Pectate lyase activity during ripening of banana fruit

Pectate lyase (PEL) activity was demonstrated in ripe banana fruits on supplementing the homogenizing medium with cysteine and Triton X-100. The enzyme was characterized on the basis of alkaline pH optimum, elimination of the activity by EDTA and activation by Ca(2+). PEL activity was not detected in preclimacteric banana fruits. PEL activity increased progressively from early climacteric and reached maximum level at climacteric peak and declined in post climacteric and over ripened fruits. Replacing pectate with pectin in PEL assay manifested enzyme activity even in preclimacteric fruits. In contrast to PEL, polygalacturonase activity progressively increased during fruit ripening even in postclimacteric fruits.     

Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f.sp. cubense

The accumulation of soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to cell wall-derived elicitor from the pathogen, Fusarium oxysporum, f.sp. cubense, race four, was investigated. The root tissue from the banana cultivar "Goldfinger" was found to respond strongly and rapidly towards the elicitor through the increased synthesis of phenolic compounds. Following elicitation, the conjugated and non-conjugated phenolic metabolites in the induced root tissue were extracted and quantified. Induced phenolic synthesis was rapid and reached near maximum values after 16 h. High-performance liquid chromatography revealed both compositional and quantitative differences between induced phenolics (p-coumaric, ferulic, and sinapic acids) and those constitutively present (p-coumaric- and ferulic acid). In addition, vanillic acid was found in the ester-bound fraction and protocatechuic acid in the cell-wall bound fraction of elicited tissue. The deposition and accumulation kinetics of polymerized phenolic monomers as lignin and lignin-like polymers was quantified over a time period of 0-36 h and found to reach maximum values after 24 h. Ionization difference UV spectra of lignin indicated compositional differences in the newly synthesized lignin fraction and correlated with increased concentrations of ferulic acid and sinapic acids esters. The results show that the increased flux through the phenylpropanoid pathway resulted in the synthesis of cinnamic acid and benzoic acid derivatives that were esterified and incorporated into the cell wall fraction as part of the anti-microbial defenses activated in the root tissue.     

Dimeric phenylphenalenones from Musa acuminata and various Haemodoraceae species. Crystal structure of anigorootin

Three fused octacyclic phenylphenalenone dimers were isolated from Musa acuminata: Anigorootin, which was first isolated from Anigozanthos flavidus and hitherto represented the only compound of that type, the new 4'-hydroxy-anigorootin, and 4',4"-di-hydroxy-anigorootin, which is a revised structure. The crystal structure of anigorootin was determined by X-ray crystallography. 3,3'-Bis-hydroxyanigorufone, a dimer of the conventional type known from Anigozanthos preissii, was also found in Musa acuminata. Phytochemical analysis of several Haemodoraceae species revealed the occurrence of anigorootin, 3,3'-bis-hydroxyanigorufone, and the novel metabolite 3,3'-bis-anigorufone. The occurrence of the same compounds in Musaceae and Haemodoraceae indicates the close chemotaxonomic relationship of both plant families.     

类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。     

Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation

A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn-HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a deltaG(H(2)O) of 1.741 kcal/mol, D1/2 of 2.3M, and m value of 0.755kcal/molM with urea and a deltaG(H(2)O) of 1.927kcal/mol, D1/2 of 1.52M, and m value of 1.27 kcal/molM with Gdn-HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 degrees C for 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a deltaCp of 0.864kcal/(molK). The deltaG(25 degrees C) at, pH 8.5, was 2.06kcal/mol and was in good agreement with the deltaG(H(2)O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.     

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.)

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.     

Endophytic fungi from Musa acuminata leaves and roots in South China

One hundred and sixty-three endophytic fungal cultures were isolated from 200 leaf samples of Musa acuminata trees, which were soaked in 36% formaldehyde solution for surface sterilization. They belonged to the genera of Gloeosporium musae (45%), Myxosporium spp. (11%), Deightoniella torulosa (8.5%), Alternaria tenuis (7.9%), Sphaceloma spp. (7.4%), Aureobasidium spp. (4.3%), Melida spp. (1.8%), Uncinula spp. (1.8%), Penicillium spp. (1.8%), Aspergillus spp. (1.2%), Sarcinella spp. (1.2%), Cladosporium sp. (0.6%), Cephalosporium sp. (0.6%) and sterile mycelium (6.7%). Sixty-eight endophytic fungal cultures were isolated from 100 root samples. They respectively belonged to the genera of Aspergillus spp. (31%), Paecilomyces spp. (16%), Penicillium spp. (15%), Fusarium spp. (10%), Gloeosporium musae (6%), yeast (3%), Deightoniella torulosa (3%), Spicaria sp. (1.4%), Cephalosporrium sp. (1.4%), Meliola sp. (1.4%) and sterile mycelium (10%). Water agar (containing 50 g chloramphenicol ml^–1 and 50 g streptomycin ml^–1) seemed to be a better medium for isolation of endophytic fungi than potato-dextrose agar (PDA, containing 50 g chloramphenicol ml^–1 and 50 g streptomycin ml^–1).     

A molecular marker-based linkage map of diploid bananas (Musa acuminata)

A partial molecular linkage map of the Musa acuminata diploid genome is presented. This map is based on 58 RFLP, four isozyme and 28 RAPD markers segregating in an F₂ population of 92 individuals. A total of 90 loci was detected, 77 of which were placed on 15 linkage groups while 13 segregated independently. Segregation distortions were shown by 36% of all loci, mostly favoring the male parent. Chromosome structural rearrangements were believed to be one of the main causes of these distortions. The use of genetic linkage data to further the genetic and evolutionary knowledge of the genus Musa, as well as to help improve the design of breeding strategies, is discussed.     

Regulatory mutations affecting the synthesis of pectate lyase in Xanthomonas campestris

Four classes of Xanthomonas campestris mutants were identified with respect to pectate lyase. Pectate lyase production in the wild-type and classes I and IIb mutants was partially dependent on the growth-phase whereas in classes IIa and III it was totally dependent. Enzyme activity in some of the mutants was constitutive and resistant to catabolite repression.     

辣椒疫霉菌果胶裂解酶PL101基因的克隆及生物信息学分析

【背景】辣椒疫病是一种世界性土传病害,严重影响世界各国辣椒生产,并带来巨大经济损失。果胶裂解酶(pectate lyase,PL)作为一类重要的细胞壁降解酶类是该病的重要致病因子。【目的】对果胶裂解酶基因进行克隆,并对其生物信息学特性进行相关分析,进一步阐明该酶的作用机制。【方法】根据辣椒疫霉菌全基因组序列,以高致病菌株SD33为模板扩增PL101基因的全长cDNA序列,并对其理化性质、跨膜区、亲疏水性、结构域等生物学特性进行分析。【结果】除获得PL101相关生物学特性信息外,还对PL101进行三维结构建模,获得可信度较高的蛋白结构,并确定PL101可能的催化位点为Asp183、Arg212、Arg272三个氨基酸。【结论】对PL101基因的克隆及相关生物学特性的分析为进一步阐明PL功能特性提供参考。     

Efficacy of RAPD,ISSR and DAMD markers in assessment of genetic variability and population structure of wild Musa acuminata colla

Physiology and Molecular Biology of Plants - North east India is considered as one of the major biodiversity hotspots worldwide and centre of origin of several plant species including Musa. Musa...     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.     

海栖热袍杆菌来源的极耐热碱性果胶裂解酶的表达、纯化及定性

将来源于极端嗜热菌属海栖热袍杆菌Thermotoga maritima MSB8的编码碱性果胶裂解酶的结构基因pelA与新型热激质粒pHsh连接, 得到重组质粒pHsh-pelA, 运用mRNA二级结构预测软件对pHsh-pelA的翻译起始区的二级结构进行优化, 得到了具有最佳mRNA二级结构及自由能的质粒pHsh-pelC。将重组质粒pHsh-pelC转入大肠杆菌JM109(DE3)进行表达, 得到了一种极耐热性碱性果胶裂解酶(PelC)。对重组酶的酶学性质研究发现, 该酶的最适反应温度为90oC, 最适反应pH为8.5, 在pH 8.2~9.8之间酶活力稳定, 95oC酶活半衰期为2 h, 并且该酶依赖Ca2+作为活性离子。在工业生产常用温度60oC下, 该酶能够长时间保持稳定, 并具有较高的酶活力。以多聚半乳糖醛酸(PGA)为底物时, 其动力学参数Km值为0.11 mmol/L, Vmax值为327 U/mg。SDS-PAGE结果显示该重组酶的分子量为43 kD, 与理论值相符。基于热激载体pHsh的重组表达系统具有诱导表达简便、诱导方式廉价的优点, 且重组酶热稳定性非常好, 这对该酶的大规模发酵应用具有重要意义。     

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.     

犬蝠对小果野芭蕉的取食及种子传播

2004年9月到12月,在西双版纳热带植物园沟谷雨林内,通过详细的野外观察和雾网实验以及种子的定时收集方法,对犬蝠(Cynopterussphinx)取食小果野芭蕉(Musaacuminata)的行为及规律进行了研究。发现在夜晚24:00以前,犬蝠取食小果野芭蕉有明显的2个活动高峰,分别发生在20:30和22:30左右,这一结果与雾网采样结果较为一致。在研究期间的雨季(9-10月)和干季(11-12月),犬蝠平均被捕获量为2.2±0.3只/d、1.4±0.3只/d,二者之间没有显著的差异;同时这两个季节收集的种子团数量分别为9.0±1.1个/d和7.2±1.4个/d,也没有显著差异。犬蝠对小果野芭蕉种子的传播受生境影响较为显著,各样地间种子传播的结果大不相同;地点和季节对犬蝠传播种子也不存在交互影响。犬蝠对小果野芭蕉种子的传播距离为50-200m,是小果野芭蕉有效的种子传播者     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

Purification and biochemical characterization of polygalacturonase II produced in semi-solid medium by a strain of Fusarium moniliforme

A strain of Fusarium moniliforme isolated from a tropical mangrove ecosystem near Mumbai, India and deposited in the National Collection of Industrial Microorganisms (NCIM) as F. moniliforme NCIM 1276. The organism produced a single extracellular polygalacturonase (PG I) [EC 3.2.1.15] at pH 5 and a single pectate lyase (PL) [EC 4.2.2.2] at pH 8 in liquid medium containing 1% citrus pectin. Growth on semi-solid medium containing wheat bran and orange pulp resulted in a three-fold increase in PG production and a two-fold increase in PL production in comparison with that in liquid medium. The increased production of PG on semi-solid media, as compared to production in liquid media was investigated. The increased production of PG was partly due to the expression of a second polygalacturonase (PG II) isoenzyme by the fungus which was biochemically different from the one produced in liquid medium. The second PG II was a 30.6kDa enzyme, had an alkaline pI of 8.6, the Km was 0.166mg ml(-1), Vmax 13.33 micromol min(-1) mg(-1) and the kcat was 403 min(-1). It had a specific activity of 18.66U mg(-1). The differences between the PGs (PG I and PG II) suggest that the two enzymes are the products of different genes. The fungus also produced the same two PGs when it infected Lycopersicon esculentum (tomato). Only one PL was produced irrespective of growth conditions.     

A simple procedure for the isolation of high quality RNA from ripening banana fruit

Isolation of high quality RNA from ripening banana fruit tissue is difficult due to high levels of polysaccharides and other substances that interfere when using conventional procedures for RNA isolation. These substances not only decrease the yield but the quality of RNA is almost unusable. We describe here a simple RNA procedure that effectively removes these contaminating substances without affecting the yield. Following this procedure, we routinely obtained 80–150 μg of total RNA per g fresh tissue. The RNA is of good quality and suitable for northern analysis, RT-PCR and cDNA library construction. NBRI publication No. 488(NS).     

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.