Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Metagenomic discovery of feruloyl esterases from rumen microflora.

Feruloyl esterases (FAEs) are a key group of enzymes that hydrolyze ferulic acids ester-linked to plant polysaccharides. The cow's rumen is a highly evolved ecosystem of complex microbial microflora capable of converting fibrous substances to energy. From direct cloning of the rumen microbial metagenome, we identified seven active phagemids conferring feruloyl esterase activity. The genomic inserts ranged from 1633 to 4143 bp, and the ORFs from 681 to 1359 bp. BLAST search reveals sequence homology to feruloyl esterases and esterases/lipases identified in anaerobes. The seven genes were expressed in Escherichia coli, and the proteins were purified to homogeneity. The FAEs were found to cover types B, C, and D in the feruloyl esterase classification system using model hydroxycinnamic acid esters. The release of ferulic acid (FA) catalyzed by these enzymes was established using natural substrates corn fiber (CF) and wheat insoluble arabinoxylan (WIA). Three of the enzymes were demonstrated to cleave diferulates and hence the capability to break down Araf-FA-FA-Araf cross-links. The wide variation in the sequence, activity, and substrate specificity observed in the FAEs discovered in this study is a confirming evidence that combined actions of a full range of FAE enzymes contribute to the high-efficiency fiber digestion in the rumen microbial ecosystem.

A novel method for rapid and sensitive metagenomic activity screening.

Direct cloning of metagenomes has proven to be a powerful tool for the exploration of the diverse sequence space of a microbial community leading to gene discovery and biocatalyst development. The key to such approach is the development of rapid, sensitive, and reliable functional screening of libraries. The majority of library screen have relied on the use of agar plates in petri dishes incorporating the target enzyme substrate for activity detection of positive clones (Iqbal et al. [1], Knietsch et al. [2], Popovic et al. [3]). In this article, a novel method is described consisting of: (1) formulation and application of substrate gel microtiter assay plates, (2) screening of libraries of clones in split pools in the wells of the assay plate, and (3) progressive enrichment and isolation of individual positive clones. The method has been successfully used in the rapid discovery of novel genes and enzymes from rumen microbial metagenome with high efficacy. •Novel substrate gel assay plates for activity screening with localized and intensified signals.•Rapid and complete screening of library clones in split pools.•Progressive enrichment scheme as a refining step for isolating target gene.

Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan.

Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic deesterification was monitored by (1)H NMR spectroscopy and evaluated on the basis of the decrease of the signal of the ester methyl group and increase of the signal of methanol. The results show for the first time the action of enzymes on polymeric substrate, which imitates more closely the natural substrate in plant cell walls than the low molecular mass artificial substrates used up to present.

Cloning of a Novel Feruloyl Esterase from Rumen Microbial Metagenome for Substantial Yield of Mono- and Diferulic Acids from Natural Substrates.

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone released ferulic acid (FA) and diferulic acid (diFA) from wheat insoluble arabinoxylan (WIA) and other natural substrates. The diFA released was confirmed by mass spectrometry. A maximum of 205±5.7 µg FA and 0.84±0.1 µg diFA were released (37°C, pH 6.5, 2 hr) when a saturating amount of RuFae4 (23 nmole for 100 mg WIA) was used. These yields represent 48.3% of FA, and 6.6% of diFAs present in the WIA substrate. Addition of GH10 endoxylanase (EX) to RuFae4 both at 1 nmole concentrations increased the release of FA and diFAs by 17 and 10 fold, respectively. Addition of GH11 EX resulted in smaller increase in the amount of both FA and diFAs. Applying additive amount of the two enzymes did not lead to additive increase in the product yields, suggesting that it was primarily the GH10 enzyme contributing synergism to FA/diFA release in mixed reactions.

Structural basis for xyloglucan specificity and α-d-Xylp(1 → 6)-D-Glcp recognition at the -1 subsite within the GH5 family.

GH5 is one of the largest glycoside hydrolase families, comprising at least 20 distinct activities within a common structural scaffold. However, the molecular basis for the functional differentiation among GH5 members is still not fully understood, principally for xyloglucan specificity. In this work, we elucidated the crystal structures of two novel GH5 xyloglucanases (XEGs) retrieved from a rumen microflora metagenomic library, in the native state and in complex with xyloglucan-derived oligosaccharides. These results provided insights into the structural determinants that differentiate GH5 XEGs from parental cellulases and a new mode of action within the GH5 family related to structural adaptations in the -1 subsite. The oligosaccharide found in the XEG5A complex, permitted the mapping, for the first time, of the positive subsites of a GH5 XEG, revealing the importance of the pocket-like topology of the +1 subsite in conferring the ability of some GH5 enzymes to attack xyloglucan. Complementarily, the XEG5B complex covered the negative subsites, completing the subsite mapping of GH5 XEGs at high resolution. Interestingly, XEG5B is, to date, the only GH5 member able to cleave XXXG into XX and XG, and in the light of these results, we propose that a modification in the -1 subsite enables the accommodation of a xylosyl side chain at this position. The stereochemical compatibility of the -1 subsite with a xylosyl moiety was also reported for other structurally nonrelated XEGs belonging to the GH74 family, indicating it to be an essential attribute for this mode of action.

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases.

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a type C feruloyl esterase. The RuFae2 alone released ferulic acid from rice bran, wheat bran, wheat-insoluble arabinoxylan, corn fiber, switchgrass, and corn bran in the order of decreasing activity. Using a saturating amount of RuFae2 for 100 mg substrate, a maximum of 18.7 and 80.0 µg FA was released from 100 mg corn fiber and wheat-insoluble arabinoxylan, respectively. Addition of GH10 endoxylanase (EX) synergistically increased the release of FA with the highest level of 6.7-fold for wheat bran. The synergistic effect of adding GH11 EX was significantly smaller with all the substrates tested. The difference in the effect of the two EXs was further analyzed by comparing the rate in the release of FA with increasing EX concentration using wheat-insoluble arabinoxylan as the substrate.

Comparative characterization of a bifunctional endo-1,4- ß-mannanase/1,3-1,4- ß-glucanase and its individual domains.

A fusion gene isolated from a microbial metagenome encodes a N-terminal endo-1,4- ß-mannanase and a C-terminal 1,3-1,4- ß -glucanase,. The full-length gene and the individual N- and C-domains were separately cloned and expressed in E coli. The purified whole enzyme hydrolyzed glucomannan, galactomannan, and ß-glucan with Km and kcat values 2.2, 2.6, 3.6 mg/ml, and 302, 130, 337 min -1 , respectively. The hydrolysis of ß-glucan by the C domain enzyme decreased significantly with added glucomannan to the reaction, suggesting inhibition effect. Analogous result was not observed with the N domain enzyme when ß-glucan was added to the reaction. The whole enzyme did not show improvement of efficiency compared to the individual or additive total hydrolysis of the two domain enzymes using single or mixed substrates.

Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity.

A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA(3)Xyl(3)), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo-ß-1,4-xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan-hydrolyzing endo-ß-1,4-glucanase EG I. XYN IV was able to degrade several different ß-1,4-xylans, but was inactive on ß-1,4-mannans and ß-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble ß-1,3-ß-1,4-xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward ß-xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4-O-methyl-D-glucuronic acid (MeGlcA) substituents as substrate specificity determinants.

Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme.

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K(m) 0.25 mM, V(max) 16.3 µM·min(-1), and k(cat) 9.27 s(-1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-ß-D-xylopyranoside as the substrate.

Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass.

The Aspergillus niger feruloyl esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium with a yield of ~2 mg/l. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interaction chromatography. The specific activity was determined to be 8,200 U/µg (pH 6.5, 20°C, 3.5 mM 4-nitrophenyl ferulate). The protein had a correct N-terminal sequence of ASTQGISEDLY, indicating that the signal peptide was properly processed. The FAE exhibited an optimum pH of 6-7 and operated optimally at 50°C using ground switchgrass as the substrate. The yeast clone was demonstrated to catalyze the release of ferulic acid continuously from switchgrass in YNB medium at 30°C. This work represents the first report on engineering yeast for the breakdown of ferulic acid crosslink to facilitate consolidated bioprocessing.

Functional cloning and expression of a novel Endo-alpha-1,5-L-arabinanase from a metagenomic library.

A novel endo-alpha-L-arabinanase gene (arn2) was isolated, and expressed in E. coli in active form. The recombinant enzyme (ARN2) had optimum activity at pH 6.0 and 45-50( degrees )C with stability between pH 5.0-8.0 and at temperatures up to 40( degrees )C. The recombinant ARN2 catalyzed internal cleavage of alpha-1,5 glycosidic bonds of CM-arabinan, debranched arabinan, linear arabinan, and sugar beet (native) arabinan at rates of decreasing order, and was inactive on wheat arabinoxylan and p-nitrophenyl-alpha-L-arabinofuranoside. Kinetic analysis showed that branching in the arabinan did not significantly affect the apparent K(m) values, and the difference in the reaction rates was likely due to the chemical step after substrate binding. The enzyme hydrolyzed arabino-oligosaccharides of DP> or =6 to smaller oligomers and mostly arabinotriose. Natural and modified arabinans were cleaved to oligomers of various chain lengths, which were progressively hydrolyzed to yield arabinotriose. The pattern of degradation revealed an endo-acting mechanism with arabinotriose as the end product.

An alpha-glucuronidase enzyme activity assay adaptable for solid phase screening.

Glucuronic acid is a common chemical moiety that decorates the xylan polymer of hemicellulose. This chemical substituent impairs both enzymatic and acidic hydrolysis of xylosidic bonds. The alpha-glucuronidase enzyme hydrolyzes the 1,2-linked glucuronic acid from the terminal, non-reducing xylose of xylo-oligosaccharides. There are relatively few alpha-glucuronidase genes in the public databases. We have developed an assay with commercially available reagents that can be used to search DNA libraries for alpha-glucuronidase genes in a high-throughput, solid phase activity screen.

Structure and action mechanism of ligninolytic enzymes.

Lignin is the most abundant renewable source of aromatic polymer in nature, and its decomposition is indispensable for carbon recycling. It is chemically recalcitrant to breakdown by most organisms because of the complex, heterogeneous structure. The white-rot fungi produce an array of extracellular oxidative enzymes that synergistically and efficiently degrade lignin. The major groups of ligninolytic enzymes include lignin peroxidases, manganese peroxidases, versatile peroxidases, and laccases. The peroxidases are heme-containing enzymes with catalytic cycles that involve the activation by H2O2 and substrate reduction of compound I and compound II intermediates. Lignin peroxidases have the unique ability to catalyze oxidative cleavage of C-C bonds and ether (C-O-C) bonds in non-phenolic aromatic substrates of high redox potential. Manganese peroxidases oxidize Mn(II) to Mn(III), which facilitates the degradation of phenolic compounds or, in turn, oxidizes a second mediator for the breakdown of non-phenolic compounds. Versatile peroxidases are hybrids of lignin peroxidase and manganese peroxidase with a bifunctional characteristic. Laccases are multi-copper-containing proteins that catalyze the oxidation of phenolic substrates with concomitant reduction of molecular oxygen to water. This review covers the chemical nature of lignin substrates and focuses on the biochemical properties, molecular structures, reaction mechanisms, and related structures/functions of these enzymes.

Biochemical characterization of a novel dual-function arabinofuranosidase/xylosidase isolated from a compost starter mixture.

The gene encoding a glycoside hydrolase family 43 enzyme termed deAX was isolated and subcloned from a culture seeded with a compost starter mixed bacterium population, expressed with a C-terminal His(6)-tag, and purified to apparent homogeneity. deAX was monomeric in solution and had a broad pH maximum between pH 5.5 and pH 7. A twofold greater k (cat)/K (m) for the p-nitrophenyl derivative of alpha-L: -arabinofuranose versus that for the isomeric substrate beta-D-xylopyranose was due to an appreciably lower K (m) for the arabinofuranosyl substrate. Substrate inhibition was observed for both 4-methylumbelliferryl arabinofuranoside and the xylopyranoside cogener. While no loss of activity was observed over 4 h at 40 degrees C, the observed t (1/2) value rapidly decreased from 630 min at 49 degrees C to 47 min at 53 degrees C. The enzyme exhibited end-product inhibition, with a K (i) for xylose of 145 mM, 18.5 mM for arabinose, and 750 mM for glucose. Regarding natural substrate specificity, deAX had arabinofuranosidase activity on sugar beet arabinan, 1,5-alpha-L-arabinobiose, and 1,5-alpha-L-arabinotriose, and wheat and rye arabinoxylan, while xylosidase activity was detected for the substrates xylobiose, xylotriose, xylotetraose, and arabinoxylan from beech and birch. Thus, deAX can be classified as a dual-function xylosidase/arabinofuranosidase with respect to both artificial and natural substrate specificity.

Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08.

The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D: -xylopyranose (4NPX) and p-nitrophenyl-alpha-L: -arabinofuranose (4NPA), and it was found that the ratio k (cat)/K (m) 4NPA/k (cat)/K (m) 4NPX was approximately 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k (cat)/K (m) muA/k (cat)/K (m) muX was approximately 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K (i) of 6.8 +/- 0.62 mM and xylose K (i) of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t (1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.

Cloning of Bacillus licheniformis xylanase gene and characterization of recombinant enzyme.

Hemicellulose is a major component of lignocellulose biomass. Complete degradation of this substrate requires several different enzymatic activities, including xylanase. We isolated a strain of Bacillus licheniformis from a hot springs environment that exhibited xylanase activity. A gene encoding a 23-kDa xylanase enzyme, Xyn11, was cloned, and the recombinant protein was expressed in an Escherichia coli host and biochemically characterized. The optimum activity of the enzyme was at pH 5-7 and 40-50 degrees C. The enzyme was stable at temperatures up to 50 degrees C. Against birchwood xylan, the enzyme had an apparent K(m) of 6.7 mg/mL and V(max) of 379 micromol/min/mg.

Cloning and characterization of a novel exo-alpha-1,5-L-arabinanase gene and the enzyme.

A novel exo-alpha-1,5-L-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0-7.0 and a pH 3.0-7.0 stability range. The temperature optimum was 50 degrees C with a stability less than or equal to 45 degrees C. The recombinant ARN3 cleaved carboxymethyl (CM)-arabinan, debranched arabinan, and linear arabinan at a decreasing rate and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent K(m) and V(max) values were determined to be 6.2+/-0.3 mg/ml and 0.86+/-0.01 mg ml(-1) min(-1), respectively (pH 7.0, 37 degrees C, CM-arabinan). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.

Cloning, expression and characterization of a glycoside hydrolase family 39 xylosidase from Bacillus halodurans C-125.

The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag, and the recombinant gene product termed BH1068(His)(6) was expressed in Escherichia coli. Of the artificial substrates tested, BH1068(His)(6) hydrolyzed nitrophenyl derivatives of beta-D-xylopyranose, alpha-L-arabinofuranose, and alpha-L-arabinopyranose. Deviation from Michaelis-Menten kinetics at higher substrate concentrations indicative of transglycosylation was observed, and k (cat) and K (m) values were measured at both low and high substrate concentrations to illuminate the relative propensities to proceed along this alternate reaction pathway. The pH maximum was 6.5, and under the conditions tested, maximal activity was at 47 degrees C, and thermal instability occurred above 45 degrees C. BH1068(His)(6) was inactive on arabinan, hydrolyzed xylooligosaccharides, and released only xylose from oat, wheat, rye, beech, and birch arabinoxylan, and thus, can be classified as a xylosidase with respect to natural substrate specificity. The enzyme was not inhibited by up to 200 mM xylose. The oligomerization state was tetrameric under the size-exclusion chromatography conditions employed.