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.     

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。     

Nucleotide and amino-acid sequences of a new-type pectate lyase from an alkaliphilic strain of Bacillus.

A pectate lyase (pectate transeliminase; EC 4.2.2.2), designated Pel-15E, was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-P15. The purified enzyme had a molecular mass of approximately 33 kDa, as determined by SDS/PAGE, and a pI of approximately pH 9.2. Pel-15E exhibited optimum activity at pH 10.5 and 50-55 degrees C in glycine/NaOH buffer. Pel-15E had an absolute requirement for Ca2+ ions for manifestation of the enzymatic activity and trans-eliminated poly(galacturonic) acid, most likely by endo-type cleavage. A gene for the enzyme, which was cloned using the shotgun method and sequenced, contained a 960-bp ORF encoding 320 amino acids. The mature enzyme (286 amino acids, 32 085 Da) from the deduced amino-acid sequence showed quite low homology to known Pels from various microorganisms with 16.1-20.4% identity. Furthermore, we were not able to find any conserved regions in the sequence of Pel-15E when aligned with the sequences of other enzymes from the established Pel superfamily. However, Pel-15E had some regions that were homologous to PelA from Azospirillum irakense with 39.8% identity. Based on their amino-acid sequence homology, Pel-15E and PelA appear to belong to a new class of Pel family, although the enzymatic properties of both enzymes were quite different.     

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.     

香蕉果胶裂解酶基因的克隆

根据已经报告的香蕉果胶裂解酶基因序列,设计了特异引物,通过RT-PCR获得果胶裂解酶的cDNA,并克隆测序,与已报告的序列进行了比较,二者核苷酸序列的同源性达99.24%;推测的氨基酸序列也具有很高的同源性,达97.7%.通过RT-PCR的方法对香蕉不同组织和不同成熟度果实的果胶裂解酶基因的表达进行了研究.结果表明该基因只在果实中表达,具有组织特异性,而且只在果实的特定发育阶段表达.     

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.     

Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768

The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

香蕉果实软化相关β-半乳糖苷酶基因的克隆及其表达分析

β-半乳糖苷酶（β-galactosidase）通过分解细胞壁半纤维素切除半乳糖键而参与果实软化。为了阐明香蕉（Musasp．）果实成熟过程中的软化与细胞壁代谢酶β-半乳糖苷酶基因表达之间的关系,采用RT-PCR方法,从成熟香蕉果实果肉中分离了编码β-半乳糖苷酶基因的部分cDNA（MA-Gal）,序列分析表明,MA-Gal包含927bp,编码309个氨基酸,包含5个β-半乳糖苷酶结构域（典型真核生物中β-半乳糖苷酶包含7个结构域）,推导的MA-Gal蛋白质中有β-半乳糖苷酶蛋白的催化活性部位GGPIILSQIENEY（F）;系统进化树分析结果表明MA-Gal属于第一类β-半乳糖苷酶基因（该类主要在果实中表达）;β-半乳糖苷酶活性和硬度的变化表明其与香蕉果实硬度变化密切相关;Northern分析显示,跃变前期的果肉中,MA-Gal基因的表达量很低,后随着果实的软化表达量不断增加,并在呼吸跃变后达到最高。所有结果表明,MA-Gal参与香蕉果实成熟过程中的软化。     

A new high-alkaline and high-molecular-weight pectate lyase from a Bacillus isolate: enzymatic properties and cloning of the gene for the enzyme

A pectate lyase (Pel; pectate transeliminase: EC4.2.2.2.), designated Pel-15H, was found in an alkaline culture of Bacillus sp. strain KSM-P15 and purified to homogeneity by sequential column chromatographies. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 70,000 and the pI was around pH 4.6. Pel-15H randomly trans-eliminated polygalacturonate in the presence of Ca2+ ions, and the maximum activity was observed at pH 11.5 and at 55 degrees C in glycine-NaOH buffer. The gene for Pel-15H was cloned and sequenced, and the structural gene contained a 2,031-bp open reading frame that encoded 677 amino acids including a possible 28-amino-acid signal sequence. The mature enzyme (649 amino acids, molecular weight 69,550) showed very low similarity to Pels from Bacillus with 12.7-18.2% identity. Interestingly, part of the amino acid sequence of Pel-15H had fairly high similarity only to an N-terminal half of PelL and a C-terminal half of PeIX from Erwinia chrysanthemi 3937, and a C-terminal half of PeIX from E. chrysanthemi EC16 (approximately 35% identity for all).     

Modulation of mango ripening by chemicals: physiological and biochemical aspects

During ripening, fleshy fruits undergo textural changes that lead to loss of tissue firmness and consequent softening due to cell wall dismantling carried out by different and specifically expressed enzymes. The effect of various chemical treatments on the ripening of mango fruit (Mangifera indica) was investigated at physiological and biochemical level. Based on changes in respiration, firmness, pH, total soluble sugar and a cell wall degrading enzyme pectate lyase (PEL) activity, treatment with 1-methylcyclopropene (1-MCP), silver nitrate (AgNO₃), gibberlic acid (GA₃), sodium metabisulphite (SMS) and ascorbic acid led to delaying of ripening process while those of ethrel and calcium chloride (CaCl₂) enhanced the process. PEL of mango was found to be inhibited by certain metabolites present in dialysed ammonium sulphate enzyme extract as well as EDTA. Mango PEL activity exhibited an absolute requirement for Ca²⁺ and an optimum pH of 8.5.     

Purification and characterization of extracellular pectate lyase from Bacillus subtilis

Bacillus subtilis strain SO113 secretes a pectate lyase which is produced during the exponential death phase of growth, just before sporulation. This extracellular pectate lyase, which produces unsaturated products from polygalacturonate, was purified 35-fold from the culture supernatant of Bacillus subtilis by a CM Sephadex chromatography. It has an isoelectric point of about 9.6 and an Mr of 42,000. Optimum activity occurred at pH 8.4 and at 42 degrees C. Calcium has a stimulative effect on the enzyme activity while EDTA leads to enzyme inactivation. The pectate lyase has a specific activity of 131 mumol of aldehyde groups per min and per mg of protein. The Km of the purified enzyme for polygalacturonic acid was 0.862 g.l-1 and the Vmax for polygalacturonic acid hydrolysis was 1.475 mumol of unsaturated products per min and per mg of protein. By using monoclonal antibodies raised against Erwinia chrysanthemi 3937 pectate lyases, it was shown that pectate lyases b and c of this strain are immunologically closely related to the Bacillus subtilis pectate lyase.     

Use of Tn5tac1 to clone a pel gene encoding a highly alkaline, asparagine-rich pectate lyase isozyme from an Erwinia chrysanthemi EC16 mutant with deletions affecting the major pectate lyase isozymes.

Erwinia chrysanthemi mutant CUCPB5047, delta(pelA pelE) delta(pelB pelC)::28bp delta(pelX) delta 4bp pehX::omega Cmr, was constructed, mutated with Tn5tac1, and screened for isopropyl-beta-D-thiogalactopyranoside-dependent pectate lyase (Pel) production. A Kmr SacI fragment from the hyperexpressing Pel+ mutant CUCPB5066 was cloned into Escherichia coli and sequenced. The gene identified, pelL, encodes a novel, asparagine-rich, highly alkaline enzyme that is similar in primary structure to PelX and in enzymological properties to PelE.     

Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate

A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from alkaliphilic Bacillus sp. strain KSM-P7, designated Pel-7, was purified to homogeneity. The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was close to or higher than pH 10.5. In the presence of Ca2+ ions, Pel-7 trans-eliminated polygalacturonate in random manner to generate oligogalacturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 65 degrees C in glycine-NaOH buffer. Mn2+ and Sr2+ ions can serve as cofactors at almost the same level of Ca2+ ions. It also exhibited a protopectinase-like activity, liberating soluble pectin and/or oligogalacturonides from cotton fibers. The pel gene was cloned and sequenced, and the deduced amino acid sequence of mature Pel-7 (302 amino acids, 33, 355 Da) showed some conserved regions in Pel superfamily, although homology to amino acid sequences of known Pels with 27 to 32% identity. Furthermore, Pel-7 appears to have similar core structure of parallel beta-helix and active site topology with other Pels as revealed by secondary structure prediction in the Pel proteins. These results suggest that Pel-7 is basically grouped into Pel superfamily although the enzymatic and molecular properties are different.     

Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16.

The pehX gene encoding extracellular exo-poly-alpha-D-galacturonosidase (exoPG; EC 3.2.1.82) was isolated from a genomic library of the pectate lyase-deficient Erwinia chrysanthemi mutant UM1005 (a Nalr Kanr delta pelABCE derivative of EC16) by immunoscreening 2,800 Escherichia coli HB101 transformants with an antibody against exoPG protein. The cloned pehX gene was expressed highly from its own promoter in E. coli, and most of the enzyme was localized in the periplasm. The nucleotide sequence of pehX revealed the presence of an amino-terminal signal peptide and an open reading frame encoding a preprotein of 64,608 daltons. The cloned pehX gene was insertionally inactivated with TnphoA and used to mutate the chromosomal pehX gene of E. chrysanthemi AC4150 (Nalr) and CUCPB5006 (Nalr Kans delta pelABCE) by marker exchange mutagenesis. Analysis of the resulting mutants, CUCPB5008 (Pel+ Peh-) and CUCPB5009 (Pel- Peh-), indicated that exoPG can contribute significantly to bacterial utilization of polygalacturonate and the induction of pectate lyase in the presence of extracellular pectic polymers. CUCPB5009 retained a slight ability to pit polygalacturonate semisolid agar and macerated chrysanthemum pith tissues when large numbers of bacteria were inoculated.     

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.