Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology

Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf‐enzyme products and their validation with 15 commercial microbial‐enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude‐leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast‐derived enzymes are stable in crude‐leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude‐extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker‐free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site‐specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf‐production platform offers a novel low‐cost approach by the elimination of fermentation, purification, concentration, formulation and cold‐chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.     

Efficacy of Melia azedarach L. extract on the malarial vector Anopheles stephensi Liston (Diptera: Culicidae)

Methanolic extracts of leaves and seeds from the chinaberry tree, Melia azedarach L. (Meliaceae) was tested against mature and immature mosquito vector Anopheles stephensi Liston (Diptera) under laboratory condition. The extract showed strong larvicidal, pupicidal, adulticidal, antiovipositional activity, repellency and biting deterency. The M. azedarach seed and leaf extracts were used to determine their effect on A. stephensi adults and their corresponding oviposition and consequent adult emergence in comparison with the control. The seed extracts showed high bioactivity at all doses, while the leaf extracts proved to be active, only in the higher dose. Results obtained from the laboratory experiment showed that the seed extracts suppressed the pupal and adult activity of A. stephensi even at low dose. In general, first and second instar larvae were more susceptible to both leaves and seed extracts. Clear dose-response relationships were established with the highest dose of 2% plant extract evoking 96% mortality. Entire development of A. stephensi was inhibited by M. azedarach treatment. Less expensive (less than 0.50 US dollars per 1 kg seed), naturally accruing bio-pesticide could be an alternative for chemical pesticides.     

A new enzyme for the interconversion of pyruvate and phosphopyruvate and its role in the C4 dicarboxylic acid pathway of photosynthesis

1. An enzyme was isolated from leaves of tropical grasses that catalyses the reversible conversion of pyruvate, ATP and orthophosphate into phosphopyruvate, AMP and pyrophosphate. A requirement for Mg(2+) could not be replaced by Mn(2+) or Ca(2+). 2. By replacing orthophosphate with [(32)P]orthophosphate or with arsenate, evidence was provided that the orthophosphate consumed appears in pyrophosphate. 3. Without Mg(2+) or 2-mercaptoethanol the enzyme was rapidly and irreversibly inactivated. EDTA only partially replaced the requirement for the thiol compound. The enzyme was considerably more unstable at 0 degrees or when frozen than at 22 degrees . Even with the best conditions devised the enzyme lost about 25% of its activity every 3hr. 4. The activities of the enzyme in leaves of the tropical grasses sugar cane (Saccharum hybrid var. Pindar), maize (Zea mays) and sorghum (Sorghum vulgare) were comparable with their maximum photosynthesis rates. The enzyme was not detectable in leaf extracts from several other plants. 5. Its role in photosynthesis is discussed.     

Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies.

Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase.     

In Vitro Stability and Inactivation of Peptide Hydrolases Extracted from Phaseolus vulgaris L.

Endopeptidase activity against azocasein had a higher temperatureoptimum (50°C) in leaf extracts than in cotyledon extracts(37°C). The temperature optima for aminopeptidase (46°C)and for carboxypeptidase (53°C) were similar in leaf andcotyledon extracts. The endopeptidase activity showed an excellentstability in crude extracts from leaves even at 37°C, whilethe endopeptidase in cotyledon extracts was less stable. Carboxypeptidasewas very stable in both leaf and cotyledon extracts. Aminopeptidasewas the least stable of the enzymes investigated and its inactivationrate depended on the source of the extract. A moderate stabilitywas observed in extracts of leaves or of ungerminated seeds,but this enzyme was rapidly inactivated in cotyledon extractsat pH 5.4. At pH 7.5 aminopeptidase remained active longer thanat pH 5.4. From experiments with mixed extracts it could beconcluded that in cotyledons an aminopeptidase inactivatingfactor was formed during germination. This factor was heat sensitive,excluded by Sephadex G-25, precipitated by 75% ammonium sulfateand inhibited by tosyl-L-lysine chloromethyl ketone. These datasuggest that the factor is a protein and considering the similarproperties it appears possible that it is the endopeptidaseformed during germination. (Received May 15, 1981; Accepted July 18, 1981)     

Lipoxygenase inhibition by novel fatty acid ester from Annona squamosa seeds

Studies on the seeds of Annona squamosa yielded a novel lipoxygenase inhibitor fatty acid ester, (+) - annonlipoxy (1). Compound 1 was screened for its enzyme inhibitory activity against lipoxygenase (E.C.1.14.18.1), exhibiting activity with IC(50) 69.05 +/- 5.06 microm. Baicalein (IC(50) 22.6 +/- 0.5 microm) was used as a positive control. Crude extracts of Annona squamosa fruit pulp and seeds were screened for its enzyme inhibitory activity against lipoxygenase and acetylcholinesterase. The crude ethanolic extract of fruit pulp and seeds of Annona squamosa also exhibited lipoxygenase activity with 22.2 and 26.7% inhibition, while the pet.ether extract of seeds of A. squamosa exhibited 52.7% inhibition at a concentration of 40 microg/200 ml. The crude ethanolic extract of seeds of Annona squamosa was also bioassayed for acetylcholinesterase inhibition and it was found inactive.     

Phosphotransacetylase from Clostridium acidiurici

The phosphotransacetylase from Clostridium acidiurici has two properties not observed for this enzyme in other bacteria: (i) it requires a divalent metal for activity, and (ii) it is not subject to uncoupling by arsenate. The enzyme has been obtained in highly purified form, with a specific activity 500-fold higher than crude extracts. Ferrous or manganous ions are required for maximal activity, with Mn(2+) being 50 to 75% as effective as Fe(2+). The acetyl group can be transferred from acetyl phosphate to coenzyme A in 20 mm arsenate without a net decrease in high-energy acyl linkages. Likewise, H(32)PO(4) (2-) will exchange with acetyl-PO(4) (2-) in the presence of arsenate without loss of acetyl phosphate. This suggests that the active site on the enzyme is capable of discriminating between phosphate and arsenate while permitting the reversible transfer of acyl groups between CoA and phosphate.     

Plant Seeds as Mineral Nutrient Resource for Seedlings--A Comparison of Plants from Calcareous and Silicate Soils

Mineral nutrients of seeds constitute a significant source ofessential elements to seedlings and developing individuals ofvascular plants. In spite of their potential ecological significance,seed nutrient pools have attracted little attention with respectto calcifuge–calcicole behaviour of plants. The objectivesof this study were, therefore, to compare concentrations of13 macro- and micronutrients (K, Rb, Mg, Ca, Mn, Fe, Co, Cu,Zn, Mo, B, P and S) in seeds and leaves of 35 mainly herbaceousvascular plant species growing on both limestone (calcareous)and silicate (non-calcareous) soils. Concentrations of Rb andCo in seeds of plants originating from limestone soils were,on average, about half of those from silicate soils. Concentrationsof Mn, Mg, Zn and P of seeds were, or tended to be, lower orslightly lower in limestone-soil plants, whereas mean Ca andMo concentrations were higher. Comparing seed and leaf concentrationsof the same species from limestone and silicate soils generallydemonstrated a high P enrichment ratio, but a particularly lowK enrichment ratio in seeds, valid for both types of soil. Itwas also apparent that Fe and Mn, micronutrients which are lessreadily solubilized and taken up by plants on limestone soils,had significantly higher seed:leaf concentration ratios in plantsfrom limestone than from silicate soils, whereas the oppositewas true for Ca. This indicates a ‘strategy’ tosatisfy the demand of seedlings for elements which are lessreadily available in the soil.Copyright 1998 Annals of BotanyCompany Seed, leaf, plant, nutrient, content, calcareous, silicate, acid, soil.     

Improvements in the detection of pea seed-borne mosaic virus by ELISA

The detection by ELISA of pea seed-borne mosaic virus (PSbMV) in pea leaves and seeds was improved by the addition of cellulase or Triton X-100 to the extraction fluid, probably because the additives aided the release of virus particles from host materials. With leaf extracts the additives were most effective at 0.1%. In initial tests cellulase was used with macerozyme, but the latter enzyme was then shown to decrease the effectiveness of cellulase. Triton X-100 was as effective as cellulase and the absorbance values obtained in ELISA of infected leaf extracts, diluted to 1/10 in extraction fluid containing the additive, were about six times greater than those of infected extracts diluted in normal extraction fluid. Five named isolates of PSbMV, in addition to the homologous isolate, were readily detected in infected leaves extracted in fluid containing Triton X-100. In tests on seeds and seedlings of seven infected seed lots of pea cv. Waverex, using Triton X-100 in the extraction fluid, PSbMV was detected in five times as many seeds as seedlings, probably mainly because in many infected seeds the virus was in the testa and not in the embryo. About 9% of infected seedlings were without recognisable symptoms 4 wk after emergence.     

Regulation of Mineral Redistribution in Pod-Bearing Soybean Explants

On the way from the roots to the seeds during reproductive developmentin soybean (Glycine max), a large proportion of the mineralspass through the leaves rather than travelling directly viathe xylem. This direct and indirect movement of mineral nutrientshas important implications for mineral redistribution, seeddevelopment and leaf senescence. Therefore, we have studiedthe role of cytokinin and mineral flux from the roots in regulatingmineral redistribution from the leaves to the seeds using explants,i.e. a leaf, a pod and a subtending stem segment, with theirbases immersed in treatment solutions. Thus, defined solutionscontaining cytokinin and/or minerals can be substituted forthe roots. When explants (excised at early-mid podfill) aresupplied H₂O only, leaf N, P, K, Mo, Mg, Zn, Fe, B, Cu, Ca,and Mn decline, ranging from 93% for Mo to 38% for Fe. In explantson H₂O, N, P, K, Mo, Mg, Zn, and Fe appear to be redistributedfrom the leaves to the seeds, while the B, Cu, Ca, and Mn lostfrom the leaves do not seem to move to the seeds. Although amixture of minerals resembling xylem sap can delay net lossof these elements from the leaves, it does not prevent the decreases.The cytokinin zeatin (4.6 µM) inhibits the loss of N,IC, Mo, Mg, Zn, Fe, B, Cu, Ca, and Mn from the leaves, but notthat of P. When combined with minerals, zeatin not only preventsthe loss of the minerals from the leaves but may even greatlyincrease them with the possible exception of Zn, Fe, and Cu.Supplying the mineral nutrient mixture increases the quantitiesof N, P, K, Mg, Cu, and B in the seeds but not Zn, Fe, Mn, Ca,and Mo. For those minerals, especially N, where zeatin inhibitsefflux from the leaves, it may reduce the amounts in the seeds,but it does not change P, K, Mg, and Ca. The accumulation andredistribution patterns of the different mineral nutrients showmany dissimilarities thereby suggesting differences in the controlof their distribution. Key words: Cytokinin, mineral transport, seed development, senescence     

Efficacy of two tropical plant extracts for the control of mosquitoes

The larvicidal activity of seed and leaf extracts of Calophyllum inophyllum and leaf extract of Rhinacanthus nasutus on the juveniles of Culex quinquefasciatus , Anopheles stephensi and Aedes aegypti was determined. Ethyl acetate (EA) soluble fractions of C. inophyllum and petroleum ether (PE) fraction of R. nasutus extracts showed very high larvicidal activity. Irrespective of the species of the mosquitoes tested, at concentrations ranging from 3.91 to 9.39 ppm, 9.04 to 35.49 ppm and 13.21 to 28.92 ppm of the active fractions of seed and leaf extracts of C. inophyllum and the leaf extract of R. nasutus , respectively, killed 50% of the treated larvae. The fractions also interfered with adult emergence. Exposure of the mosquitoes at any stage of their larval development to less than 0.86, 5.49 and 6.81 ppm of the active fractions of seed and leaf extracts of C. inophyllum and the leaf extract of R. nasutus , respectively, inhibited 50% of the treated larvae from emerging as adults. Experiments under laboratory and semi-field conditions showed that the activity of the extracts persisted for up to 10 days.     

A thermo-stable dehalogenase in the extracts fromPseudomonas sp (strain 19S)

Summary A thermo-stable dehalogenase was demonstrated in the crude extracts fromPseudomonas sp (19S). The ability of the enzyme to catalyze the dehalogenation of various halogen-substituted organic acids was investigated and the highest activity was found with monochloroacetate. The enzyme followed Michaelis-Menten kinetics, and the K_m for monochloroacetate was 0.2mM. Maximum activity was found at pH 10.5 and 60°C. The enzyme activity in the cell-free extract was unaffected by EDTA or by Mn, Zn, or Cu ions, but was dramatically reduced by HgCl₂ (70%) and Pb (NO₃)₂ (80%).     

Lipoxygenase inhibition by novel fatty acid ester from Annona squamosa seeds

Studies on the seeds of Annona squamosa yielded a novel lipoxygenase inhibitor fatty acid ester, (+) - annonlipoxy (1). Compound 1 was screened for its enzyme inhibitory activity against lipoxygenase (E.C.1.14.18.1), exhibiting activity with IC₅₀ 69.05 ± 5.06 μm. Baicalein (IC₅₀ 22.6 ± 0.5 μm) was used as a positive control. Crude extracts of Annona squamosa fruit pulp and seeds were screened for its enzyme inhibitory activity against lipoxygenase and acetylcholinesterase. The crude ethanolic extract of fruit pulp and seeds of Annona squamosa also exhibited lipoxygenase activity with 22.2 and 26.7% inhibition, while the pet.ether extract of seeds of A. squamosa exhibited 52.7% inhibition at a concentration of 40 μg/200 ml. The crude ethanolic extract of seeds of Annona squamosa was also bioassayed for acetylcholinesterase inhibition and it was found inactive.     

Fixation of carbon dioxide in the dark by the malic enzyme of bean and oat stem rust uredospores

Malic enzyme was found in both bean rust and cat stem rust uredospores. In bean rust uredospores it was shown to catalyze the formation of pyruvic acid from l-malic acid and to synthesize malic acid from pyruvic acid and CO₂. The malic enzyme from bean rust uredospores was specific for NADP and dependent on manganous ions for activity. The specific activity of the bean rust malic enzyme in crude extracts of ungerminated uredospores was approximately 6 times greater than that found in crude extracts obtained from germinated uredospores. The malic enzyme was also found in extracts obtained from healthy and rust-infected bean leaves. The specific activity of the enzyme was approximately 2 to 5 times greater in partially purified extracts obtained from the infected bean tissue at 6 days after inoculation. The specific activity of the malic enzyme in crude extracts obtained from oat stem rust uredospores was 2 times greater than the specific activity of this enzyme in crude extracts obtained from bean rust uredospores. Phosphoenolpyruvate carboxylase activity could not be demonstrated in crude extracts obtained from the ungerminated uredospores of the bean rust fungus.     

Effect of Eugenia winzerlingii Extracts on Bemisia tabaci and Evaluation of its Nursery Propagation

The development of plant-derived products to control Bemisia tabaci Genn. (Hemiptera: Aleyrodidae) is an urgent need for production of horticultural crops. Plant extracts and essential oils of several species of the genus Eugenia (Myrtaceae) have shown insecticidal activity. In southern Mexico, leaf extracts from Eugenia winzerlingii showed nematicidal effect but its insecticidal properties have not been explored. Therefore, the objective of this study was to evaluate the insecticidal effect of aqueous and organic extracts from E. winzerlingii leaves on B. tabaci egg, nymph and adult stages, and else to explore its nursery propagation. Then, extracts of this species were obtained by maceration with different polarity solvents. Bioassays were carried out on Capsicum chinense leaves. Mortality assays showed that aqueous and total crude ethanol (TCE) extracts necrosed the eggs (LC50 = 0.21% w/v and 4.68 mg/mL, respectively), whereas hexane, ethyl acetate (ETA), residual ethanol and TCE extracts affected the nymphs (LC50 = 0.25 - 4.85 mg/mL). In adults, oviposition inhibition by free choice assay indicated that TCE and ETA extracts had major activity (EC50 = 14.62 and 27.86 μg/cm2, respectively). On other hand, the sexual and vegetative propagation of E. winzerlingii showed that this species can be easily cultivated by seeds. In conclusion, extracts of E. winzerlingii leaves are highly effective in controlling B. tabaci. TCE extract, in particular, was toxic to three stages of B. tabaci. This plant could be a potential alternative to develop a novel botanical insecticide to manage this destructive pest.     

Role of leaf extracts of some medicinal plants in the management of fungal diseases of okra (Abelmoschus esculentus (L.) Moench)

In order to manage the fungal pathogens in okra, seeds of variety Arka Anamika were subjected to soaking treatment with the aqueous leaf extracts of Coleus aromaticus, Adathoda vesica, Vitex negundo, Solanum nigrum, Leucas aspera, Ocimum sanctum and Catharanthus roseus. Among the extracts used, C. aromaticus, V. negundo extracts were found superior in reducing the incidence of mycoflora. These leaf extract treatments also resulted in increased seed germination and vigour of the seedlings. Both in green-house and field conditions also these extracts were proved their efficient in the enhancement of biomass, number of leaves, number of seeds per fruit, seed density and ascorbic acid content in the raw fruits.     

A note on the nutrient and mineral composition of Leucaena leucocephala in Western Nigeria

Analysis of Leucaena leucocephala from various localities in Western Nigeria shows that young leaves, pods and seeds contain more crude protein but lower crude fibre and ether extract than mature ones. The crude fat component was higher in the seeds (4.6% and 5.5% for green and ripe seeds, respectively) than in any other edible part of the plant, while the green and brown seed coats contain the least amount of ether extract and total ash. The mineral composition of the dry matter of mature leaves is 2.8% calcium, 0.26% phosphorus, 0.37% magnesium, 1.78% potassium, 0.21% sodium and 0.12% iron.     

Biochemical Characterization of Lipoxygenase Lacking Mutants,L-l-less,L-2-less,and L-3-less Soybeans

In this paper, lipoxygenase lacking mutants were characterized in comparison with normal soybeans. The three lipoxygenase isozymes (L-l, L-2, and L-3) in crude seed extracts of normal soybeans were resolved clearly by an improved SDS-polyacrylamide gel electrophoresis. As expected, the three mutant types, L-l-less (P. I. 408251 and 133226), L-2-less (P. I. 86023), and L-3 less (Wasenatsu and Ichigowase) soybeans did not give L-l, L-2, and L-3 protein bands, respectively on a single dimension SDS gel.

An anti L-2 serum obtained from a rabbit reacted not only with the purified L-2 protein, but also partially with the purified L-l and L-3 proteins. By double immunodiffusion and immuno-disc gel electrofocusing analyses using the anti L-2 serum, L-l, L-2, and L-3 isozymes could not be detected in crude seed extracts from P.I. 408251, P. I. 86023, and Wasenatsu soybeans, respectively.

Three lipoxygenase activity peaks (L-l, L-2, and L-3 enzyme peaks) and a small unknown activity peak eluted right after the L-l peak were fractionated by DEAE-Sephacel column chromatography of crude seed extracts of Raiden (normal) soybeans. The chromatographic analyses have demonstrated that both the L-l and the unknown enzyme activities disappear completely in the L-l-less type soybean seeds, and that the L-2 and L-3 enzyme activities disappear completely in P. I. 86023 and the L-3-less type soybean seeds, respectively.     

Plants have isoforms for acyl carrier protein that are expressed differently in different tissues

Two closely related isoforms of acyl carrier protein (I and II) have been purified from spinach leaves. Differences in the N-terminal amino acid sequence and amino acid composition indicate that these proteins are coded by different genes. The two spinach leaf isoforms have been resolved and characterized by ion-exchange high-performance liquid chromatography, by thin layer isoelectric focusing, and by differences in mobility upon polyacrylamide gel electrophoresis. Both isoforms are effectively bound by antibodies raised to acyl carrier protein I. However, in competition experiments isoform II is only about 40% effective in blocking isoform I binding to antibody. Therefore, the isoforms are immunologically related but hold only some antigenic sites in common. Immunoblot analysis ("Western blotting") of crude spinach leaf tissue extracts probed with antibody to acyl carrier protein I reveals both isoforms. In addition, both forms of acyl carrier protein are present in dark-grown leaf tissue and in isolated chloroplasts. However, in spinach seeds and roots only acyl carrier protein II can be detected. Similar results are observed with extracts of castor oil plant leaf and seed. Therefore, the expression of the two acyl carrier protein isoforms is tissue specific.     

Nuclear and cytoplasmic "stay-green" mutations of soybean alter the loss of leaf soluble proteins during senescence

In soybean ( Glycine max [L.] Merr.) the homozygous combination of the recessive alleles dI and d2 (i.e., dldld2d2 ) at two different nuclear loci or the cytoplasmic gene cytG inhibit chlorophyll degradation during senescence; i.e. their leaves are green when they are shed. The main objectives of the present work were: (J) to determine whether these "stay-green" genes also interfere with the loss of the bulk of leaf soluble proteins and ribulose bisphospnate carboxylase/oxygensase (Rubisco; EC 4.1.1.39) during senescence and (2) to relate this to alterations in leaf proteolytic activity. Leaves of the normal. Yellowing cvs Clark and Harosoy lost about 90% of their soluble proteins before abscission. The abscising leaves of these cultivars contained no detectable Rubisco. By contrast, protein degradation was significantly less in leaves of near-isogenic lines of Clark and Harosoy carrying dIdId2d2 , with or without G (a dominant nuclear gene in a third locus causing green seed coats). These leaves still retained 50% of the soluble protein and large amounts of both subunits of Rubisco at the time of abscission. Alone, neither dl nor d2 had any effect. The cytoplasmic gene cytG slowed the loss of Rubisco. although eventually when leaves were shed they contained as little Rubisco as Clark. Despite inhibition (i.e. dIdId2d2 and GGdIdId2d2 ) or retardation (i.e. cytG ) of protein loss, these mutant genotypes did not differ from Clark in the breakdown of endogenous Rubisco by leaf extracts ("autodigestion"). The wild-type alleles in the dI and d2 loci may control a central regulatory process of the senescence syndrome.