The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi

The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel beta-helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure. Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex. Although the primary calcium ion binds substrate in subsite -1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite +1. The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-beta-elimination mechanism. The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.     

Expression and thermostability of Paenibacillus campinasensis BL11 pectate lyase and its applications in bast fibre processing

An open reading frame (ORF) with 963 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in Escherichia coli. It encodes a pectate lyase (EC 4.2.2.2) of 35.6 kDa, denominated Pel‐BL11. The recombinant Pel‐BL11 was fused with His‐tag and purified. An optimal activity of 1623 IU mg^?1 was exhibited at 50°C, pH 10. Significant activities of Pel‐BL11 are demonstrated between 40 and 70°C and from a pH of 7–11. The observed half‐lives are 103 min at 70°C and 288 min at 40°C. Compared to other published acid and alkaline pectate lyases, Pel‐BL11 demonstrated exceptional thermostability and wider pH adaptability. Temperature effects on the cleavage of the pectate α‐1,4‐glycosidic bond by Pel‐BL11 were examined. Continuous cleavage occurred for the first 3 h at 30 and 50°C. However, at 70°C, the majority of the cleavage occurred during the first 10 min. Weight loss in gampi and paper mulberry fibres after enzyme treatment validate the potential of this treatment in fibre degumming.     

麻类脱胶高效菌株果胶裂解酶基因克隆与表达

【目的】克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究。【方法】根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达。选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质。【结果】克隆到果胶裂解酶基因pel(GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸。pACYCDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL。其最适反应温度为50°C,最适pH为9.0;保温1 h,酶活稳定温度≤45°C,稳定pH为9.0?10.0。酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L;Zn2+、Ca2+和NH4+促进酶活力,Fe3+和Pb2+严重抑制酶活力;聚半乳糖醛酸钠为该酶的最适底物。【结论】从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景。     

Crystal structure of polysaccharide lyase family 20 endo-β-1,4-glucuronan lyase from the filamentous fungus Trichoderma reesei

The crystal structure of endo-β-(1→4)-glucuronan lyase from Trichoderma reesei (TrGL) has been determined at 1.8 Å resolution as the first three-dimensional structure of polysaccharide lyase (PL) family 20. TrGL has a typical β-jelly roll fold, which is similar to glycoside hydrolase family 16 and PL7 enzymes. A calcium ion is bound to the site far from the cleft and appears to contribute to the stability. There are several completely conserved residues in the cleft. Possible catalytic residues are predicted based on structural comparison with PL7 alginate lyase A1-II′.     

A novel thermophilic pectate lyase containing two catalytic modules of Clostridium stercorarium

The Clostridium stercorarium F-9 pel9A gene encodes a pectate lyase Pel9A consisting of 1,240 amino acids with a molecular weight of 135,171. The mature form of Pel9A is a modular enzyme composed of two family-9 catalytic modules of polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. Pel9A showed an overall sequence similarity to the hypothetical pectate lyase PelX of Bacillus halodurans (sequence identity 53%), and CM9-2 showed moderate sequence similarities to some pectate lyases of family 9. Sequence identity between CM9-1 and CM9-2 was 21.3%. The full-length Pel9A lacking the N-terminal signal peptide was expressed, purified, and characterized. The enzyme required Ca(2+) ion for its enzyme activity and showed high activity toward polygalacturonic acid but lower activity toward pectin, indicating that Pel9A is a pectate lyase. Immunological analysis using an antiserum raised against the purified enzyme indicated that Pel9A is constitutively synthesized by C. stercorarium F-9.     

Stimulation of berberine secretion and growth in cell cultures of Thalictrum minus

Summary An endo-pectate lyase (PL; EC 4.2.2.2), originally cloned fiom the phytopathogenic bacterium Erwinia chrysanthemi EC16, was expressed in recA ^– E. coli strain DK1, purified to a single band by isoelectric focusing and used to induce berberine production in established plant suspension cultures of Thalictrum minus L. subsp. saxatile. Addition of 10^–9M pectate lyase c (PLc) stimulated berberine production and enhanced secretion of the alkaloid into the medium. A lower concentration of PLc, 10^–11M, stimulated a transient two-fold increase in cell growth rate relative to untreated cultures. Parallel changes in L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity with the rate of berberine synthesis and the inverse relationship between cell growth and berberine synthesis imply that berberine synthesis is stress-related in this cell line.     

Characterization of a family 3 polysaccharide lyase with broad temperature adaptability, thermo-alkali stability, and ethanol tolerance

The 774-bp pectate lyase gene plyAI4 from Bacillus sp. I4 was cloned and expressed in E. coli. The gene encodes a 257-residue polypeptide (PlyAI4, 28.3 kDa) with the highest identities of 97.3% with a putative pectate lyase from Bacillus subtilis BSn5 (ADV94306) and 60.3% with an identified pectate lyase of the polysaccharide lyase family (PL) 3 from Paenibacillus amylolyticus 27C64 (ADB78774). The purified recombinant PlyAI4 (rPlyAI4) exhibited apparently optimal activity at pH 10.5 ?? 11.0 and 50°C. Compared with the majority of reported alkaline pectate lyases, rPlyAI4 exhibited more residual enzyme activity at 20°C (??45%) or at 70°C (??50%) and better thermostability at 70°C (??60 min half-life at 70°C). In the presence of 20% (v/v) ethanol, pectate lyase activity was enhanced by 0.2 fold. After incubation in 40% (v/v) ethanol at 37°C and pH 8.5 for 1 h, the purified rPelAI4 retained more than 75% of the initial activity. Sequence analysis proposed a new signature block, A-D-G-[V/I]-H, for PL 3 pectate lyases. These properties may prove to be important with regards to PlyAI4 for basic research and industrial application.     

Immobilization of recombinant pectate lyase from Clostridium thermocellum ATCC‐27405 on magnetic nanoparticles for bioscouring of cotton fabric

Recombinant pectate lyase from family 1 polysaccharide lyase (PL1B) was immobilized on synthesized magnetic nanoparticles (MNPs) after 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride activation. At 70 mg/mL MNPs 100% binding of 1 mg/mL PL1B was achieved. The immobilized PL1B‐MNP displayed activity of 20.3 and 18.2 U/mg against polygalacturonic acid and citrus pectin, respectively, which was higher than the activity of free PL1B, on the same substrates of 17.8 and 16.2 U/mg. The immobilized PL1B‐MNP showed 32 fold and 14 fold enhanced thermal stability at 80°C and 90°C, respectively as compared with free PL1B at same temperatures. At high temperature the immobilized PL1B‐MNP retained its activity for a longer duration than free PL1B. The immobilized PL1B‐MNP could be reused till five cycles and after that it retained 70% of initial activity. It could be easily recovered from the reaction mixture with the help of a magnet. Bioscouring of cotton fabric was carried out with immobilized PL1B‐MNP which showed efficient removal of pectin from the fabric surface. The enhanced wettability of fabric resulted in the decrease of the water absorbing time period from 3 min taken by the free PL1B treated fabric to 15 s taken by the immobilized PL1B‐MNP treated fabric. As per our knowledge this is the first attempt of bioscouring of coarse cotton fabric by pectinase immobilized on magnetic nanoparticles. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:231–244, 2017     

Overexpression of the <Emphasis Type="Italic">plg</Emphasis>1 gene encoding pectin lyase in <Emphasis Type="Italic">Penicillium griseoroseum</Emphasis>

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml⁻¹ respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.     

Microbial enzyme production in a membrane bioreactor

Summary Erwinia chrysanthemi cells were used to study the possibility of producing bacterial enzymes in a bioreactor coupled with a membrane filtration unit. Continuous fermentations with total cell recycle failed to give good production of pectate lyase (PL). Enzymatic, mechanical and physico-chemical damages were involved in this phenomenon. With a sequential recycle mode, we obtained productivity of 1.5 units·h^–1·1^–1 with a high PL concentration. Protease accumulation occurred when the bioreactor was coupled to a filtration unit. Moreover we have observed no loss of activity due to high shear stress caused by pumping. Offprint requests to: P. Boyaval     

Bifunctional pectinolytic enzyme with separate pectate lyase and pectin methylesterase domains from an alkaliphilic Bacillus

The gene for a novel enzyme having pectate lyase (Pel) and pectin methylesterase (Pme) activities found in the genome of an alkaliphilic Bacillus, KSM-P358, was sequenced. The structural gene contained a long open reading frame of 4314 bp corresponding to a 32-amino-acid signal peptide and a 1406-amino-acid mature enzyme with a molecular mass of 155,666. The mature enzyme contained two uncontiguous regions at amino acids 800–1051 and 1105–1406 exhibiting homology to a Pel from a Bacillus strain with 43.7% and a Pme from Erwinia chrysanthemi with 33.4% identity, respectively. The recombinant enzyme expressed in Bacillus subtilis cells had a molecular mass of 160 kDa and exhibited pH and temperature optima for Pel activity of 10 and 40 °C and those for the Pme activity of 8.5 and 45 °C. The genes for the domains for the Pel and Pme could be separately expressed in Escherichia coli cells, and the catalytic properties of the respective protein fragments were essentially identical to those of the intact enzyme. This novel enzyme is mosaic in that some regions before the two domains exhibited limited but substantial similarity to some regions of carbohydrate-active enzymes. The regions contained parts of a gene for Pels from a Bacillus sp. and Pseudomonas fluorescens, a xylanase from P. fluorescens subsp. cellulosa, a 1,4--mannanase from a Pyromyces sp., a putative Pel from a Streptomyces coelicolor cosmid, a (1,3-1,4)--glucanase from Clostridium thermocellum.     

Efficient secretory expression of an alkaline pectate lyase gene from Bacillus subtilis in E. coli and the purification and characterization of the protein

The gene encoding pectate lyase (PL) from Bacillus subtilis WSHB04-02 was amplified by PCR, fused with a periplasmic secretion signal peptide sequence, pelB, from pET22b(+), cloned and expressed in Escherichia coli cells using a temperature control vector, pHsh. The recombinant E. coil was grown in a 5 l fermentor. PL was secreted in broth at 22 U l⁻¹ after 20 h when temperature was increased from 30°C to 42°C. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. It was optimally active at pH 9.4 and 50°C over 30 min. Analysis of polygalacturonic acid (PGA) degradation products by electrospray ionization (ESI)-mass spectrometry (MS) indicated that PL produced a mixture of unsaturated oligo-galacturonides including unsaturated tri-galacturonic acid and unsaturated bi-galacturonic acid but not unsaturated mono-galacturonic acid.     

来自Yeosuana marina sp.JLT21内切型海藻酸裂解酶的异源表达及酶学表征

褐藻寡糖有着丰富的生物学功能,酶法制备功能性褐藻寡糖具有重要实践应用价值.为发掘高活性及稳定性的褐藻寡糖制备酶,对浅海热液嗜热菌Yeosuana marina sp.JLT21中的海藻酸裂解酶YMA-1的基因在大肠杆菌中进行表达、纯化及酶活鉴定.结果发现YMA-1由306个氨基酸残基构成,是多糖裂解酶家族7(PL7)新...     

Structure and dynamics analysis of a new member heparinase II/III of family 12 polysaccharide lyase from Pseudopedobacter saltans by computational modeling and small-angle X-ray scattering

Abstract

The structure of heparinase II/III belonging to family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was generated by homology modeling. Multiple sequence alignment showed conserved (Asn216, Tyr270 and His400) and semi-conserved active site amino acid residues. The modeled structure of PsPL12a displayed α/α toroid domain at N-terminal and antiparallel β sheets at C-terminal domain. The modeled structure was similar to those of heparinases from polysaccharide lyase 12 and 21 families. Validation of PsPL12a model by Ramachandran plot showed 94.6% of residues in the favored region, 5.2% of residues in the allowed region and only 0.2% of residues in the outlier region. The area and volume computed for PsPL12a displayed nearly a closed conformation of the active site, similar to HepIII from Bacteroides thetaiotaomicron. The charge calculation on the surface of the PsPL12a structure showed the higher distribution of positive charge in the active site cleft as compared with other homologous structures. Molecular docking study of MD-simulated PsPL12a structure with heparin oligosaccharide showed high binding affinity as compared with heparan sulfate oligosaccharides. Comparison of the active site of modeled PsPL12a with other homologous heparinases revealed putative catalytic triad involving the residues Asn216, His400 and Tyr270. Small-angle X-ray scattering analysis of PsPL12a displayed a fully folded and boxing glove-like envelop.

Communicated by Ramaswamy H. Sarma     

Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and EGTA in vitro

Mature plant cell walls lose their ability to expand and become unresponsive to expansin. This phenomenon is believed to be due to cross-linking of hemicellulose, pectin, or phenolic groups in the wall. By screening various hydrolytic enzymes, we found that pretreatment of nongrowing, heat-inactivated, basal cucumber (Cucumis sativus) hypocotyls with pectin lyase (Pel1) from Aspergillus japonicus could restore reconstituted exogenous expansin-induced extension in mature cell walls in vitro. Recombinant pectate lyase A (PelA) and polygalacturonase (PG) from Aspergillus spp. exhibited similar capacity to Pel1. Pel1, PelA, and PG also enhanced the reconstituted expansin-induced extension of the apical (elongating) segments of cucumber hypocotyls. However, the effective concentrations of PelA and PG for enhancing the reconstituted expansin-induced extension were greater in the apical segments than in the basal segments, whereas Pel1 behaved in the opposite manner. These data are consistent with distribution of more methyl-esterified pectin in cell walls of the apical segments and less esterified pectin in the basal segments. Associated with the degree of esterification of pectin, more calcium was found in cell walls of basal segments compared to apical segments. Pretreatment of the calcium chelator EGTA could also restore mature cell walls' susceptibility to expansin by removing calcium from mature cell walls. Because recombinant pectinases do not hydrolyze other wall polysaccharides, and endoglucanase, xylanase, and protease cannot restore the mature wall's extensibility, we can conclude that the pectin network, especially calcium-pectate bridges, may be the primary factor that determines cucumber hypocotyl mature cell walls' unresponsiveness to expansin.     

Effect of Dionaea muscipula extract and plumbagin on maceration of potato tissue by Pectobacterium atrosepticum

Pectobacterium atrosepticum (Pba) is a plant pathogen that causes major crop losses. Dionaea muscipula extracts and their antibacterial constituent, plumbagin, inhibit Pba growth in vitro. However, this effect is reduced when the extracts are added to bacterial cultures present on potato tubers or suspended in potato tuber filtrate (PF). To explain this, we examined the response mechanism of Pba cells to Dionaea extract and plumbagin and compared it with the effect of a bactericidal peptide – CAMEL. The addition of the extract and plumbagin to a Pba1043 culture in stationary phase increased the extracellular pectate lyase (Pel) activity in the presence of PF. While the addition of the Dionaea extract and plumbagin caused a dramatic reduction in RNA and protein synthesis in Pba1043, it did not result in cellular damage. PF alone increased the expression of Pba genes encoding protein components of cellular efflux pump systems: ompX, acrA and emrA. Application of both PF and plumbagin resulted in a synergistic stimulation of acrA gene expression. Plumbagin added to potato tubers inoculated with a field isolate Pba5A/1/2005 increased extracellular Pel activity and reduced tissue maceration but did not affect bacterial counts per gram of tissue. These results show that plumbagin in the presence of compounds from potato tuber stimulates Pel production/secretion in Pba cells and increases the expression of the acrA gene. This may be the molecular basis for the less pronounced effects of Dionaea extract on Pba in planta relative to those observed in vitro.     

Exopolygalacturonate lyase from Thermotoga maritima: cloning,characterization and organic synthesis application

A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.