Paenibacillus Barcinonensis Research Articles

Changes of supramolecular cellulose structure and accessibility induced by the processive endoglucanase Cel9B from Paenibacillus barcinonensis

A newly identified cellulase with a high polysaccharide degrading potential and a processive mode of action, has been evaluated on cellulose fibers. Cellulase Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with the domain structure GH9-CBM3c-Fn3-CBM3b, consisting of a family nine catalytic module GH9, an auxiliary module CBM3c, a fibronectin-like module Fn3, and a functional cellulose binding module CBM3b. The whole cellulase Cel9B (E1) and two truncated forms of the enzyme that consist of the catalytic module linked to the auxiliary module, GH9-CBM3c (E2), and of the cellulose binding module of the enzyme, CBM3b (CBD), were applied to softwood dissolving pulp. The changes in the supramolecular structure and morphology of the fibres after the enzymatic treatment were evaluated by viscosimetry, X-ray diffraction (XRD), thermogravimetric analysis, differential scanning calorimetry and scanning electron microscopy (SEM). XRD studies provided the crystallite size, interplanar distances and crystallinity index of the samples before and after the enzymatic treatment. The treatment with cellulases E1 and E2 decreased the degree of polymerization and increased the crystallinity index of the pulp. Both E1 and E2 had a pronounced capacity for removing fuzz and improved the smoothness and surface appearance of the fibers, as shown by SEM. On the other hand, CBD proved to be less effective under the tested conditions. Moreover, the solubility of dissolving pulp in alkaline solutions has been evaluated as an indirect measure of cellulose accessibility. A notable enhancement in alkaline solubility of the samples treated with the cellulases was observed.

Enhanced Production of Endo-β-1,4-xylanase from Paenibacillus sp. HX-1 Newly Isolated from Soil Samples at Hambak Mountain in Yongin city, Korea

A xylanase-producing bacterium was isolated from a soil sample collected in Yongin city, Korea. The strain was aerobic and gram positive, and grew between pH 5.0 and 11.0, forming a yellow-colored colony. The strain was classified as a novel subspecies bacterium of Paenibacillus barcinonensis by 16S rRNA gene sequence similarity, phylogenetic analysis, phenotypic, and biochemical characteristics, and thus named Paenibacillus sp. HX-1. This strain produced extracellular endo--1,4-xylanase, and the best xylanolytic activity (205.17 unit/ml) was obtained at 96 h in an optimized TNX medium containing 1% (w/v) bacto tryptone, 1% (w/v) NaCl, and 0.7% (w/v) beechwood xylan at pH 7.0, and 200 rpm. The endo--1,4-xylanase produced by the strain HX-1 yielded xylobiose as the end product from beechwood xylan hydrolysis. The enzyme exhibited optimum pH and temperature at pH 7.0 and , respectively. The remarkable enhancing effect of the TNX medium on xylanase production by HX-1, in spite of its simple formula, may have great advantages for industrial applications of xylanase.

Effectiveness of novel xylanases belonging to different GH families on lignin and hexenuronic acids removal from specialty sisal fibres

AbstractBACKGROUNDThe effectiveness of xylanases on lignin removal from pulps differs widely depending on the enzyme family, the type of pulp and the bleaching sequence among other factors. Xylanases can also reduce the presence of undesirable hexenuronic acids in the papermaking fibers. The performance of non‐commercial xylanases belonging to families GH10, GH30, GH30‐CBM35 and GH11, and of the multicomponent xylanase from Paenibacillus barcinonensis for lignin and hexenuronic acids removal from sisal (Agave sisalana) has been evaluated.RESULTSSisal pulps were bleached by an XP sequence, where X denotes the enzyme treatment and P a hydrogen peroxide extraction stage. Kappa number, brightness, viscosity and hexenuronic acid content of samples were determined. Sugars released from sisal pulps, other non‐wood fibres and also eucalyptus fibres, by the treatment with xylanases were also analysed. The best results were obtained with the GH10 xylanase and with crude supernatants of P. barcinonensis, which produced a lignin removal of 23% and a reduction of 25% in the hexenuronic acid content of sisal pulps without a significant loss of viscosity.CONCLUSIONThe release of sugars in the effluents from the X stage applied to sisal correlated with the effectiveness of the xylanases tested. The xylan content of wood and non‐wood fibres, the type of xylan and its accessibility also had an influence on the xylanase activity on pulps. © 2013 Society of Chemical Industry

Antibacterial Ability and Molecular Characterization of Probionts Isolated from Gut Microflora of Cultured Red Tilapia

Four bacterial isolates, which proved to have the potential to be used as probiotics, were used to study their genotypic characterization using analysis of 16S rRNA gene sequence, antagonistic ability and safety when applied on tilapia via immersion. Gram staining showed that all 4 probionts were Gram positive bacteria. Three probionts were rod in shape and the fourth was cocci in shape and arranged in grape-like cluster. These 4 probionts were also identified using 16S rRNA gene sequencing and their identities were Paenibacillus barcinonensis strain D12, Paenibacillus sp. strain D14, Staphylococcus cohnii strain Bll and Bacillus megaterium strain E28, respectively. All of the probionts were examined for their antagonistic ability against pathogenic bacteria (Vibrio alginolyticus ATCC 33839, Aeromonas salmonicida and Aeromonas hydrophila ATCC 35654) under in vitro conditions by using cross-streaking method. P. barcinonensis strain D12 and Paenibacillus sp. strain D14 have shown stronger antagonistic ability than S. cohnii strain Bll and B. megaterium strain E28 in the antagonism test. P. barcinonensis strain D12 and Paenibacillus sp. strain D14 were chosen to test their safety on tilapia due to their better performance in antagonism test. Both probionts, P. barcinonensis strain D12 and Paenibacillus sp. strain D14, were safe for tilapia.

Purification and Characterization of a Thermophilic Cellulase from a Novel Cellulolytic Strain, Paenibacillus barcinonensis

A novel bacterial strain, MG7, with high cellulase activity was isolated and identified by morphological characteristics and molecular phylogeny analysis as Paenibacillus barcinonensis. Maximum production of cellulase by MG7 was observed at pH 7.0 and 35°C. The enzyme was purified with a specific activity of 16.88 U/mg, the cellulase activity was observed in a zymogram, and its molecular mass (58.6 kDa) was confirmed by SDS-PAGE. The purified enzyme showed maximum activity at pH 6.0 and 65°C and degraded cellulosic substrates such as carboxy methyl cellulose (CMC), Avicel, filter paper, and beta-glucan. The enzyme showed stability with 0.5% concentration of various surfactants. The K(m) and V(max) of cellulase for CMC and Avicel were found to be 0.459 mg/ml and 10.46 mg/ml/h, and 1.01 mg/ml and 10.0 mg/ml/h, respectively. The high catalytic activity and its stability to temperature, pH, surfactants, and metal ions indicated that the cellulase enzyme by MG7 is a good candidate for biotechnological applications.

Biomodification of cellulose flax fibers by a new cellulase

The potential of the new cellulase Cel9B from Paenibacillus barcinonensis for the modification of cellulose flax fibers was for the first time assessed in this work. Cel9B treatments were found to enhance the development of properties during the refining of fibers, the precise effects depending on the enzyme dose used. Scanning electron micrographs showed the treatments to alter flax fiber surface and cause defibrillation, peeling and fiber wall removal. Zero-span tensile strength was determined to elucidate the changes in intrinsic resistance of cellulose fibers and solubilization of carbohydrates evaluated by HPLC. Low Cel9B doses resulted in improved tensile strength in handsheets and can thus be used to save energy in the refining process. Also, on equal tear resistance, handsheets had a higher tensile index. High doses of Cel9B raised the collapsibility and bonding potential of cellulose flax fibers and produced heavier handsheets. Cel9B also improved air permeance at low refining intensities. The results obtained in this work are useful in understanding the effects brought by cellulase Cel9B on flax fibers and provide a proof of a biotechnological platform for the development of added-value fiber products.

CLONING, EXPRESSION, AND CHARACTERIZATION OF AN ALKALOPHILLIC ENDO-1,4-BETA-XYLANASE FROM PAENIBACILLUS SP. HPL-002

The biochemical properties of a purified enzyme of a new alkalophillic endo-1,4-beta-xylanase gene, KRICT PX2 (GU967374), which was isolated from Paenibacillus sp. HPL-002 (KCTC11410BP) and expressed in E. coli, were investigated. The specific activity of the purified xylanase was 51.26 μmol/min/mg proteins. The Km and Vmax values of the protein for birch wood xylan were also verified to have 0.061 μM and 55.3 μmol/min/mg proteins, respectively. The optimum pH and temperature for the activity of the enzyme were pH 8~9 and 50oC, respectively, and, the activity was stably maintained at 40oC. Most metallic salts, ethylenediamine tetra-acetic acid, 2-mercaptoethanol, phenylmethane-sulphonyl fluoride, and furfural, have no impact on the enzyme’s activity at 1 mM. The simulated 3-D structure of this xylanase is similar to Xyn10B from Paenibacillus barcinonensis. Further research on the degradation of different-origin xylans and enzyme production will be necessary for practical applications.

Cloning, expression, and characterization of an alkalophillic endo-1,4-beta-xylanase from Paenibacillus sp. HPL-002

The biochemical properties of a purified enzyme of a new alkalophillic endo-1,4-beta-xylanase gene, KRICT PX2 (GU967374), which was isolated from Paenibacillus sp. HPL-002 (KCTC11410BP) and expressed in E. coli, were investigated. The specific activity of the purified xylanase was 51.26 μmol/min/mg proteins. The Km and Vmax values of the protein for birch wood xylan were also verified to have 0.061 μM and 55.3 μmol/min/mg proteins, respectively. The optimum pH and temperature for the activity of the enzyme were pH 8~9 and 50oC, respectively, and, the activity was stably maintained at 40oC. Most metallic salts, ethylenediamine tetra-acetic acid, 2-mercaptoethanol, phenylmethane-sulphonyl fluoride, and furfural, have no impact on the enzyme’s activity at 1 mM. The simulated 3-D structure of this xylanase is similar to Xyn10B from Paenibacillus barcinonensis. Further research on the degradation of different-origin xylans and enzyme production will be necessary for practical applications.

Some aspects of the reactivity of pulp intended for high-viscosity viscose

The biochemical properties of a purified enzyme of a new alkalophillic endo-1,4-beta-xylanase gene, KRICT PX2 (GU967374), which was isolated from Paenibacillus sp. HPL-002 (KCTC11410BP) and expressed in E. coli, were investigated. The specific activity of the purified xylanase was 51.26 μmol/min/mg proteins. The Km and Vmax values of the protein for birch wood xylan were also verified to have 0.061 μM and 55.3 μmol/min/mg proteins, respectively. The optimum pH and temperature for the activity of the enzyme were pH 8~9 and 50oC, respectively, and, the activity was stably maintained at 40oC. Most metallic salts, ethylenediamine tetra-acetic acid, 2-mercaptoethanol, phenylmethane-sulphonyl fluoride, and furfural, have no impact on the enzyme’s activity at 1 mM. The simulated 3-D structure of this xylanase is similar to Xyn10B from Paenibacillus barcinonensis. Further research on the degradation of different-origin xylans and enzyme production will be necessary for practical applications.

Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae

The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase.

Rational Protein Design of Paenibacillus barcinonensis Esterase EstA for Kinetic Resolution of Tertiary Alcohols

AbstractProtein engineering is a very powerful tool to optimize enzymes for specific applications and thus provide important chiral building blocks such as tertiary alcohols. By use of structural comparisons, esterase from Paenibacillus barcinonensis (EstA) was engineered to convert tertiary alcohol esters with excellent enantioselectivity. Whereas the wild‐type enzyme converts 1,1,1‐trifluoro‐2‐phenylbut‐3‐yn‐2‐yl acetate with very low activity and enantioselectivity (E=12, at 4 °C), several mutants show a significantly increased enantioselectivity, for example E>100 for mutant EstA–AGA, under the same reaction conditions. Furthermore, the range of tertiary alcohols obtained in enantiopure form was also broadened for EstA mutants.

Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp

The modular endoglucanase Cel9B from Paenibacillus barcinonensis is a highly efficient biocatalyst, which expedites pulp refining and reduces the associated energy costs as a result. In this work, we set out to identify the specific structural domain or domains responsible for the action of this enzyme on cellulose fibre surfaces with a view to facilitating the development of new cellulases for optimum biorefining. Using the recombinant enzymes GH9-CBD3c, Fn3-CBD3b, and CBD3b, which are truncated forms of Cel9B, allowed us to assess the individual effects of the catalytic, cellulose binding, and fibronectin-like domains of the enzyme on the refining of TCF kraft pulp from Eucalyptus globulus. Based on the physico-mechanical properties obtained, the truncated form containing the catalytic domain (GH9-CBD3c) has a strong effect on fibre morphology. Comparing its effect with that of the whole cellulase (Cel9B) revealed that the truncated enzyme contributes to increasing paper strength through improved tensile strength and burst strength and also that the truncated form is more effective than the whole enzyme in improving tear resistance. Therefore, the catalytic domain of Cel9B has biorefining action on pulp. Although cellulose binding domains (CBDs) are less efficient toward pulp refining, evidence obtained in this work suggests that CBD3b alters fibre surfaces and influences paper properties as a result.

Recombinant Expression of an Alkali Stable GH10 Xylanase from Paenibacillus barcinonensis

Xylanase A from Paenibacillus barcinonensis, a new species isolated from a rice field, has been cloned and expressed in Escherichia coli. Purified recombinant xylanase showed high activity on xylans from hardwoods and cereals, and exhibited K(m) and V(max) of 2.93 mg/mL and 50.67 U/mg on birchwood xylan. Xylanase A was highly active at 60 degrees C in alkaline pH values up to 9.5 and remained stable for at least 3 h in alkaline conditions. The amino acid sequence deduced from xynA revealed that it is a single domain xylanase belonging to the GH10 family. Thin layer chromatography analysis showed that the enzyme released a mixture of hydrolysis products including substituted xylooligomers from cereal arabinoxylans, while xylose, xylobiose, and aldotetraouronic acid were the main products released from glucuronoxylan from birchwood. The enzyme released a complex mixture of xylooligomers for acetylated xylan from eucalyptus, revealing its potential to depolymerize this widely used resource in the pulp and paper industry.

Structural Insights into the Specificity of Xyn10B from Paenibacillus barcinonensis and Its Improved Stability by Forced Protein Evolution

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (beta/alpha)(8) barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the beta/alpha motifs. One of these loops -L7- located at the beta7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His(249)-Glu(250), which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.

Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture

Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with a novel molecular architecture among family 9 enzymes that comprises a catalytic domain (GH9), a family 3c cellulose-binding domain (CBM3c), a fibronectin III-like domain repeat (Fn3(1,2)), and a C-terminal family 3b cellulose-binding domain (CBM3b). A series of truncated derivatives of endoglucanase Cel9B have been constructed and characterized. Deletion of CBM3c produced a notable reduction in hydrolytic activity, while it did not affect the cellulose-binding properties as CBM3c did not show the ability to bind to cellulose. On the contrary, CBM3b exhibited binding to cellulose. The truncated forms devoid of CBM3b lost cellulose-binding ability and showed a reduced activity on crystalline cellulose, although activity on amorphous celluloses was not affected. Endoglucanase Cel9B produced only a small ratio of insoluble products from filter paper, while most of the reducing ends produced by the enzyme were released as soluble sugars (91%), indicating that it is a processive enzyme. Processivity of Cel9B resides in traits contained in the tandem of domains GH9-CBM3c, although the slightly reduced processivity of truncated form GH9-CBM3c suggests a minor contribution of domains Fn3(1,2) or CBM3b, not contained in it, on processivity of endoglucanase Cel9B.

Rational protein design of Paenibacillus barcinonensis esterase EstA for kinetic resolution of tertiary alcohols

Rational protein design of Paenibacillus barcinonensis esterase EstA for kinetic resolution of tertiary alcohols

Paenibacillus taichungensis sp. nov., from soil in Taiwan

Among a large collection of Taiwanese soil isolates, a novel Gram-variable, rod-shaped, motile, endospore-forming bacterial strain, strain V10537(T), was subjected to a polyphasic study including 16S rRNA and gyrB gene sequence analysis, DNA-DNA hybridization experiments, cell wall peptidoglycan type, cellular fatty acid composition analysis and comparative phenotypic characterization. 16S rRNA gene sequence analysis indicated that the organism belonged to the genus Paenibacillus. Strain V10537(T) possessed meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan. It contained menaquinone MK-7 as the predominant isoprenoid quinone and anteiso-C(15 : 0) (53.6 %) and C(16 : 0) (19.0 %) as the major fatty acids. Phylogenetically, the most closely related species to strain V10537(T) were Paenibacillus pabuli, Paenibacillus xylanilyticus, Paenibacillus amylolyticus, Paenibacillus barcinonensis and Paenibacillus illinoisensis, with 16S rRNA gene sequence similarities of 99.5, 98.8, 98.3, 98.2 and 98.1 % to the respective type strains. The gyrB gene sequence similarities between strain V10537(T) and these strains were 76.9-85.0 %. DNA-DNA hybridization experiments showed levels of relatedness of 8.5-45.6 % between strain V10537(T) and these strains. The DNA G+C content of strain V10537(T) was 46.7 mol%. Strain V10537(T) was clearly distinguishable from other Paenibacillus species and thus represents a novel species of the genus Paenibacillus, for which the name Paenibacillus taichungensis sp. nov. is proposed. The type strain is V10537(T) (=BCRC 17757(T) =DSM 19942(T)).

Paenibacillus provencensis sp. nov., isolated from human cerebrospinal fluid, and Paenibacillus urinalis sp. nov., isolated from human urine

Gram-negative, spore-forming rods were isolated from a human urine sample (strain 5402403(T)) and a human cerebrospinal fluid sample (strain 4401170(T)). Based on genotypic characteristics, these strains belonged to the genus Paenibacillus and were closely related. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that they clustered with Paenibacillus massiliensis 2301065(T) (95.9 and 94.3 % 16S rRNA gene sequence similarity, respectively, for strains 5402403(T) and 4401170(T)), Paenibacillus illinoisensis NRRL NRS-61356(T) (90.6 and 93.8 %), Paenibacillus xylanilyticus XIL14(T) (95.3 and 95.4 %), Paenibacillus barcinonensis BP-23(T) (94.3 and 94.0 %), Paenibacillus pabuli NCIMB 12781(T) (89.1 and 92.3 %) and Paenibacillus amylolyticus NRRL NRS-290(T) (94.2 and 93.8 %). The predominant fatty acids were 15 : 0 anteiso (49.0 and 55.3 %, respectively, for strains 5402403(T) and 4401170(T)), 16 : 0 iso (15.4 and 13.5 %), 16 : 0 (7.6 and 3.6 %), 15 : 0 (6.3 and 2.8 %), 17 : 0 anteiso (5.7 and 7.5 %), 14 : 0 iso (4.1 and 2.7 %) and 15 : 0 iso (4.1 and 3.4 %). 16S rRNA gene sequence similarity between strain 4401170(T) and strain 5402403(T) was 98.4 %, but the DNA-DNA reassociation rate between the two strains was 53.2 %. So, considering the recommendations of the ad hoc committee, they do not belong to the same species. On the basis of phenotypic data and genotypic inference, it is proposed that the strains should be assigned to the novel species Paenibacillus urinalis sp. nov. (type strain 5402403(T) =CIP 109357(T) =CCUG 53521(T)) and Paenibacillus provencensis sp. nov. (type strain 4401170(T) =CIP 109358(T) =CCUG 53519(T)).

Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts

Xylanase B from Paenibacillus barcinonensis was cloned in shuttle vectors for Escherichia coli and Bacillus subtilis, and expressed in Bacillus hosts. Several recombinant strains were constructed, among which B. subtilis MW15/pRBSPOX20 showed the highest production. This recombinant strain consists of a protease double mutant host containing P. barcinonensis xynB gene under the control of a phage SPO2 strong promoter. Maximum production was found when the strain was cultured in nutrient broth supplemented with xylans. Analysis of xylanase B location in B. subtilis MW15/pRBSPOX20 showed that the enzyme remained cell-associated in young cultures, consistent with its intracellular location in its original host, P. barcinonensis, and the lack of a signal peptide. However, when cultures reached the stationary phase, xylanase B was released to the external medium as a result of cell lysis. The amount of enzyme located in the supernatants of old cultures could account for 50% of total xylanase activity. Analysis by SDS–PAGE showed that xylanase B is an abundant protein found in the culture medium in late stationary phase cultures.

Pectate lyase C from Bacillus subtilis: a novel endo-cleaving enzyme with activity on highly methylated pectin

The gene yvpA from Bacillus subtilis was cloned and expressed in Escherichia coli. It encoded a pectate lyase of 221 amino acids that was denominated PelC. The heterologously expressed enzyme was purified by His-tag affinity chromatography and characterized. PelC depolymerized polygalacturonate and pectins of methyl esterification degree from 22 % to 89 %, exhibiting maximum activity on 22 % esterified citrus pectin. It showed an absolute Ca2+ requirement and the optimum temperature and pH were 65 degrees C and pH 10, respectively. The deduced amino acid sequence of PelC showed 53 % identity to pectate lyase PelA from Paenibacillus barcinonensis, which was also characterized. Similarly to PelC, purified PelA showed activity on polygalacturonate and pectins with a high degree of methyl esterification. The two enzymes cleaved pectic polymers to a mixture of oligogalacturonates, indicating an endo mode of action. Analysis of activity on trigalacturonate showed that PelC cleaved it to galacturonic acid and unsaturated digalacturonate, whereas PelA did not show activity on this substrate. PelC and PelA showed high homology to a few recently identified pectate lyases of family 3 and form with them a cluster of small-sized pectate lyases from non-pathogenic micro-organisms.