants emit characteristic fragrances and are used for traditional foods and folk medicines. From their fragrant tissues, i.e., sprouts of Araliaceae plants and developing rhizomes of zedoary, total RNAs were extracted and reverse transcribed. The resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. From the contig sequences obtained, full-length Tps genes were amplified by PCR with newly synthesized primer sets. The isolated full-length genes were introduced into engineered Escherichia coli cells, which can utilize acetoacetate to synthesize farnesyl diphosphate, the substrate for TPSs, through the mevalonate pathway. TPS products synthesized in the transformed E. coli cells were analysed by gas chromatography-mass spectrometry, nuclear magnetic resonance, and optical rotation. Consequently, the isolated Tps genes were found to encode β-caryophyllene synthase, germacrene D synthase, linalool/(3S)-(+)-nerolidol synthase, β-eudesmol synthase, and germacrene B synthase. These results lead us to expect that some of the effective ingredients in folk medicines are volatile terpenes and that intake of traditional foods including these edible plants would have some positive effects on our health.

High-power ultrasound is a versatile technology which can potentially be used in many food processing applications including food preservation. This is part 2 of a series of review articles dealing with the effectiveness of nonthermal food processing technologies in food preservation focusing on their effect on enzymes. Typically, ultrasound treatment alone does not efficiently cause microbial or enzyme inactivation sufficient for food preservation. However, combined with mild heat with or without elevated pressure (P ≤ 500 kPa), ultrasound can effectively inactivate enzymes and microorganisms. Synergistic effects between ultrasound and mild heat have been reported for the inactivation of both enzymes and microorganisms. The application of ultrasound has been shown to enhance the rate of inactivation of quality degrading enzymes including pectin methylesterase (PME), polygalacturonase (PG), peroxidase (POD), polyphenol oxidase (PPO), and lipoxygenase (LOX) at mild temperature by up to 400 times. Moreover, ultrasound enables the inactivation of relatively heat-resistant enzymes such as tomato PG1 and thermostable orange PME at mild temperature conditions. The extent to which ultrasound enhances the inactivation rate depends on the type of enzyme, the medium in which the enzyme is suspended, and the processing condition including frequency, ultrasonic intensity, temperature, and pressure. The physical and chemical effects of cavitation are considered to be responsible for the ultrasound-induced inactivation of enzymes, although the dominant mechanism depends on the structure of the enzyme.

Tea oil tree (Camellia oleifera, Co) provides a fine edible oil source in China. Tea oil from the seeds is very beneficial to human health. Fructose-1,6-bisphosphate aldolase (FBA) hydrolyzes fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, two critical metabolites for oil biosynthesis. The objectives of this study were to identify FBA genes and investigate the relationship between FBA gene expression and oil content in developing seeds of tea oil tree. In this paper, four developmentally up-regulated CoFBA genes were identified in Camellia oleifera seeds based on the transcriptome from two seed developmental stages corresponding to the initiation and peak stages of lipid biosynthesis. The expression of CoFBA genes, along with three key oil biosynthesis genes CoACP, CoFAD2 and CoSAD were analyzed in seeds from eight developmental stages by real-time quantitative PCR. The oil content and fatty acid composition were also analyzed. The results showed that CoFBA and CoSAD mRNA levels were well-correlated with oil content whereas CoFAD2 gene expression levels were correlated with fatty acid composition in Camellia seeds. We propose that CoFBA and CoSAD are two important factors for determining tea oil yield because CoFBA gene controls the flux of key intermediates for oil biosynthesis and CoSAD gene controls the synthesis of oleic acid, which accounts for 80% of fatty acids in tea oil. These findings suggest that tea oil yield could be improved by enhanced expression of CoFBA and CoSAD genes in transgenic plants.

Folate deficiency is a global health problem affecting many people in the developing and developed world. Current interventions (industrial food fortification and supplementation by folic acid pills) are effective if they can be used but might not be possible in less developed countries. Recent advances demonstrate that folate biofortification of food crops is now a feasible complementary strategy to fight folate deficiency worldwide. The genes and enzymes of folate synthesis are sufficiently understood to enable metabolic engineering of the pathway, and results from pilot engineering studies in plants (and bacteria) are encouraging. Here, we review the current status of investigations in the field of folate enhancement on the eve of a new era in food fortification.

Pigeonpea ( Cajanus cajan ) urease was immobilized on 1 cmx1 cm DEAE-cellulose paper strips. The optimum immobilization (51% activity) was observed at 4 degrees C, with a protein concentration of 1.0 mg/strip. The apparent optimum pH shifted from 7.3 to 6.8. Immobilized urease showed an optimal stability temperature of 67 degrees C, compared with 47 degrees C for the soluble urease. Time-dependent kinetics of the thermal inactivation of the immobilized urease were examined and found to be monophasic as compared with the soluble enzyme, which was biphasic. The Michaelis constant ( K (m)) for the DEAE-cellulose-immobilized urease was found to be 4.75 mM, 1.5 times higher than the soluble enzyme. Immobilized strips stored at 4 degrees C showed an increased half-life ( t (1/2)=150 days). There was practically no leaching of the enzyme from the immobilized strips over a period of 2 weeks. These strips were used for estimating the urea content of blood samples; the results obtained matched well with those obtained in a clinical laboratory through an Autoanalyzer(R) (Zydus Co., Rome, Italy). The easy availability of pigeonpea urease, the ease of its immobilization on DEAE-cellulose strips and the significantly lower cost of urease described in the present study makes it a suitable product for future applications in diagnostics.

Vitamin E has been touted as a panacea for age-related diseases, including cardiovascular disease and Alzheimer's disease and, thus, the demand for this nutraceutical has increased dramatically in recent years. This demand has, in turn, driven research to increase vitamin E production from plant sources. We have summarized the cumulative work of several groups in this area, describing the current status of efforts to bioengineer plants for elevated vitamin E content.

This review summarizes the most recent advances in our understanding of the haem enzyme ascorbate peroxidase. The aim is to show how the combined applications of protein engineering, mechanistic and structural studies can be used to provide an overall picture of enzyme catalysis, and how this information can be used to provide new insight into other, more well-characterized peroxidases (in particular cytochrome c peroxidase). It contains 212 references and covers literature up to March 2003.

Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy. The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation. Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features.

Plants continuously have to defend themselves against life-threatening events such as drought, mechanical damage, temperature stress, and potential pathogens. Nowadays, more and more similarities between the defense mechanism of plants and that of animals are being discovered. In both cases, the lipoxygenase pathway plays an important role. In plants, products of this pathway are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity. The first step in the lipoxygenase pathway is the reaction of linoleic or linolenic acids with molecular oxygen, catalyzed by the enzyme lipoxygenase. The hydroperoxy fatty acids thus formed are highly reactive and dangerous for the plant and therefore further metabolized by other enzymes such as allene oxide synthase, hydroperoxide lyase, peroxygenase, or divinyl ether synthase. Recently, these enzymes have been characterized as a special class of cytochrome P450 enzymes. Hydroperoxide lyases cleave the lipoxygenase products, resulting in the formation of omega-oxo acids and volatile C6- and C9-aldehydes and -alcohols. These compounds are major contributors to the characteristic "fresh green" odor of fruit and vegetables. They are widely used as food flavors, for example, to restore the freshness of food after sterilization processes. The low abundance of these compounds in nature and the high demand make it necessary to synthesize them on a large scale. Lipoxygenase and hydroperoxide lyase are suitable biocatalysts for the production of "natural" food flavors. In contrast to lipoxygenase, which has been extensively studied, little is yet known about hydroperoxide lyase. Hydroperoxide lyases from different organisms have been isolated, and a few genes have been published lately. However, the structure and reaction mechanism of this enzyme are still unclear. The identification of this enzyme as a cytochrome P450 sheds new light on its structure and possible reaction mechanism, whereas recombinant expression brings a biocatalytic application into sight.

The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of alpha-amylase, beta-amylase, exo-(1-->3, 1-->4)-beta-D-glucanase, and endo-(1-->3)-beta-D-glucanase were detected in these leaves. The endo-(1-->3)-beta-D-glucanase (EC 3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pI > or = 10.3. Its optimal activity occurred at pH 4.5 and 50 degrees C. Kinetic analysis gave Vmax, kcat, and Km values of 659 U/mg, 395 s(-1), and 0.42 mg/mL, respectively, for laminarin as substrate. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance liquid chromatography revealed that the enzyme hydrolyzes not only soluble but also insoluble (1-->3)-beta-glucan chains in an endo fashion. This property is unusual for endo-acting (1-->3)-beta-D-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.

Fatty acids are accumulated in triacylglycerols (TAGs), in specialized organelles of seeds named oil bodies. The major site of TAG accumulation is detected in developing seed and mesocarp of certain species. We have isolated two cDNAs encoding DGAT enzymes from olives. The deduced polypeptides differ by 26 amino acids in size. However, they have high homology and almost identical hydropathy profiles. The DGAT gene is expressed in all tissues that synthesize TAGs. However, higher levels of DGAT transcripts have been detected in seed tissues of developing olive drupe. DGAT expression and mRNA accumulation in drupe tissues is developmentally regulated. Each DGAT transcript shows a distinct profile of accumulation. The existence of two different DGAT transcripts might reflect two different enzymes with discrete function and/or localization.

A palmitoyl-acyl carrier protein (ACP) thioesterase cDNA clone was isolated from an oil palm cDNA library. The cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and a crude bacterial extract was assayed for acyl-CoA-hydrolysing activity. The recombinant enzyme was able to hydrolyse medium- and long-chain acyl-CoAs. Northern-blot analysis showed a high level of gene expression in leaf, flower and 15-, 17- and 18-week mesocarp tissues. Low-level gene expression was detected in germinated seedlings and 8- and 12-week mesocarp tissues, but no transcript was detected in any kernel tissues. Southern-blot analysis indicated the presence of a single gene and we have also isolated a genomic clone using the cDNA as a probe. Two genomic fragments were subcloned and a 7 kb contiguous stretch of the oil palm genome was sequenced. Comparison of this sequence with the cDNA sequence identified a putative 93 amino acid transit peptide, most of which is missing from the cDNA. The coding region of the gene consisted of seven exons and six introns.

The oil palm mesocarp contains an endogenous lipase which is strongly activated at low temperature. Lipase activity is thus very conveniently assayed by prior exposure of the fruits to low temperature. More than 100 oil palm samples from the germplasm collection of the Palm Oil Research Institute of Malaysia (now known as the Malaysian Palm Oil Board) were screened for non-esterified fatty acid activity using both the low-temperature activation assay and a radioactivity assay. The results showed good correlation between assay procedures. The different samples had a very wide range of lipase activity. Elaeis oleifera samples had significantly lower lipase activity compared with E. guineensis (var. tenera) samples. Even within E. guineensis (var. tenera), there was a wide range of activity. The results confirmed that lipase activity is genotype-dependent. Selection for lipase genotypes is thus possible and this will have obvious commercial value.

Lipoxygenase activity is critical for the development of flavours and aromas in olive oils. We have partly purified isoforms of molecular mass 95 kDa that have activity against linoleic or alpha-linolenic acids by a simple procedure from olive callus cultures.

Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H+ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes: the 14-3-3 content in the membranes increased 2- to 3-fold, while the H+-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H+-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H+ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins.

In the western Sahel and many other regions of sub-Saharan Africa, wild edible plants contribute significantly to human diets, not only during periods when cereal staples are scarce, but also when they are readily available. Although there have been published reports regarding the nutrient contents of these plant foods, little attention has been devoted to their content of antinutrients such as calcium chelators and inhibitors of the pancreas-derived proteases, trypsin and chymotrypsin, which are required for the efficient digestion and absorption of dietary proteins. In this study, aqueous extracts of 61 different leaves, seeds, fruits and flowers of edible plants gathered in the Republic of Niger were analyzed for their content of trypsin inhibitory substances using alpha-N-benzoyl-DL-arginine-p-nitroanilide as the substrate and bovine trypsin as the enzyme source. Twelve of these plant foods contained more antitrypsin activity than soybeans (1.34-8.18 vs. 1.32 microg trypsin inhibited/mg dry weight). Boiling for 3 min did not inactivate the antitrypsin activity in most of the plant extracts. These data confirm that more than half of the wild edible plant foods widely consumed by various populations who inhabit the western Sahel contain significant quantities of heat-stable trypsin inhibitor that could possibly compromise the bioavailability of proteins present in the diets of these populations.

Protein breakdown in the rumen is generally regarded as a two-stage process in which proteases produced by rumen microorganisms cleave plant protein into peptides and amino acids. However, many of the fiber-degrading cellulolytic species in the rumen are not in fact proteolytic, and the proteolytic activity of the entire rumen microbial population is only moderate when compared to the gastric and pancreatic secretions in the abomasum. Moreover, plant cell walls remain largely intact after initial chewing (particularly in cattle), presenting a physical barrier that must be breached prior to their effective colonization. The present study considers the hypothesis that the plant enzymes are at least partly responsible for herbage protein degradation in grazing ruminants. Ryegrass, red clover, white clover, and bird's-foot trefoil were incubated in the presence and absence of rumen microorganisms. The production of volatile fatty acids indicated the level of microbial activity, whereas the relative disappearance of the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco LSU) indicated proteolytic activity. In all incubations, the relative abundance of the Rubisco LSU decreased as the incubation progressed. When rumen microorganisms were absent, low molecular weight peptides (below 20 kDa) accumulated as the incubation progressed. This accumulation was not observed in the presence of rumen microorganisms. Therefore we suggest that the intrinsic plant proteases contribute to the initial stages of proteolysis of grazed herbage.

We have discovered a multienzymatic complex in fresh young sugarcane leaves. This complex is constituted of three enzymes: PEPcase, NADP-MDH and malic enzyme. After successive molecular sieving chromatography, we have obtained a highly purified sample of the complex which has a molecular weight of 711 kDa. Its functional interest has been evaluated by comparing the kinetic properties of the enzymes in their free forms to those in their complexed form. We show that the association of the three enzymes leads to important changes in their respective kinetic properties.

Peroxidase-rich fruits and vegetables are reputed to interfere with guaiac-based fecal occult blood tests. We added horseradish peroxidase to fecal samples and tested them with Hemoccult, Hemoccult SENSA(R), and hydrated Hemoccult. Positivity rates with Hemoccult and Hemoccult SENSA decreased rapidly as the time between smearing (preparation) and development increased, whereas they remained high with hydrated Hemoccult. For samples with added blood, positivity rates did not decrease with time. When 61 volunteers were tested on a standard restriction and on a challenge diet high in plant peroxidase, no positive results occurred during standard restriction. During the challenge diet, one volunteer was positive with Hemoccult and Hemoccult SENSA when development was delayed 24 h, and no volunteers were positive when it was delayed 48 h and 72 h. However, with hydrated Hemoccult, positives occurred in 13 of 61 volunteers at 24 h, 8 of 61 at 48 h, and 5 of 61 at 72 h. Thus, peroxidase-rich plant foods do not need to be excluded from the diet with Hemoccult and Hemoccult SENSA if development is delayed for at least 48 h after smearing. A delay of this duration will not solve the problem of plant peroxidase interference with hydrated Hemoccult.

Papain exhibits the characteristics of molten globule under acidic conditions as seen by circular dichroism, fluorescence and ANS binding. Between pH 2.0-2.5 the protein exhibits substantial secondary structure as indicated by far-UV CD spectrum but loses the persistent tertiary interactions of the native state. Enhanced binding of ANS to the state at pH 2.0 in relation to the native and unfolded states at neutral pH indicates a considerable exposure of aromatic side chains. Temperature and guanidine hydrochloride induced unfolding of papain in this state is noncooperative and the transition curves are biphasic in nature. As papain molecule consists of two domains, the results suggest that the domains unfold independently and sequentially., (Copyright 1998 Academic Press.)

Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.

Urease from pigeonpea was entrapped in polyacrylamide gel with 50% immobilization at 10% total monomer (containing 5% cross-linker) with high mechanical stability of the gel. Approximately 0.61 mg of protein could be loaded per 5 ml of gel. The immobilized enzyme had a t1/2 of approx. 200 days when stored in 0.1 M Tris/acetate buffer, pH 6.5, at 4 degrees C. The gel strips were used 4-5 times for urea assay over a period of 6 h with less than 2% loss of activity. Approximately 50% immobilization of urease in calcium alginate was observed at 3% alginate with 0.12 mg protein/ml alginate. The resultant enzyme beads showed a t1/2 of approx. 75 days when stored in 0.1 M Tris/acetate buffer, pH 6.5, at 4 degrees C. The beads were used 4-5 times for urea assay over a period of 6 h with about 40% loss of activity. In both cases, the enzyme activity was directly proportional to the amount of immobilized enzyme. There was practically no leaching of the entrapped enzyme over a period of 48 h from either of the polymers. Both the immobilized enzyme preparations were used to analyse the blood urea of some clinical samples from the University hospital. The results obtained compared favourably with those obtained by the usual method employed in the clinical pathology laboratory.

Two cDNA clones, pSoSPS1 and pSoSPS2, encoding sucrose-phosphate synthase (SPS) of sugarcane were isolated from a leaf cDNA library. Northern analysis revealed the transcript of SoSPS1 to be predominant in leaves, but that of SoSPS2 to be distributed not only in leaves but also in roots at a similar level. The transcript of SoSPS1 was markedly accumulated during greening of etiolated leaves, whereas that of SoSPS2 was constitutively expressed. These findings indicate that SPS in sugarcane is encoded by multiple genes, which show organ-specificity and are differentially regulated in response to light.

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of alpha-and beta-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.

A molecular biological study on limonene synthase that catalyzes the cyclization of geranyldiphosphate to yield the olefin 4(S)-limonene, an intermediate in the biosynthesis of a monoterpenoid, perillaldehyde, in Perilla frutescens Britton has been carried out. We isolated and characterized 10 cDNAs homologous to spearmint limonene synthase cDNA from an expression library constructed from cotyledons of a Perilla strain homozygous for two pairs of dominant genes, G and H, which are responsible for the formation of 4(S)-limonene. Two of these cDNA clones were functionally expressed in Escherichia coli, yielding enzymes which were catalytically active in generating 4(S)-limonene from geranyldiphosphate. The longest open reading frame in the representative cDNA clone PFLC1 consisted of 1812 nucleotides corresponding to 603 amino acids. Its identity to the ORFs of spearmint limonene synthase, tobacco 5-epi-aristolochene synthase, and castor bean casbene synthase were 65, 35, and 30%, respectively. Genomic Southern blot analyses of various genotypes (GGHH, GGhh, ggHH, and gghh) of P. frutescens suggested that more than one copy of the PFLC1 DNA exists in strains having the HH genotype. In contrast, no PFLC1 DNA sequences were found in the genomes of strains with the hh genotype that are incapable of producing cyclohexanoid monoterpenes for lack of limonene synthase activity. Northern blot analysis, using a PFLC1 3'-flanking region as a hybridization probe, showed that PFLC1 mRNA accumulated in all the aerial parts of the GGHH plants, particularly in the leaves. In the ggHH plants, on the other hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx.

We have isolated two cDNA clones (CDES01) and 04) encoding stearoyl-acyl carrier protein desaturase (SACPD; EC 1.14.99.6) from immature sesame seeds, and have analyzed accumulation levels of the corresponding mRNAs at different stages and organs in sesame. Clone CDES01 contains an open reading frame coding for a 396-amino acid protein of 45 kDa. CDES04 encodes a partial sequence of 141-amino acids. Deduced amino acid sequences of both clones exhibit a high identity to those of other plant SACPD cDNAs. Northern blots probed with CDES01 and CDES04 indicate that both messages accumulate in a seed-specific manner with a peak at 21 days after anthesis. However, expression patterns also indicate that regulation between CDES01 and CDES04 are slightly different. The CDES01 message accumulates at a low level in young leaves in addition to seeds, whereas accumulation of the RNA transcript corresponding to CDES04 is restricted to seeds. This observation implies the presence of at least two isozymes of SACPD having overlapping but slightly distinct functions in sesame.

A papaya cystatin (Cst)-encoding cDNA clone was isolated from a papaya leaf cDNA library and the active protein produced in Escherichia coli. The amino-acid sequence reveals a protein of 11,262 Da with over 40% identity to other published plant Cst. Unique features of the papaya Cst include a single Cys residue, variation in the papain-binding region, and the first reported inhibition of papaya proteinase IV by a Cst.

The multiplicity of DHA reducing proteins has been observed by means of a native-PAGE technique in several plant species, thus demonstrating, in accordance with recent literature, that several differently evolved proteins are likely to perform DHA reduction. Moreover, a research strategy coupling the use of native-PAGE with chromatographic separation procedure, tentatively performed in Solanum tuberosum, proved to be a useful tool for the separation and partial identification of the proteins involved in DHA reduction.

Immunoglobulins anti-endonuclease 32 kDa inhibit DNA synthesis. We observed that low concentrations of IgGs (about 50 micrograms IgG per 1 x 10(6) cell nuclei) temporary inhibit DNA synthesis. This inhibition concerns only the synthesis of DNA bound to the nuclear matrix (associated with isolated nuclear matrix). Preincubation of cell nuclei of White bush with IgG generates longer DNA fragments than in controls. Involvement of the 32 kDa endonuclease or an endonuclease-65 kDa protein complex from the nuclear matrix in replication or structural organisation of replication is considered.

It was shown that two of main enzymatic activities of plant nucleus and nuclear matrix, namely RNA-polymerasic and DNA-nucleolytic are susceptible to modulation with free fatty acids. The effects observed were dependent to both fatty acid length and degree of unsaturation. In nuclei a stimulation of nuclease activity was observed whereas in matrices short chain fatty acids inhibited the studied activity. The effect of fatty acids on RNA-polymerase was also different in nuclei and matrices. In nuclei all fatty acids studied inhibited polymerasic activity whereas in matrices short chain fatty acids stimulated this activity by up to 80% and the long chain fatty acids inhibited by up over 70%. The overall alteration of studied activities in nuclei and matrices by unsaturated fatty acids was similar. Nucleolytic activity was stronger inhibited and polymerasic activity was stimulated when the effects of linoleic and linolenic acids were studied. The results suggest possible importance of lipid component in nuclear matrix biological function.

A sugarcane (Saccharum sinensis Roxb.) cell line R932 resistant to growth inhibition by the proline analogue hydroxyproline was selected. R932 showed greater tolerance to PEG and low temperature stress than the donor. The line R932 showed larger accumulation of proline (x3.2) than the sensitive donor. In vitro enzymic analysis of gamma-glutamyl kinase involved in the proline biosynthesis showed that the enzyme in the R932 was less sensitive to inhibition by 50-mM exogenous proline than that in the donor. The results suggested that the change in gamma-glutamyl kinase properties might lead to excessive accumulation of proline, and the elevated proline contents might in turn lead to an increase in tolerance to environmental stress.

Chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme in the photosynthetic CO2 fixation pathway of C4-plants. The presence of a histidine at its active site has been proposed, based on sequence alignment with nonchloroplastic NAD-dependent malate dehydrogenases. In order to investigate this hypothesis, the effect of diethylpyrocarbonate on the sorghum leaf enzyme has been tested. Diethylpyrocarbonate strongly inhibited NADP-MDH activity, its effect being dramatically decreased in the presence of substrates and reversed by hydroxylamine. When diethylpyrocarbonate-inactivated NADP-MDH was cleaved with trypsin, one peptide with increased absorbance at 240 nm was detected. Sequencing of this peptide and analysis by mass spectrometry demonstrated that histidine 229 was modified by diethylpyrocarbonate. This amino acid was changed to an alanine by site-directed mutagenesis, and the modified protein was produced in Escherichia coli. It was similar to the plant enzyme except that it was totally inactive. Taken together, these results indicate that His229 is an essential residue in the active site of sorghum NADP-MDH.

Protopectinase-T (PPase-T), isolated from the culture broth of Trametes sanguinea, was able to release pectic substances from sugar beet by degrading rhamnogalacturonan. The smallest polysaccharide (SPS) that can serve as a substrate for PPase-T was prepared from sugar beet pulp by extraction with NaOH and digestion with alpha-L-arabinofuranosidase, alpha-L-arabinase, and beta-(1-->4)-D-galactanase. The reaction products of SPS with PPase-T were isolated by chromatography on DEAE-Toyopearl 650M and Toyopearl HW40-S columns, and analyzed by labeling the reducing ends with [3H]NaBH4 and by 13C NMR spectroscopy. The results indicated that PPase-T cleaved galactopyranosyluronic-rhamnopyranosyl linkages in SPS. Moreover, it was confirmed that the chemical structure of SPS contains a backbone based on units of [-->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rha p-(1-->].

The gene encoding neomycin phosphotransferase II (NPTII) has been used routinely as a selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the same coding sequence used for plant transformation was introduced into Escherichia coli to produce gram quantities of this protein. A unique, simple, rapid and efficient purification method was developed to purify thirty grams of NPTII protein. The microbially produced NPTII was shown to be chemically and functionally equivalent to the NPTII protein expressed in and purified from genetically engineered cotton seed, potato tubers and tomato fruit. Microbially produced and plant produced NPTII proteins have comparable molecular weights, immuno-reactivities, epitope structures, amino terminal amino acid sequences, biological activities and both lack glycosylation. Demonstrating the equivalence of NPTII protein from these sources establishes the validity of using the microbially produced NPTII to assess the safety of the NPTII protein produced in genetically engineered crops.

ATP and Mg2+ caused a transient increase in the intrisinc fluorescence of Rubisco activase which was inhibited by the presence of ADP. Only minor changes in fluorescence were observed with ATP or Mg2+ alone. The fluorescence increase was stabilized by addition of an ATP regenerating system or by substitution of ATP with a non-hydrolyzable analog, adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S). The initial rate of increase in fluorescence also depended on the concentration of protein in a manner consistent with second-order kinetics. The concentration dependence for the effect of ATP gamma S was sigmoidal, although at pH 8 the half-saturation requirements for both ATP gamma S (12 microM) and Mg2+ (1.5 mM) were not too dissimilar to the binding affinities (6 microM and 2 mM, respectively) determined indirectly with the fluorescent probe, 1-anilinonapthalene-8-sulfonate. However, the concentration dependence of ATP was about 5-fold higher than its binding affinity, also sigmoidal and quite similar to the concentration responses of ATP hydrolysis and activation of Rubisco by the protein. These characteristics of the intrinsic fluorescence indicate that it monitors a conformational change in the protein occurring after binding of the nucleotide and associated with increased aggregation. Direct evidence of increased aggregation in the presence of Mg2+ and ATP or ATP gamma S was obtained by gel-filtration chromatography. However, the apparent molecular mass was heterogeneous and also varied with temperature. The increased aggregation of the protein resulted in altered kinetic properties. The ATP hydrolysis activity of the protein increased and the half-maximal ATP concentration decreased as the protein concentration was increased in the assay. Also, a brief pretreatment of the protein with ATP and Mg2+ to increase aggregation eliminated the otherwise observed time delays in the Rubisco activation and ATP hydrolysis kinetics.

The reconstruction of the proteolytic system responsible for the profound hydrolysis of the main storage protein of buckwheat seeds, 13S globulin, has been carried out in vitro. It was found that subsequent action of a metalloproteinase from dry buckwheat seeds and cysteine proteinase and carboxypeptidase from growing seedlings led to a profound hydrolysis of the storage protein. The 13S globulin that was not hydrolyzed with metalloproteinase was not degraded by the proteolytic enzymes of growing seedlings. A scheme is proposed, which describes the functioning of the proteolytic system hydrolyzing the storage protein in growing buckwheat seedlings in vivo.

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.

Using the purification procedure of Fickenscher and Scheibe (Biochim. Biophys. Acta 749 (1983), 249-254) and a modification of the method, we produced a series of NADP-MDH forms from spinach and pea-leaf extracts that were characterized by a stepwise shortening of the N-terminal sequences. Limited proteolysis of the enzymes resulted in the generation of even shorter forms. Immunoprecipitation of the NADP-MDH from crude extracts revealed that the sequences of the intact enzymes from pea, spinach and maize started at a position (Ser) identical with that established for the Sorghum enzyme (Crétin, C., et al. (1990) Eur. J. Biochem. 192, 299-303). Spinach NADP-MDH isolated by conventional methods was shown to represent the intact form. Thus, the kinetic, regulatory and structural properties of the various truncated forms could be compared with those of an intact form. Removal of 5 or 11 amino acids, as occurred during isolation of the pea NADP-MDH, was without any significant effect. The enzymes were all dimeric and still exhibited the characteristic redox-regulatory properties. However, removal of 31 and 37 amino acids using aminopeptidase K resulted in the formation of active monomers characterized by only slightly lowered affinities towards the substrates, a shift of their pH optimum from 8 to 7, the loss of oxaloacetate inhibition and an increased maximal velocity. Although these forms lacked most or all of the N-terminal extra-peptide, including the 2 cysteines involved in redox-modification, they were still sensitive to the redox-potential. However, the low concentration of thiol required for immediate and complete restoration of any lost activity (40 mM beta-mercaptoethanol) suggested that this reaction might not be relevant for redox-regulation in vivo.

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48,539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.

A study of the biosynthesis of coenzyme A (CoA), a critical cofactor in the metabolism of lipids and other molecules in higher plants, was initiated. Pantothenate kinase was partially purified from spinach leaves. This enzyme was predominantly localized in the chloroplast with very little activity observed in the mitochondria or cytosol. DEAE-agarose chromatography resolved two pantothenate kinase activity peaks which differed in their requirement for reductant, stability upon boiling, and reactivity in the presence of spinach holo-acyl carrier protein (ACP) I. One active peak of this enzyme was further purified on Cibacron blue 3GA to yield a preparation containing pantothenate kinase enriched to 20% of the total protein within the fraction. Pantothenate kinase was inhibited by malonyl-CoA, but not by CoASH or acetyl-CoA, and the activity was stabilized by the phosphatase inhibitors sodium molybdate, sodium tungstate, and the phosphatase substrate glycerol 2-phosphate, but was inhibited by sodium fluoride. Further experiments demonstrated a linear increase in pantothenate kinase activity during spinach seed germination, consistent with a role for this enzyme in the developmental utilization of seed triacylglycerol.

The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.

Single crystals of ribonuclease Mc, a new class of plant ribonuclease from the seeds of the bitter gourd, were obtained from solutions of polyethylene glycol 8000 by the hanging-drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 67.28 A, b = 75.21 A, c = 38.54 A. The assumption of one monomer per asymmetric unit gives rise to a Vm value of 2.29 A3/Da. The crystals diffract beyond 2.0 A resolution and are suitable for high resolution X-ray structure analysis.

Polyclonal antibodies have been raised against the acyl-CoA elongase purified from leek epidermal cells. The antibodies recognize the fractions containing the elongating activity after DEAE or Ultrogel chromatography and their response with the other fractions is very low. The immune complex is immunoprecipitable with Protein A-Sepharose. 1% of the solubilized proteins from leek epidermis microsomes are immunoprecipitated. The immunoprecipitate contains an elongating activity which is 86 +/- 20-times that of the unbound fraction.

Protease inhibitor was isolated and purified from pigeon pea Cajanus capan. By using gel filtration analysis the inhibitor was found to have an Mr of 18,200. It inhibits trypsin competitively with a specific inhibitor constant Ki of 1.53 x 10(-7) M. The purified inhibitor produced a marked reduction in aflatoxin B1-induced beta-galactosidase activity in Escherichia coli PQ37. This reduction is independent of whether the protease inhibitor was added to the reaction medium prior to or after aflatoxin B1 activation. The observed reduction may therefore be a result of the inhibitor's activity on the RecA protease produced in response to aflatoxin B1-induced DNA damage in the bacteria.

Inhibition of ATPase activity by vanadate, having K1/2 of 0.5 mM, was demonstrated in the CF1-ATPase. The Ca(2+)-dependent ATPase activity of the isolated enzyme was inhibited in an allosteric manner by vanadate with a Hill coefficient of 3.19 +/- 0.6. Vanadate also inhibited ATPase and Pi-ATP exchange activities of the chloroplast membrane-bound enzyme. Using 51V NMR it was demonstrated that ATP caused partial release of about 1.87 equivalents while ADP caused additional binding of approximately 1.46 equivalents of vanadate, when added to a solution containing CF1 equilibrated with vanadate. The relevance of these results to a possible involvement of a pentacovalent phosphate as transition state intermediate in the hydrolysis of ATP by CF1-ATPase is discussed.