Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Expression and Characterization of Fifteen <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> 99-880 Polygalacturonase Enzymes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Polygalacturonase (PG) enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae has a large PG gene family with 15 of 18 genes encoding unique active enzymes. The PG enzymes, 12 endo-PG and 3 exo-galacturonases, were expressed in Pichia pastoris and purified enabling biochemical characterization to gain insight into the maintenance of this large gene family within the Rhizopus genome. The 15 PG enzymes have a pH optima ranging from 4.0 to 5.0. Temperature optima of the 15 PG enzymes vary from 30 to 40°C. While the pH and temperature optima do little to separate the enzymes, the specific activity of the enzymes is highly variable ranging from over 200 to less than 1 μmol/min/mg. A general pattern related to the groupings found in the phylogentic tree was visible with the group containing the exo-PG enzymes demonstrating the lowest specific activity. Finally, the progress curves of the PG enzymes, contained within the phylogenetic group that includes the exo-PG enzymes, acting on trigalacturonic acid lend additional support to the idea that the ancestral form of PG in Rhizopus is endolytic and exolytic function evolved later.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Enzymology in vivo using NMR and molecular genetics

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substates and effectors concentrations and possible interaction with other cellular macromolecules, which may modify their kinetic properties. These is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non-invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measured the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo     

Thermostable enzymes for industrial applications

Summary The variety of thermostable (TS) enzymes has been steadily increasing for use in industrial applications, mainly as replacements for thermolabile (TL) enzymes. For example, TS amylases fromBacillus licheniformis andBacillus stearothermophilus have replaced TL amylases fromBacillus subtilis. TS enzymes also have advantages in new areas such as cyclodextrin production. The TS cyclodextrin glycosyl transferase (CGTase) fromThermoanaerobacter sp. (95°C optimum) gives a higher productivity than the CGTase fromBacillus macerans (55°C optimum). In the area of enzymatic bleach boosting of wood pulps, a TS xylanase (Myceliophera thermophila) would be advantageous over a TL xylanase (Trichoderma reesei), due to the high temperature of the incoming pulp. Not all TS enzymes are from thermophiles; the mesophileCandida antarctica produces a TS lipase which has a temperature optimum of 90°C when immobilized. The characterization of these enzymes will be described along with comparisons to some newly described TS enzymes.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM THE NERVOUS SYSTEM OF DIFFERENT INVERTEBRATES

—Choline acetyltransferase has been purified from three invertebrate species, namely snail (Helix aspersa), cockroach (Periplaneta americana) and horse shoe crab (Limulus polyphemus.) All three enzymes followed a Theorell-Chance enzyme mechanism with a sequential addition of the substrates. All three enzymes were activated by sodium and potassium chloride and inhibited by high concentrations of magnesium or calcium chloride. The apparent K_m for choline and acetyl-CoA was for snail: K_mch= 370 μm ,K_mAcetyl-CoA= 51μm ; cockroach:K_mCh= 550 μm , K_mAcely-CoA= 16 μm horse shoe crab:K_mCn= 2700 μm K_mAcctyl-coA= 68 μm CoA inhibited the enzymes competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibited the enzymes competitively with respect to choline and non-competitively with respect to acetyl-CoA. All the enzymes were inhibited strongly by 5,5′-dithiobis (2-nitrobenzoate), iodoacetate, acryloylcholine, chloracetylcholine and 3-bromacetonyltrimethyl-ammonium. The enzymes were only weakly inhibited by the styrylpyridine derivatives. The isoelectric points were 5.3 and 5.0 for the horse shoe crab and cockroach enzymes respectively. All three enzymes showed low affinity for a cation-exchanger (CM-Sephadex).     

Signal peptidases and signal peptide hydrolases

Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two fromE. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the –3, –1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes inEscherichia coli that can degrade a signal peptidein vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptidein vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

二氧化碳固定酶固碳效率及其对地衣芽孢杆菌代谢的影响

【背景】随着代谢工程与合成生物学的快速发展,通过对异养微生物进行代谢改造,利用生物法进行二氧化碳固定成为一个新的趋势。生物代谢途径中存在着大量固碳酶,这些酶尚待挖掘与应用,不同的酶固碳效率之间也缺少比较。【目的】在体外和体内对固碳功能和效率进行评价。【方法】选取3种固碳酶,即核酮糖1,5-二磷酸羧化加氧酶(ribose 1,5-diphosphate carboxylation oxygenase, RuBisCo)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PCK)和乙酰辅酶A羧化酶(acetyl coenzyme A carboxylase, ACC)在大肠杆菌中异源表达并纯化。测定纯酶的酶活,并建立无细胞催化实验-液质联用评价酶固碳能力的方法。在厌氧发酵条件下检测代谢指标,比较过表达固碳酶的地衣芽孢杆菌相较于原始菌的代谢差异。【结果】3种酶均实现可溶性表达,纯酶的比酶活分别为66.43、1.16和12.52 U/mg。通过体外无细胞催化实验,ACC在3种酶中表现出最高的固碳效率。分别过表达了PCK、ACC的重组地衣芽孢杆菌,厌氧发酵主产物乳酸的转化率从48.6%分别提升至58.1%和59.7%。【结论】可以通过体外、体内结合的方式对固碳酶的效率进行评价,该研究可为固碳酶在微生物遗传改造中理性、精准地应用提供参考。     

Identification and characterization of novel esterases from a deep-sea sediment metagenome

A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909 bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.     

Modified chitosan as a spacer for protein immobilization

Abstract

Three new, water-soluble, N-modified chitosan derivatives containing poly(ethylene glycol), dextran or inulin side chains were used as spacers for enzyme immobilization on a natural silk carrier. Amylolytic enzymes Maltogenase L and Promozyme D2, lipolytic enzyme Resinase HT and a complex of proteolytic enzymes from Streptomyces flavus 197 were immobilized. The activity of the immobilized enzymes and their stability during storage were similar to that obtained with synthetic polyamine—poly(ethylene imine) as a spacer. High operational stability of co-immobilized amylolytic enzymes Maltogenase L and Promozyme D2 in a continuous flow mini-reactor was demonstrated.     

Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus

Different strains of the thermophilic ascomycetous fungus Thermoascus aurantiacus have been reported in the literature to produce high levels of a variety of industrial interest enzymes (i.e. amylases, cellulases, pectinases and xylanases), which have been shown to be remarkably stable over a wide range of temperatures and appear to have tremendous commercial potential. Most studies on enzyme production by T. aurantiacus are carried out in chemically defined liquid medium, under conditions suitable for induction of a particular enzyme. A few studies have investigated the production of some enzymes by T. aurantiacus by solid-state fermentation, using lignocellulosic materials. The present review focuses on the enzymes produced by T. aurantiacus, their main kinetic parameters, and the effect of different culture conditions on production and enzyme activity. It also provides a view of the possible applications of T. aurantiacus enzymes, considering that this thermophilic fungus could comprise a potential source of thermostable enzymes.     

Primordial‐like enzymes from bacteria with reduced genomes

The first cells probably possessed rudimentary metabolic networks, built using a handful of multifunctional enzymes. The promiscuous activities of modern enzymes are often assumed to be relics of this primordial era; however, by definition these activities are no longer physiological. There are many fewer examples of enzymes using a single active site to catalyze multiple physiologically‐relevant reactions. Previously, we characterized the promiscuous alanine racemase (ALR) activity of Escherichia coli cystathionine β‐lyase (CBL). Now we have discovered that several bacteria with reduced genomes lack alr, but contain metC (encoding CBL). We characterized the CBL enzymes from three of these: Pelagibacter ubique, the Wolbachia endosymbiont of Drosophila melanogaster (wMel) and Thermotoga maritima. Each is a multifunctional CBL/ALR. However, we also show that CBL activity is no longer required in these bacteria. Instead, the wMel and T. maritima enzymes are physiologically bi‐functional alanine/glutamate racemases. They are not highly active, but they are clearly sufficient. Given the abundance of the microorganisms using them, we suggest that much of the planet's biochemistry is carried out by enzymes that are quite different from the highly‐active exemplars usually found in textbooks. Instead, primordial‐like enzymes may be an essential part of the adaptive strategy associated with streamlining.     

Vacuolar (lysosomal) trehalase ofSaccharomyces cerevisiae

In the yeastSaccharomyces cerevisiae thePEP4 gene product, protease A, is responsible for activating all soluble vacuolar (lysosomal) enzymes. These vacuolar enzymes remain inactive inpep4 mutants. Vacuolar trehalase activity was diminished in such mutants as well. This suggests that the vacuolar (lysosomal) trehalase is processed in a manner similar to other vacuolar enzymes inS. cerevisiae.     

Haemonchus contortus: The in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes

Kaur R. and Sood M. L. 1982. Haemonchus contortus: the in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes. International Journal for Parasitology 12: 585–588. Various enzymes of glycolysis (hexokinase, phosphoglucomutase, phosphoglucoisomerase, adolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase-enolase-pyruvate kinase and lactate dehydrogenase) have been detected in adult Haemonchus contortus. Low pyruvate kinase and lactate dehydrogenase activities suggested an alternate pathway from phosphoenolpyruvate. In vitro incubation had no significant effects on these enzymes and the worm was able to maintain normal metabolism for 12 h. Varying degrees of inhibition of glycolytic enzymes were observed with 50 μg/ml of dl-tetramisole and rafoxanide. The enzymes were inhibited to a greater extent by dl-tetramisole. These effects may block the glycolytic pathway and deprive the parasite of its ATP production.     

Enzymes of SPZ7 phage: Isolation and properties

Bacteriophage enzyme preparations exolysin and endolysin were studied. Exolysin (a phage-associated enzyme) was obtained from tail fraction and endolysin from phage-free cytoplasmic fraction of disintegrated Salmonella enteritidis cells. A new method for purification of these enzymes was developed, and their molecular masses were determined. The main catalytic properties of the studied enzymes (pH optimum and specificity to bacterial substrates) were found to be similar. Both enzymes lyse Escherichia coli cells like chicken egg lysozyme, but more efficiently lyse S. enteritidis cells and cannot lyse Micrococcus luteus, a good substrate for chicken egg lysozyme. Similar properties of exolysin and endolysin suggest that these enzymes are structurally similar or even identical.     

Molecular Cloning of the Genes for Pyruvate Kinase of Two Bacilli,Bacillus psychrophilus and Bacillus licheniformis,and Comparison of the Properties of the Enzymes Produced in Escherichia coli

The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by A TP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.     

Activity of bacteriolytic enzymes adsorbed to clays

Myxococcus virescens is able to produce extracellular bacteriolytic enzymes that are rapidly adsorbed on montmorillonite. These adsorbed enzymes are active and can be assayed by measuring the release of UV-absorbing materials in mixtures containingMicrococcus luteus cells. The activity of the clay-adsorbed enzymes is, however, considerably lower than that of the unadsorbed enzymes. Both unadsorbed and adsorbed enzymes have their maximum activity at approximately the same pH. At lower clay-enzyme concentrations, the activity is proportional to the concentration. If, however, increasing amounts of clay are added to a fixed volume of clay-enzyme suspension, the activity remains almost unchanged until a definite limit is reached, then the activity decreases rapidly. This limit was dependent only on the ratio of the amounts of enzyme and clay and not on the absolute concentration of the enzyme. The montmorillonite-adsorbed bacteriolytic enzymes fromM. virescens were not active against gram-negative bacteria, and no activity against purified cell walls fromM. luteus could be measured. Montmorillonite-adsorbed egg white lysozyme was not active onM. luteus cells.     

Broad-specificity GH131 β-glucanases are a hallmark of fungi and oomycetes that colonize plants

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β-1,4, β-1,3 and mixed β-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.