Proteolytic activites in cell extracts of Trypanosoma cruzi.

Cell extracts of culture forms of Trypanosoma cruzi are capable of hydrolysing substances belonging to 4 different groups of protease substrates: (a) substrates for trypsin-like enzymes: benzoyl-arginine-p-nitroanilide and benzoylarginine-naphtylamide; (b) substrates for aminopeptidases: leucyl, lysl and glutamyl-beta-naphtylamide; (c) a substrate fochymotrypsin-like enzymes: carbobenzoxy-L-tyrosine-p-nitorphenylester, and (d) a nonspecific substrate for a broad range of proteases: azocasein. Some physico-chemical characteristics of each enzymic reaction were studied. They were found to be distint enought to allow attributing each hydrolytic activity to a separate enzyme.     

Distinct substrate specificities and unusual substrate flexibilities of two hydroxycinnamoyltransferases,rosmarinic acid synthase and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl-transferase,from <Emphasis Type="Italic">Coleus blumei</Emphasis> Benth.

cDNAs and genes encoding a hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase (CbRAS; rosmarinic acid synthase) and a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CbHST) were isolated from Coleus blumei Benth. (syn. Solenostemon scutellarioides (L.) Codd; Lamiaceae). The proteins were expressed in E. coli and the substrate specificity of both enzymes was tested. CbRAS accepted several CoA-activated phenylpropenoic acids as donor substrates and d-(hydroxy)phenyllactates as acceptors resulting in ester formation while shikimate and quinate were not accepted. Unexpectedly, amino acids (d-phenylalanine, d-tyrosine, d-DOPA) also yielded products, showing that RAS can putatively catalyze amide formation. CbHST was able to transfer cinnamic, 4-coumaric, caffeic, ferulic as well as sinapic acid from CoA to shikimate but not to quinate or acceptor substrates utilized by CbRAS. In addition, 3-hydroxyanthranilate, 3-hydroxybenzoate and 2,3-dihydroxybenzoate were used as acceptor substrates. The reaction product with 3-aminobenzoate putatively is an amide. For both enzymes, structural requirements for donor and acceptor substrates were deduced. The acceptance of unusual acceptor substrates by CbRAS and CbHST resulted in the formation of novel compounds. The rather relaxed substrate as well as reaction specificity of both hydroxycinnamoyltransferases opens up possibilities for the evolution of novel enzymes forming novel secondary metabolites in plants and for the in vitro formation of new compounds with putatively interesting biological activities.     

Purification and properties of p-hydroxybenzoate hydroxylases from Rhodococcus strains

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl^– and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions.     

Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).     

Structure-function relationship of substrate length specificity of dextran glucosidase from <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an α-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms of substrate chain length specificity. SMDG efficiently hydrolyzes both short-and long-chain substrates, while O16G acts on only short-chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short β → α loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced by a smaller amino acid, alanine, asparagine or proline; and (ii) short β → α loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates, as implied by structural analysis.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage:concentrate ratios

Batch cultures of mixed rumen micro-organisms were used to study the effects of three fibrolytic enzymes (xylanase from Trichoderma viride (XYL) and fibrolytic enzymes from Aspergillus niger (ASP) and Trichoderma longibrachiatum (TR)) on the fermentation of three substrates composed of grass hay:concentrate in the proportions (dry matter (DM) basis) of 0.7:0.3 (HF), 0.5:0.5 (MF) and 0.3:0.7 (LF). Enzymes were characterized for xylanase, endoglucanase, exoglucanase and amylase activities, and were supplied at rates of 40 and 80 enzymatic units/g substrate DM. In 8 h incubations, all enzymes increased (P=0.048 to P<0.001) the true degradability of substrate DM and the production of acetate, propionate, total volatile fatty acids (VFA) and gas. After 24 h incubation, some of the observed effects disappeared, but all enzymes still increased (P=0.028 to P<0.001) the degradability of substrate acid detergent fibre and the production of acetate, propionate and total VFA. For all enzymes, the effects on ruminal variables were less marked at 24 than at 8 h of incubation. Only few significant (P=0.044 to P=0.001) enzyme × substrate interactions were detected, although the magnitude of the response for each substrate varied with the enzyme. When considering the amount of organic matter apparently fermented (OMAF) and the methane:OMAF ratio as main variables, TR80 produced the greatest increase in OMAF (17.0%) for HF substrate, with ASP80 and TR40 having similar values (11.1 and 12.6%), and XYL and ASP40 showing no effects (P>0.05). A decrease (P<0.05) of methane:OMAF ratio was only found for TR80 at 8 h (17.4%). All enzymes, with the exception of ASP40, increased (P<0.05) OMAF at 8 h for MF substrate (11.3–25.4%), TR80 showing the greatest response. After 24 h of incubation, both doses of XYL and TR increased (P<0.05) OMAF (mean value 8.2%) and decreased methane:OMAF ratio (mean value 9.5%). All enzymes increased significantly OMAF with LF substrate at 8 h (7.5–19.9%), but after 24 h no effect (P>0.05) was detected on OMAF and methane:OMAF ratio. In general, few differences were detected between both doses of enzymes, which indicate than the used enzymes would be effective in enhancing ruminal degradation of substrates at a dose lower than 80 enzymatic units/g substrate DM.     

Tendency of using different aromatic compounds as substrates by 2,4-DNT dioxygenase expressed by pJS39 carrying the gene dntA from Burkholderia sp. strain DNT

The substrate range of 2,4-dinitrotoluene (DNT) dioxygenase was investigated by measuring substrate-dependent O₂ uptake and maximum growth (expressed in A₆₀₀) on substrate-containing minimal medium. The control for each strain had no added particular substrate. The following aromatic compounds: catechol, α-naphthalene acetic acid, β-dimethylaminobenzaldehyde, 3,4-dinitrosalicylic acid, p-nitrophenol, naphthanol, o-anisic acid, salicylic acid, toluene, and benzoic acid, were tried as possible substrates. Considering all substrates used, only p-nitrophenol showed zero oxygen uptake rate and zero growth. This indicates that it was rather unlikely that p-nitrophenol is a substrate analog for 2,4-DNT. Catechol was clearly used as a sole carbon source by both wild-type Escherichia. coli (JM103) and the dnt transformant (JS39). Using α-naphthalene acetic acid and β-dimethylaminobenzaldehyde as substrates resulted in DNT dioxygenase oxygen uptake rates of 11.8 and 14?μM/hr/mg protein, respectively. However, using both compounds as a carbon source, JS39 had twice the growth rate of E. coli JM103. For the remaining six substrates tested (3, 4-dinitrosalicylic acid, p-nitrophenol, o-anisic acid, salicylic acid, toluene, and benzoic acid), there appeared to be growth advantages for JS39 (even though the growth in the presence of substrate was less than the controls) suggesting a situation similar to that described for α-naphthalene and β-dimethylaminobenzaldehyde above. Combining results from our assay with respirometry and growth-based experiments will allow a better understanding of the biochemical consequences of these interactions. These results suggest that DNT dioxygenase gene, dntA carried by JS39, and those potential genes for substrates-degraded enzyme(s) system could have a common root.     

A New Metabolite,Parasiticolide A,from Aspergillus parasiticus

Two β-glucosidases, G1 and G2, were purified from the culture supernatant of Penicillium herquei Banier and Sartory. Both the purified enzymes were homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights of G1 and G2 were estimated to be 125,000 and 122,000, respectively, by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. G1 and G2 contained 12.7% and 16.1% carbohydrate as glucose, and had isoelectric points of 5.02 and 5.24, respectively. Both enzymes had optimum pHs of 4.0~4.5 and optimum temperatures at 60°C, but pH - and thermo-stabilities of G1 were higher than those of G2. Both enzymes were active not only on p-nitrophenyl β-d-glucopyranoside, salicin, and the p-glucobioses tested but also on laminarin. CM-Cellulose was a very poor substrate for both enzymes. The activities of G1 toward the substrates except for laminarin and CM-cellulose were apparently higher than those of G2. Both enzymes acted on cellobiose to produce a transfer product.     

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.     

Functional studies of single-nucleotide polymorphic variants of human glutathione transferase T1-1 involving residues in the dimer interface

Glutathione transferase T1-1 catalyses detoxication and bioactivation processes in which glutathione conjugates are formed from endogenous and xenobiotic substrates, including alkylating agents and halogenated alkanes. Although the common null polymorphism of the human GSTT1 gene has been studied extensively, little is known about the consequences of GSTT1 single-nucleotide polymorphisms (SNPs). Here, we have examined the effects of two SNPs that alter amino acid residues in the dimer interface of the GST T1-1 protein and one that causes a conservative substitution in the core of the subunit. Variant proteins were expressed in an Escherichia coli strain in which the metabolism of ethylene dibromide to a glutathione conjugate leads to lacZ reversion mutations. We measured the kinetic properties of the enzymes with the characteristic substrate 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and determined the specific activities with several other substrates. Circular dichroism spectroscopy was used to measure protein thermal denaturation profiles. Variant T104P, which has been reported as inactive, showed weak but detectable activity with each substrate. Variant R76S was expressed at lower levels and showed much-reduced thermal stability. The results are interpreted in the context of the three-dimensional structure of human GST T1-1.     

Production of extracellular enzymes by different isolates of Pochonia chlamydosporia

For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p < 0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p < 0.05), and significant differences were found between isolates (p < 0.001) and the amounts of enzymes produced (p < 0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.     

Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation

Protease inhibitors represent a major class of drugs, even though a large number of proteases remain unexplored. Consequently, a great interest lies in the identification of highly sensitive substrates useful for both the characterization and the validation of these enzyme targets and for the design of inhibitors as potential therapeutic agents through high-throughput screening (HTS). With this aim, a synthetic substrate library, in which the highly fluorescent (L)-pyrenylalanine residue (Pya) is efficiently quenched by its proximity with the p-nitro-(L)-phenylalanine (Nop) moiety, was designed. The cleavage between Pya and Nop leads to a highly fluorescent metabolite providing the required sensitivity. This library, characterized by a water-soluble primary sequence Ac-SGK-Pya-(X)_n-Nop-GGK-NH₂, X being a mixture of 10 natural amino acids (A, I, L, K, F, W, E, Q, T, P) and n varying from 0 to 3, was validated using enzymes belonging to the four main types of hydrolases: serine-, metallo-, cystein-, and aspartyl-proteases. The selectivity of substrates belonging to this library was evidenced by characterizing specific substrates for the isoenzymes NEP-1 and NEP-2. This library easily synthesized is of great interest for the identification and development of selective and specific substrates for still uncharacterized endoproteases.     

Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase

Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry.     

Growth performance and pathway flux determine substrate preference of Alcaligenes eutrophus during growth on acetate plus aromatic compound mixtures

During batch growth of Alcaligenes eutrophus on various aromatic compounds in the presence of acetate, several distinct behaviour patterns were observed. The utilization of substrates of the meta pathway (phenol or p-cresol) was inhibited by acetate. When the aromatic was a substrate of the p-hydroxybenzoate branch of the ortho pathway, growth was mixotrophic, i.e. both substrates were consumed simultaneously. For the substrates of the gentisate pathway or the benzoate branch of the ortho pathway, substrate preference was governed by growth performance. Aromatic compounds enabling growth rate and yields higher than those obtained on acetate alone (i.e. benzoate, benzaldehyde, m-hydroxybenzoate and gentisate) inhibited acetate utilization, while acetate was the substrate consumed preferentially in mixtures containing aromatic compounds supporting only slow growth (i.e. benzoyl formate and 4-fluorobenzoate). Received: 18 April 1996 / Received revision: 9 July 1996 / Accepted: 15 July 1996     

Bioconversion of Pinoresinol Diglucoside and Pinoresinol from Substrates in the Phenylpropanoid Pathway by Resting Cells of Phomopsis sp.XP-8

Pinoresinol diglucoside (PDG) and pinoresinol (Pin) are normally produced by plant cells via the phenylpropanoid pathway. This study reveals the existence of a related pathway in Phomopsis sp. XP-8, a PDG-producing fungal strain isolated from the bark of the Tu-chung tree (Eucommiaulmoides Oliv.). After addition of 0.15 g/L glucose to Phomopsis sp. XP-8, PDG and Pin formed when phenylalanine, tyrosine, leucine, cinnamic acid, and p-coumaric acid were used as the substrates respectively. No PDG formed in the absence of glucose, but Pin was detected after addition of all these substrates except leucine. In all systems in the presence of glucose, production of PDG and/or Pin and the accumulation of phenylalanine, cinnamic acid, or p-coumaric acid correlated directly with added substrate in a time- and substrate concentration- dependent manner. After analysis of products produced after addition of each substrate, the mass flow sequence for PDG and Pin biosynthesis was defined as: glucose to phenylalanine, phenylalanine to cinnamic acid, then to p-coumaric acid, and finally to Pin or PDG. During the bioconversion, the activities of four key enzymes in the phenylpropanoid pathway were also determined and correlated with accumulation of their corresponding products. PDG production by Phomopsis sp. exhibits greater efficiency and cost effectiveness than the currently-used plant-based system and will pave the way for large scale production of PDG and/or Pin for medical applications.     

REVISITING RUBISCO AS A PROTEIN SUBSTRATE FOR INSECT MIDGUT PROTEASES

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.     

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi

The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller’s method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.     

Cloning,screening and characterization of ester hydrolases with enantioselectivity in typical bacteria

A broad exploitation of ester hydrolases from 7 typical bacteria was reported in this study. Thirty-two predicted esterases and hydrolases were cloned based on published genomic information. The catalytic activity of obtained clones was tested with p-nitrophenyl esters at various temperatures and pH values. The results indicated that eight enzymes presented with typical esterase activity on p-nitrophenyl butylate and caprylate. The result also showed that despite their great sequence difference, the eight enzymes shared similar properties (substrate specificity, optimal pH and temperature) with each other. Phylogenetic analysis revealed a close relationship between these eight enzymes and “true esterases”. As there was no information on enantioselectivity of these enzymes reported, the enantioselectivity of these enzymes to various chiral substrates was investigated for the first time. In comparison with commercial enzyme, Candida rugosa lipase (CRL), enzymes E12, E14, E18, E21 and E24 presented with equal or higher activity and enantioselectivity to the substrates. Furthermore, enzyme E14 (predicted carboxylesterase from Rhodobacter sphaeroides), E21 (S-formylglutathione hydrolase from Pseudomonas putida) and E24 (carboxylesterase from P. putida) presented with enantioselectivity in the resolution of methyl mandelate, 1-phenyethyl acetate and 2-octanol. These findings suggested that the novel ester hydrolases with high activity and enantioselectivity could be obtained from alpha/beta hydrolase family.     

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.