Purification and Characterization of Hyaluronate Lyases Produced by Two Types of Bacteria

Hyaluronate lyases were obtained from two types of naturally isolated bacterial strains Paenibacillus yunnanensis and Paennarthrobacter nicotinovorans.PyHL(form P.yunnanensis)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 74 kDa by SDS-PAGE and the maximal activity at pH 5.0,35℃.PyHL maximally degraded hyaluronate by an endo-type manner,and showed low degradation activity toward chondroitin sulfates.Dermatan sulfate was not the substrate.PnHL(from P.nicotinovorans)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 70 kDa by SDS-PAGE and the maximal activity at pH 6.0,30℃.Genomic analysis of P.nicotinovorans on the bases of the internal amino acid sequences of PnHL.

S1P lyase在肝癌中的表达及对其预后的影响

目的探讨S1P lyase在肝癌中的表达情况,及其对肝癌预后的影响。方法选取桂林医学院附属医院54例肝细胞癌患者,real-time PCR分析S1P lyase在54对肝细胞癌癌组织及癌旁组织中的表达,运用统计学方法探讨S1P lyase表达与肝癌临床病理特征的关系;利用Kaplan-Meier分析S1P lyase对肝癌预后的影响;应用STRING数据库初步探讨神经鞘脂信号通路中可能与S1P lyase相互作用的信号分子。结果 S1P lyase在肝癌组织中的表达明显低于癌旁组织(P<0.05)。S1P lyase在最大径≥10cm的肝癌病例中的表达量低于5cm<最大径<10cm及最大径<5cm的肝癌病例(P<0.05),AJCC分期(Ⅲ+Ⅳ)期肝癌患者中S1P lyase的表达低于(Ⅰ+Ⅱ)期患者(P<0.05)。Kaplan-Meier分析显示S1P lyase表达量越低,肝癌病人预后越差(HR=0.65,P=0.017)。而STRING数据库数据表明S1P lyase可能与神经鞘脂代谢通路中CERS2、CERS4、CERS5、CERS6、SGPP1、SGPP2、ORMDL1、ORMDL2及ORMDL3存在潜在相互作用的可能。结论 S1P lyase在肝癌中低表达,S1P lyase低表达预示着肝癌患者的不良预后。

Enhancement of Phenylalanine Ammonia Lyase,Polyphenoloxidase,and Peroxidase in Cucumber Seedlings by Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae) Infestation

In this study, the activities of phenylalanine ammonia lyase （PAL）, polyphenoloxidase （PPO）, and peroxidase （POD） were assayed in cucumber seedlings （Cucumis sativus L.） at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci （Gennadius） using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.

Pectate lyase is a factor in the adaptability for Heterodera glycines infecting tobacco

The soybean cyst nematode, Heterodeara glycines, is a serious pathogen of soybean, and reported to be the host of a wide range of Fabaceae. In the present study, the host specificity and reproductivity of two populations of H. glycines collected from soybean and tobacco were identified and characterized. The comparative identity between β-1,4-endoglucanase, pectate lyase and chorismate mutase of H. glycines parasitizing on soybean and tobacco were 99, 97 and 98%, respectively. The qR T-PCR analysis indicated that the expression of pectate lyase 2 gene was significantly higher in second-stage juveniles of H. glycines Henan population parasitizing on tobacco than that of H. glycines Shanxi population parasitizing on soybean. In addition, the pectic acid content of cell wall was significantly higher(45%) in the roots of tobacco than the roots of soybean. Our results indicate that the changes in transcript parasitism genes may be a result of long-term evolution illustrating how a plant-parasitic nematode adapts to the host environment for optimal infestation and survival.

ATP-citrate lyase regulates stemness and metastasis in hepatocellular carcinoma via the Wnt/β-catenin signaling pathway

Background: Hepatocellular carcinoma(HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells(LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase(ACLY), as a key enzyme for de novo lipogenesis, has been reported to be upregulated in various tumors. However, its expression and role in HCC and LTICs remain unknown. Methods: The expressions of ACLY in HCC tissues were detected by quantitative real-time PCR(q RT-PCR), Western blotting and immunohistochemistry. Kaplan-Meier curves and Chi-square test were used to determine the clinical significance of ACLY expression in HCC patients. A series of assays were performed to determine the function of ACLY on stemness, migration and invasion of HCC cells. Luciferase reporter assay, Western blotting and immunoprecipitation were used to study the regulation of the Wnt/β-catenin signaling by ACLY. Rescue experiments were performed to investigate whether β-catenin was the mediator of ACLY-regulated stemness and migration in HCC cells. Results: ACLY was highly expressed in HCC tissues and LTICs. Overexpression of ACLY was significantly correlated with poor prognosis, progression and metastasis of HCC patients. Knockdown of ACLY remarkably suppressed stemness properties, migration and invasion in HCC cells. Mechanistically, ACLY could regulate the canonical Wnt pathway by affecting the stability of β-catenin, and Lys49 acetylation of β-catenin might mediate ACLY-regulated β-catenin level in HCC cells. Conclusions: ACLY is a potent regulator of Wnt/β-catenin signaling in modulating LTICs stemness and metastasis in HCC. ACLY may serve as a new target for the diagnosis and treatment of HCC.

Screening Peptide Inhibitors Using Phage Peptide Library with Isocitrate Lyase in Mycobacterium tuberculosis as Target

When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase（ICL） is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB（TB：tubercular） drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.

Optimization of Culturing Condition and Medium Composition for the Production of Alginate Lyase by a Marine Vibrio sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the produc- tion of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL–1. The best inoculum volume and inoculum age were 10% and 12 h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had nega- tive effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had posi- tive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of algi- nate lyase production under the optimized condition was described. The optimal harvest time was 48 h.

Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium Pseudoalteromonas sp. NJ-21

In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.

Phenylalanine ammonia-lyase gene families in cucurbit species: Structure, evolution, and expression

Phenylalanine ammonia-lyase （PAL）, the first enzyme of phenylpropanoid pathway, is always encoded by multigene families in plants. In this study, using genome-wide searches, 13 PAL genes in cucumber （CsPAL1-13） and 13 PALs in melon （Cm- PALl-13） were identified. In the corresponding genomes, ten of these PAL genes were located in tandem in two clusters, while the others were widely dispersed in different chromosomes as a single copy. The protein sequences of CsPALs and CmPALs shared an overall high identity to each other. In our previous report, 12 PAL genes were identified in watermelon （CIPAL1-12）. Thereby, a total of 38 cucurbit PAL members were included. Here, a comprehensive comparison of PAL gene families was performed among three cucurbit plants. The phylogenetic and syntenic analyses placed the cucurbit PALs as 11 CsPAL-CmPAL-CIPAL triples, of which ten triples were clustered into the dicot group, and the remaining one, CsPAL1-CmPAL8-CIPAL2, was grouped with gymnosperm PALs and might serve as an ancestor of cucurbit PALs. By comparing the syntenic relationships and gene structure of these PAL genes, the expansion of cucurbit PAL families might arise from a series of segmental and tandem duplications and intron insertion events. Furthermore, the expression profiling in different tissues suggested that different cucurbit PALs displayed divergent but overlapping expression profiles, and the CsPAL-CmPAL-CIPAL orthologs showed correlative expression patterns among three cucurbit plants. Taken together, this study provided an extensive description on the evolution and expression of cucurbit PAL gene families and might facilitate the further studies for elucidating the functions of PALs in cucurbit plants.

Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Isolation of Protoplasts from Undaria pinnatifida by Alginate Lyase Digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28 ℃ for 2 h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5 g fresh thalline with NaCl 50 and at the shaking speed of 150 r min -1 during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca 2+ and slightly enhanced by Fe 2+ and Mn 2+ at concentrations of 0.05, 0.08 and 0.10 mol L -1.

Lyase Activities of Heterologous CpcS and CpcT for Phycocyanin Holo-β-subunit from Arthrospira platensis in Escherichia coli

Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin(PCB)-producing genes(hox1 and pcyA) while the other contained the phycobiliprotein gene(cpcB) and the lyase gene(either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit(β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.

Cloning and characterization of a new endo-type polyG-specific alginate lyase from bacteria Vibrio sp. QD-5

Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp. QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome sequencing, and the result showed that the Vibrio sp. QD-5 containing an alginate lyase gene cluster. One of these genes, aly-IV, was cloned and characterized for the first time. After overexpression, Aly-IV, with a molecular mass of about 62 kDa and a theoretical isoelectric point (pI) of 5.12, was purified to a specific activity of 1 256.78 U/mg and showed highest activity at 35°C in the Tris-HCl buffer at pH of 8.9. Moreover, the enzyme activity was enhanced by the metal ions of Na+, K+ and Mg2+ under certain concentration. Aly-IV degraded favorably polyG blocks in an endo-type, yielding monomer and dimer as the main products. Due to its high substrate specificity, Aly-IV could be used as a potential tool for production of polyG oligosaccharides with low degree of polymerization (DP) and for determining the fine structure of alginate.

Organomercury Captured by Lyase Overexpressed <i>Escherichia coli</i>and Its Evaluation by <i>In-Cell</i>Radiometry*

Organomercury lyase (MerB) overexpressed in Escherichia coli captured and decomposed organomercury compounds, and it has been detected by radioactive analysis with neutron irradiation. Genetically modified E. coli captures a lot of mercury from a cultivation solution with about 80% recovery, when the bacteria are growing during 24 to 72 hours. Since the modified E. coli has no additive gene for mercury metabolism, the bacteria could hold mercury tightly by the MerB enzyme in their cell and do not release them into medium. In the later, 72 hours after, bacteria have less recovery ratio;it may be affected by undecompsed mercury compounds in bacteria growth. The recovery ability of the bacteria would not be changed by addition of the MerB producing reagent (IPTG). A quantitative value of mercury atom is estimated by an emission of γ-ray by reactor neutron from a dried cell or solution on a filter paper, which is available for nondestructive testing of bacteria holding mercury atoms. In this method an efficient recovery system of toxic mercury from a polluted solution has been archived without destruction of samples, so called in-cell analysis.

Expression and purification of recombinant lyase gp17 from the LSB-1 phage in Escherichia coli

Dear Editor,In recent years,owing to the fact that antibiotic-resistant bacteria have become more and more prevalent,there has been a resurgence of interest in the use of bacteriophages.However,bacteriophage therapy remains an underutilized option in modern medicine due to technical hurdles such as limited host range,narrow spectrum of

Effect of Pectin Lyase Enzyme on Fermentation and Drying of Cocoa (Theobroma cacao L.): An Alternative to Improve Raw Material in the Industry of Chocolate

Cocoa (Theobroma cacao), in all its presentations, is consumed all over the world and is one of the main drivers of the economic in several countries. The world’s Cocoa tendency is focused on developing special beans. This category is subject to postharvest processes of utmost importance such as the fermentation and dry, which are currently carried out with traditional and poorly effective devices, which need to be improved to obtain a high quality product. The aim of this study was to evaluate the influence of the pectin lyase enzyme (E.C.4.2.2.10) on the postharvest cocoa process. We evaluated the enzyme dosage (1.0% and 0.5%) in fermentation and its effect on the variables temperature, acidity and drying time by convection at 60°C. The Pectin lyase activity during fermentation does not cause a significant effect on the variables of temperature and acidity;however, the drying process time required to achieve 7.0% moisture was reduced. The enzyme dosage of 1.0% was the best result, the amount of exudate obtained (115 ml) during fermentation and the best degree of fermentation (77% ± 3.8) were increased and further shows a change in porosity facilitating the scale surface and internal moisture diffusion. The drying rate (Nw) expressed in kgwater/m2 * min was determined based on the empirical model of Newton, where the higher speed was obtained during the falling period. In conclusion, enzyme dosage 1% was the best concentration evaluated because weaken grain husk, which allowed an adequate fermentation,and subsequent time drying reduction until 10.8 h.

Child with adenylosuccinate lyase deficiency caused by a novel complex heterozygous mutation in the ADSL gene:A case report

BACKGROUND Adenylosuccinate lyase(ADSL)deficiency is a rare autosomal-recessive defect of purine metabolism caused by mutation of the ADSL gene.It can cause severe neurological impairment and diverse clinical manifestations,including epilepsy.CASE SUMMARY Here,we describe a 3-year-old Chinese boy who had both psychomotor retardation and refractory epilepsy.Magnetic resonance imaging showed myelin hypoplasia.Electroencephalography findings supported a diagnosis of epilepsy.Whole-exon sequencing revealed the presence of a novel complex heterozygous mutation in the ADSL gene:The splicing mutation c.154-3C>G and the missense mutation c.71C>T(p.Pro24Leu).Considering the patient’s clinical presentation and genetic test results,the complex heterozygous mutation was predicted to prevent both ADSL alleles from producing normal ADSL,which may have led to ADSL deficiency.Finally,the child was diagnosed with ADSL deficiency.CONCLUSION We identified a novel complex heterozygous mutation in the ADSL gene associated with ADSL deficiency,thus expanding the known spectrum of pathogenic mutations that cause ADSL deficiency.Additionally,we describe epilepsy that occurs in patients with ADSL deficiency.

Evaluation of the Hydroxynitrile Lyase Activity in Cell Cultures of Capulin (Prunus serotina)

Enzymatic preparations obtained from young plants and cell cultures of capulin were screened for hydroxynitrile lyase activity. The threeweek old plants, grown under sterile conditions, were used to establish a solid cell culture. Crude preparations obtained from this plant material were evaluated for the transformation of benzaldehyde to the corresponding cyanohydrin (mandelonitrile). The results show that the crude material from roots, stalks, and leaves of young plants and calli of roots, stalks, internodes and petioles biocatalyzed the addition of hydrogen cyanide (HCN) to benzaldehyde with a modest to excellent enantioselectivity.

Genetic diversity and population structure analysis of Pistacia species revealed by phenylalanine ammonia-lyase gene markers and implications for conservation

Information on population genetic structure and crop genetic diversity is important for genetically improving crop species and conserving threatened species. The PAL gene sequence is part of a multigene family that can be utilized to design DNA marker systems for genetic diversity and population structure investigation. In the current study, genetic diversity and population structure of 100 accessions of wild Pistacia species were investigated with 78 PAL markers. A protocol for using PAL sequences as DNA markers was developed. A total of 313 PAL loci were recognized, showing 100% polymorphism for PAL markers. The PAL markers produced relatively more observed and effective alleles in Pistacia falcata and Pistacia atlantica, with a higher Shannon＇s information index and expected heterozygosity in P. atlantica, Pistacia vera and Pistacia mutica. Pairwise assessment of Nei＇s genetic distance and genetic identity between populations revealed a close association between geographically iso- lated populations of Pistacia khinjuk and Pistacia chinensis. The accessions of wild Pistacia species had more genetic relationship among studied groups of species. Analysis of molecular variance indicated 19% among- population variation and 81% within-population variation for the PAL gene based DNA marker. Population structure analysis based on PAL revealed four groups with high genetic admixture among populations. The results establish PAL markers as a functional DNA marker system and provide important genetic information about accessions from wild populations of Pistacia species.

Bioscouring Knitted Cotton Fabric with an Experimental Pectate Lyase

An experimental pectate lyase enzyme was used to scour knitted cotton fabric and the emphasis was on pectin removal.Using an enzyme dosage of 0.2 g/L at temperature 55℃ and pH 6.35 for 30 min,good scouring properties were obtained.When appropriate concentrations of 1-Hydroxy Ethylidene-1,1-Diphosphonic Acid(HEDP)and CaCl2 were added,the percentage pectin removal improved significantly.