Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30?u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50?°C for 16?h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299?u/mg) with 300?mM Ca(2+) and was stable up to 60?°C with 150?mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.     

Characterization of a new cutinase from Thermobifida alba for PET-surface hydrolysis

A new cutinase from Thermobifida alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7° to 45.0°. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed Km values of 213 and 1933 μM and k_cat values of 2.72 and 6.03 s^?1 respectively. The substrate specificity was investigated with bis(benzoyloxyethyl)terephthalate (3PET) and Tha_Cut1 was shown to release primarily 2-hydroxyethyl benzoate. This contrasts with the well-studied Humicula insolens cutinase which preferentially liberates terminal benzoic acid from 3PET.     

Cutinolytic esterase activity of bacteria isolated from mixed-plant compost and characterization of a cutinase gene from Pseudomonas pseudoalcaligenes

The objective of the current study was to examine cutinolytic esterase (i.e., cutinase) activity by pseudomonads and bacteria isolated from mixed-plant compost. Approximately 400 isolates representing 52 taxa recovered from mixed-plant compost using cuticle baits, along with 117 pseudomonad isolates obtained from a culture collection (i.e., non-compost habitats), were evaluated. The ability of isolates to degrade the synthetic cutin polycaprolactone (PCL) was initially measured. Isolates from 23 taxa recovered from the compost degraded PCL. As well, isolates from 13 taxa of pseudomonads cleared PCL. Secondary screening measured esterase activity induced by the presence of apple cuticle using the chromogenic substrate p-nitrophenyl butyrate. Eighteen isolates representing four taxa (Alcaligenes faecalis , Bacillus licheniformis , Bacillus pumilus , and Pseudomonas pseudoalcaligenes) recovered from compost exhibited substantial esterase activity when grown with cuticle. In contrast, none of the pseudomonad isolates from the culture collection produced appreciable esterase activity. Although degradation of PCL was not correlated with esterase activity, isolates that were unable to degrade PCL failed to produce measureable esterase activities. Zymogram analysis indicated that the esterases produced by bacteria from compost ranged in size from 29 to 47 kDa. A gene from P. pseudoalcaligenes (cutA) was found to code for a cutin-induced esterase consisting of 302 amino acids and a theoretical protein size of 32 kDa. The enzyme was unique and was most closely related to other bacterial lipases (≤48% similarity).     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Crystallographic analysis of a thermoactive nitrilase

The nitrilase superfamily is a large and diverse superfamily of enzymes that catalyse the cleavage of various types of carbon-nitrogen bonds using a Cys-Glu-Lys catalytic triad. Thermoactive nitrilase from Pyrococcus abyssi (PaNit) hydrolyses small aliphatic nitriles like fumaro- and malononitryl. Yet, the biological role of this enzyme is unknown. We have analysed several crystal structures of PaNit: without ligands, with an acetate ion bound in the active site and with a bromide ion in the active site. In addition, docking calculations have been performed for fumaro- and malononitriles. The structures provide a proof for specific binding of the carboxylate ion and a general affinity for negatively changed ligands. The role of residues in the active site is considered and an enzymatic reaction mechanism is proposed in which Cys146 acts as the nucleophile, Glu42 as the general base, Lys113/Glu42 as the general acid, WatA as the hydrolytic water and Nζ_Lys113 and N_Phe147 form the oxyanion hole.     

A thermoactive uropygial esterase from chicken: purification, characterisation and synthesis of flavour esters

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.     

An airborne actinobacteria Nocardiopsis alba isolated from bioaerosol of a mushroom compost facility

Actinobacteria are widely distributed in many environments and represent the most important trigger to the occupant respiratory health. Health complaints, including hypersensitivity pneumonitis of the workers, were recorded in a mushroom compost facility (MCF). The studies on the airborne bacteria were carried out to find a possible microbiological source of these symptoms. Culture analysis of compost bioaerosols collected in different location of the MCF was performed. An assessment of the indoor microbial exposure revealed bacterial flora of bioaerosol in the mushroom compost facility represented by Bacillus, Geobacillus, Micrococcus, Pseudomonas, Staphylococcus spp., and actinobacterial strain with white aerial mycelium. The thermotolerant actinobacterial strain of the same morphology was repeatedly isolated from many locations in MCF: air, compost sample, and solid surface in production hall. On the base of complex morphological, chemotaxonomic, and phylogenetic characteristics, the isolate has been classified as Nocardiopsis alba. Dominant position of N. alba in microbial environment of the mushroom compost facility may represent an indicator microorganism in compost bioaerosol. The bioavailability of N. alba in mushroom compost facility creates potential risk for the health of workers, and the protection of respiratory tract and/or skin is strongly recommended.     

Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library

A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 °C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze β-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.     

Identification,molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus

Biotechnology Letters - We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family...     

Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost

Abstract During the first stage of the preparation of mushroom compost oxygen is believed to be readily available. However we measured methane in the evoking air above the compost piles and were able to isolate thermophilic methanogenic bacteria from this compost. The isolates grow only on H₂ and CO₂ as energy and carbon source and do not require complex factors for growth. On the basis of nutritional and morphological characteristics these methanogens were identified as strains of Methanobacterium thermoautotrophicum .     

Selection and characterization of feather-degrading bacteria from canola meal compost

Canola meal that contains a high level of protein (40% crude protein) was used as compost material for the isolation of feather-degrading bacteria. After 7 and 14 days, bacteria were isolated from compost amended and unamended with soil. Eighty bacterial isolates from canola meal compost were then grown on milk-agar and isolates that produced proteolytic enzymes were identified by the formation of clear haloes around the colonies. A feather medium was chosen for a secondary selection of feather-degrading isolates. Of the eight isolates that hydrolyzed milk protein, five isolates hydrolyzed feathers. Their keratinolytic activities were subsequently confirmed by an assay using azo-keratin as substrate. Seven of the eight bacteria that hydrolyzed milk protein were Bacillus spp, and all five isolates that hydrolyzed feathers were strains of Bacillus licheniformis. Protease inhibition studies indicated that serine proteases are the predominant proteolytic enzymes produced by these feather-degrading isolates. Received 02 April 1999/ Accepted in revised form 17 June 1999     

Establishment of phage and bacteria in a sterilized compost in a glasshouse

Summary Four strains ofRhizobium trifolii were individually inoculated to pots containing sterilized sand vermiculite mixture, half of which were seeded with red clover and half not. Pots were maintained in an ordinary glasshouse and watered with tap water.Phage was first detected after 4 months, and almost all pots contained one or more phages againstRhizobium trifolii after 9 months. The presence of plants increased the titer of phages in some pots inoculated withR. trifolii, but had no effect on the number of different phages.The pots also contained phages against soil bacteria other than Rhizobium indicating that phages are spread readily and constitute a normal part of the life cycle of soil bacteria.The number of different phages isolated from the pots was affected by the strain of Rhizobium used as inoculum.     

Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant

A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94 U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72 h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11 U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12 h and then with the AXE esterase for an additional 12 h. Ferulic acid accumulated to 396 μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24 h.     

Biochemical and molecular characterisation of a thermoactive, alkaline and detergent-stable lipase from a newly isolated Staphylococcus aureus strain

A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180 kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part of the gene encoding the mature SAL3 is cloned and sequenced. The deduced polypeptide sequence, corresponding to the mature SAL3, was very similar to the mature Staphylococcus simulans lipase sequence with two additional amino acid residues (LK) at the N-terminus of SAL3. The lipase activity is maximal at pH 9.5 and 55 °C. The specific activity of about 4200 U/mg or 3500 U/mg was measured using tributyrin or olive oil emulsion as substrate, respectively, at pH 9.5 and 55 °C.In contrast to other staphylococcal lipases previously characterised, SAL3 is found to be stable between pH 5 and 12 after 24 h incubation. The enzyme retained 50% of its activity after 60 min incubation at 60 °C. This novel lipase is able to hydrolyse its substrate in presence of various oxidizing agents as well as some surfactants and some commercial detergents, then SAL3 can be considered as a good candidate for industrial and biotechnological applications.     

Microbial analysis of a composted product of marine animal resources and isolation of bacteria antagonistic to a plant pathogen from the compost

A composting product of marine animal resources has been used as a fertilizer and a soil conditioner in Japan. This compost was produced by a repeated fed-batch fermentation system with three successive aerobic bioreactors. Composting temperature reached about 75 degrees C without heating. The bacterial diversity in this compost was investigated by denaturing gradient gel electrophoresis (DGGE) and sequence determination of the V3 region in the 16S rRNA genes. The sequence analysis showed that a majority of retrieved sequences corresponded to those of Bacillaceae, and we frequently found sequences similar to the 16S rDNA sequences of Bacillus thermocloacae and Bacillus thermoamylovorans. In addition, a bacterium antagonistic to a Fusarium strain was isolated from the compost. The isolate (Bacillus sp. NP-1) produced an antifungal compound, iturin A. These results suggest that this compost serves as a valuable source of plant growth-promoting rhizobacteria including the antifungal bacteria.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

Diversity analysis of antagonists from rice-associated bacteria and their application in biocontrol of rice diseases

Aims:  To understand the diversity, taxonomy and antagonistic potential of rice-associated bacteria, and to discover new bacteria for biocontrol of rice foliar pathogens.
Methods and Results:  Amplified ribosomal DNA restriction analysis (ARDRA), BOX-PCR and 16S rRNA gene sequence analysis were used to identify the diversity of 203 rice-associated antagonistic bacteria. Eleven potential biocontrol bacteria were used to test their biological control of rice blast in a natural field experiment. Eleven different genera were encountered in five divisions, including Bacilli , Alphaproteobacteria , Betaproteobacteria , Gammaproteobacteria and Deinococci . The most prominent genus in all microenvironments was Bacillus (68·5%). The efficacy of rice leaf blast biocontrol was 64·35% for strain 1Pe2, 57·86% for strain 2R37 and 56·44% for strain 1Re14.
Conclusions:  Biocontrol data from the field experiments demonstrated no positive correlation between antagonism, physiological characteristics and biocontrol efficacy. There was significant diversity among the rice-associated bacteria isolated from different microenvironments. The most prominent genus of all microenvironments was Bacillus . Brevibacillus brevis strain 1Pe2 and Deinococcus aquaticus strain 1Re14 have good potential for field application and commercial use.
Significance and Impact of the Study:  This is the first attempt to study the diversity and identification of rice-associated antagonistic bacteria from different microenvironments, and endophytic bacteria Deinococcus aquaticus strain 1Re14, Acidovorax sp. isolate 3Re21 and Brevibacillus brevis strain 1Pe2 are first reported as rice-associated bacteria.     

Identification of a novel carbohydrate esterase from Bjerkandera adusta: Structural and function predictions through bioinformatics analysis and molecular modeling

A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three‐dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p‐nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg^?1 protein on 2‐naphthyl acetate. No activity was detected on p‐nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N‐acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin. Proteins 2015; 83:533–546. © 2015 Wiley Periodicals, Inc.     

Flow cytometric detection of viable bacteria in compost

Abstract Flow cytometry employing several vital stains was used to study the colonisation of sterile compost by Bacillus subtilis 168 (pAB224). The dyes used included rhodamine 123 (Rh123), carboxyfluorescein diacetate (CFDA) and chemchrome B. The results demonstrated the ability of flow cytometry to detect and enumerate viable bacteria in filtered compost extracts. Flow cytometry was also used to detect and study the viability of an indigenous compost community. Although it was possible to detect a viable bacterial population, the numbers of viable bacteria estimated were significantly different to those estimated from cfu.     

节节麦的酯酶同工酶分析

对 30份不同来源的节节麦进行 4个时期的酯酶同工酶分析。结果表明 :不同来源节节麦的酯酶同工酶存在较大差异 ,共分成 1 5种基本类型。我国黄河流域的 1 0份节节麦被划分为 2个基本类型 ,但二者关系极为相近 ;新疆节节麦与之有一定差异 ,但在相似系数≤ 0 .82 0时可视为一类。所有材料在 4个时期之间没有出现一个完全相同的酶带类型 ,说明酯酶同工酶随发育时期而不断变化。