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.

Cis- and Trans-Cinnamic Acids Have Different Effects on the Catalytic Properties of Arabidopsis Phenylalanine Ammonia Lyases PAL1, PAL2, and PAL4

Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.

Recalcitrant cell wall of Ulva lactuca seaweed is degraded by a single ulvan lyase from family 25 of polysaccharide lyases

Green macroalgae,e.g.,Ulva lactuca,are valuable bioactive sources of nutrients;but algae recalcitrant cell walls,composed of a complex cross-linked matrix of polysaccharides,can compromise their utilization as feedstuffs for monogastric animals.This study aimed to evaluate the ability of pre-selected Carbohydrate-Active enZymes(CAZymes)and sulfatases to degrade U.lactuca cell walls and release nutritive compounds.A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U.lactuca cell wall polysaccharides.The enzymes were incubated with the macroalga,either alone or in combination,to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls.The individual action of a polysaccharide lyase family 25(PL25),an ulvan lyase,was shown to be the most efficient in cell wall disruption.The ulvan lyase treatment,in triplicate measures,promoted the release of 4.54 g/L(P<0.001)reducing sugars,a mono-and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga(P<0.01),respectively,and a decrease of 41.7%(P<0.001)in cell wall fluorescence,in comparison to control.The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated.A release of some monounsaturated fatty acids was observed,particularly the health beneficial 18:1c9(P<0.001).However,no significant release of total fatty acids(P>0.05),proteins(P?0.861)or pigments(P>0.05)was found.These results highlight the capacity of a single recombinant ulvan lyase(PL25 family)to incompletely disrupt U.lactuca cell walls.This enzyme could enhance the bioaccessibility of U.lactuca bioactive products with promising utilization in the feed industry.

Catalytic Characteristics of Lyases in Gloeobacter violaceus PCC 7421

In Gloeobacter violaceus PCC 7421, three possible lyase genes glr1191, glr1182 and gll1188 were selected by Blast sorting. The coded proteins of these three genes were co-expressed with their substrate protein in E. coli, respectively, and some chromoproteins were obtained. The fluorescence spectra showed that high fluorescence intensity was observed in the three experimental groups that involved the lyase genes, but little fluorescence intensity was observed in negative control groups. The ratio of relative fluorescence intensity in the experimental group with glr1191 was 64.8%. The result of SDS-PAGE indicated that the molecular weights of the three chromoproteins were 22.0 10 3 , 23.6 10 3 and 22.1 10 3 , respectively. The result of zinc-induced fluorescence re- vealed that the phycobilin in the three chromoproteins was covalently coupled to their apo-proteins. The result also showed that the coded proteins of these three genes （CpeS1 , CpeT1 , CpeY ）could cata- lyze the covalent coupling of different phycobilins to their apo- proteins and formed active chromoproteins.

Genome-wide identification of the pectate lyase(PEL)gene family members in Malvaceae,and their contribution to cotton fiber quality

Pectin is a major constituent of the plant cell wall.Pectate lyase(PEL,EC 4.2.2.2)uses anti-β-elimination chemistry to cleave theα-1,4 glycosidic linkage in the homogalacturonan region of pectin.However,limited information is available on the comprehensive and evolutionary analysis of PELs in the Malvaceae.In this study,we identified 597PEL genes from 10 Malvaceae species.Phylogenetic and motif analyses revealed that these PELs are classified into six subfamilies:Clades I,II,III,IV,Va,and Vb.The two largest subfamilies,Clades I and II,contained 237 and222 PEL members,respectively.The members of Clades Va and Vb only contained four or five motifs,far fewer than the other subfamilies.Gene duplication analysis showed that segmental duplication played a crucial role in the expansion of the PEL gene family in Gossypium species.The PELs from Clades I,IV,Va,and Vb were expressed during the fiber elongation stage,but nearly all PEL genes from Clades II and III showed no expression in any of the investigated fiber developmental stages.We further performed single-gene haplotype association analysis in 2,001G.hirsutum accessions and 229 G.barbadense accessions.Interestingly,14 PELs were significantly associated with fiber length and strength traits in G.barbadense with superior fiber quality,while only eight GhPEL genes were found to be significantly associated with fiber quality traits in G.hirsutum.Our findings provide important information for further evolutionary and functional research on the PEL gene family members and their potential use for fiber quality improvement in cotton.

Investigating the mechanisms of isochorismate synthase:An approach to improve salicylic acid synthesis and increase resistance to Fusarium head blight in wheat

Salicylic acid(SA),a vital endogenous hormone,plays a crucial role in plant growth and the response to abiotic and biotic stress.Isochorismate synthase(ICS)and phenylalanine ammonia lyase(PAL)are critical rate-limiting enzymes for SA synthesis.Fusarium head blight(FHB)seriously threatens the safety of wheat production,but increasing the content of SA can enhance FHB resistance.However,the pathway of SA synthesis and regulation in wheat remains unknown.In this study,three wheat ICS(TaICSA,TaICSB,and TaICSD)were identified,and their functions were validated in vitro for isomerizing chorismate to isochorismate.The mutation of one or two homoeoalleles of TaICSA,TaICSB,and TaICSD in the wheat variety‘Cadenza’reduced SA levels under ultraviolet treatment and Fusarium graminearum infection,further enhancing sensitivity to FHB.Overexpression of TaICSA can significantly enhance SA levels and resistance to FHB.To further study SA synthesis pathways in wheat and avoid interference with pathogenicity related genes,the leaves of wild-type Cadenza and different TaICS mutant lines were subjected to ultraviolet treatment for transcriptomic analysis.The results showed that 37 PALs might be involved in endogenous SA synthesis,and 82 WRKY and MYB family transcription factors may regulate the expression of ICS and PAL.These results were further confirmed by RT-PCR.In conclusion,this study expands our knowledge of SA biosynthesis and identifies TaICSA,as well as several additional candidate genes that encode transcription factors for regulating endogenous SA levels,as part of an efficient strategy for enhancing FHB resistance in wheat.

Highly Aromatic Norditerpenoid Heterodimers and Monomers from Trigonostemon fragilis

Four new norditerpenoid heterodimers with different dimerization patterns-namely,trigofragiloids A-C(denoted as compounds 1-3)and(+)-and(-)-trigofragiloid D(compound 4)-and three new phenanthrenone norditerpenoids-namely,trigofragiloids E-G(compounds 5-7)-were isolated from Trigonostemon fragilis.Compounds 1 and 2 feature a novel heterodimeric carbon skeleton formed by the conjugation of a tetra-norditerpenoid and an ennea-norditerpenoid;they have been identified as class 2 atropisomers by means of quantum chemical calculations.Compound 3 is an unprecedented phenylpropanoid-norditerpenoid adduct with a new dimerization pattern.Compounds(+)-and(-)-4 are the first example of S-shaped 1,4-dioxane-fused norditerpenoid dimers.Inspired by the structure elucidation of compound 4,two co-occurring analogues,actephilol A and epiactephilol A,were structurally revised as a pair of geometrical isomers and were identified as two pairs of enantiomers,(+)-and(-)-8 and(+)-and(-)-9,respectively.Their structures were characterized using a combined method.Notably,compound 7 exhibits remarkable adenosine triphosphate-citrate lyase(ACLY)inhibition with a halfmaximal inhibition concentration(IC50)value of(0.46±0.11)lmol·L^(-1),as active as the positive control BMS-303141,and a molecular docking study offers deep insight into the interaction between compound 7 and ACLY.

Activities of lipoxygenase and phenylalanine ammonia lyase in poplar leaves induced by insect herbivory and volatiles

A study was conducted to explore the defense response in woody plants after insect herbivory. The activities of two enzymes, lipoxygenase （LOX）, a key enzyme ofjasmonate （JA） pathway, and phenylalanine ammonia lyase （PAL）, a rate-limiting enzyme of phenyl- propanoid pathway, were measured in the leaves of one-year-old poplar （Populus simonii × P. pyramidalis ＇Opera 8277＇） cuttings after Clostera anachoreta larvae attack. The results show that the increased activities of LOX and PAL were found not only in the leaves wounded by C. anachoreta larvae but also in their tipper systemic leaves, indicating that JA and phenylpropanoid pathways were activated, and the defense response was stimulated systemically. The increase in LOX and PAL activities in neighboring intact poplar cuttings sug- gested that there exists the interplant communication between poplar plants mediated by the herbivore-induced volatiles. Methyl jasmonate （MeJA） was also proved to be an airborne signal to induce defense response in P simonii × P pyramidalis ＇Opera 8277＇ cuttings.

Enzymatically prepared alginate oligosaccharides improve broiler chicken growth performance by modulating the gut microbiota and growth hormone signals

Background Alginate oligosaccharide(AOS)holds great potential as a novel feed supplement in farm animals.However,the effects of AOS on chicken health and the underlying mechanisms are not fully understood.This study aimed to optimize the enzymatic preparation of AOS by using bacterial alginate lyases expressed in yeast,investigate the effects of the prepared AOS on the growth performance and gut health of broiler chickens,and reveal the underlying mechanisms.Results Five alginate lyases from bacteria were cloned into Pichia pastoris GS115 and the alginate lyase PDE9 was expressed at relatively high yield,activity and stability in P.pastoris.Animal trials were carried out using 3201-day-old male Arbor Acres broilers(four groups;8 replicates/group×10 chicks/replicate)receiving either a basal diet or the same diet supplemented with 100,200 and 400 mg/kg PDE9-prepared AOS for 42 d.The results showed that dietary supplementation of 200 mg/kg AOS displayed the highest activity in promoting the birds’ADG and ADFI(P<0.05).AOS ameliorated the intestinal morphology,absorption function and barrier function,as indicated by the enhanced(P<0.05)intestinal villus height,maltase activity,and the expression of PEPT,SGLT1,ZNT1,and occludin.AOS also increased serum insulin-like growth factor-1,ghrelin(P<0.05),and growth hormone(P<0.1).Moreover,the concentrations of acetate,isobutyrate,isovalerate,valerate,and total SCFAs in cecum of birds fed AOS were significantly higher than the control birds(P<0.05).Metagenomic analysis indicated that AOS modulated the chicken gut microbiota structure,function,and microbial interactions and promoted the growth of SCFAs-producing bacteria,for example,Dorea sp.002160985;SCFAs,especially acetate,were found positively correlated with the chicken growth performance and growth-related hormone signals(P<0.05).We further verified that AOS can be utilized by Dorea sp.to grow and to produce acetate in vitro.Conclusions We demonstrated that the enzymatically produced AOS effectively promoted broiler chicken growth performance by modulating the chicken gut microbiota structure and function.For the first time,we established the connections among AOS,chicken gut microbiota/SCFAs,growth hormone signals and chicken growth performance.

Cloning, Expression and Analysis of pal, a Positive Gene Involved in Capsaicin Biosynthesis in Capsicum chinense Jacq

The cDNA sequence of Capal gene was cloned from Capsicum chinense Jacq by RT-PCR and sequenced. Bioinformatics analysis showed that Capal en- codes a putative polypeptide of 683 amino acids with a calculated molecular mass of 74.2 kD and a theoretical pl of 6.9. Multiple sequence alignments and phyloge- netic tree analyses showed that Capal protein of C. chinense is similar to that of Capsicum annuum var. conoides, with an overall sequence similarity of 96%. The prokaryotic expression vector pET-32a-pal was constructed and induced to express in E. coil BL21. The SDS-PAGE analysis showed that the relative molecular mass of the induced new protein is about 74 kD, which was basically identical with that predicted by DNAMAN software （74.3 kD）, Real-time PCR analysis showed that ex- ogenous jasmonic acid （JA） promoted pal expression. The accumulation of capsaicin in pepper was analyzed by high performance liquid chromatography （HPLC）, and the results indicated that exogenous jasmonic acid （JA） can promote the synthesis of capsaicin. This study will provide valuable experimental basis for the research of transcription regulation and explaining the gene function of pal.

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。

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.

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.

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.

Optimization of solid fermentation of cellulase from Trichoderma koningii

To exploit peashrub resources in Ordos as fodders, it is very crucial to realize industrial production of cheap cellulase of high activity by optimizing culture technology, especially culture substrate. In this study, a new prescription experiment based on uniform design ideal was invented and successfully applied in the solid fermentation of Trichoderma koningii F244, which was performed with two different temperature degrees. The activities of FPA, cotton lyase, CMCase and β-glucosidase were assayed and then mathematical models of enzymatic activities, which were figured out by Unconstraint Mathematical Programming, were developed by Multivariate Regression Program of SPSS10.0. Enzymatic activities of optimized substrate prescriptions corresponding to mathematical models were forecasted to determine an ideal substrate prescription. It is revealed that in solid fermentation, Tween80 has negative effect on cellulase production. Furthermore, the ideal prescription for cellulase complex production by Trichoderma koningii F244 was straw powder 16.9%,wheat bran 26.5%, (NH 4) 2SO 4 9.5% and water 47.1%, whose corresponding cellulase activity was expected to be at the same high level with that of Trichoderma reesei Q9414 on its own recommended substrate. Especially, goats mainly fed on peashrub tissues mixed with cellulase complex of this prescription and culture technology, got an incremental ratio of 0.3 kg/d, which brought a very promising feeding prospect for local peashrub resource. By populization of this cellulase complex, it can integrate living standard, economic construction of local residents into vegetational restoration tightly and thus this paper will be very meaningful to be use for reference for western China like Ordos to realize its sustainable development of economy, society and environment.

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.

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.

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.

Key genes expression of reductive tricarboxylic acid cycle from deep-sea hydrothermal chemolithoautotrophic Caminibacter profundus in response to salinity, pH and O_2

CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid （rTCA） cycle. However, genes fragments of cbbL and cbbMencoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase （ACL）, pyruvate： ferredoxin oxidoreductase （POR） and 2-oxoglutarate： ferredoxin oxidoreductase （OOR） were also present in C. profun- dus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% （optimum 3.0%）, pH 5.0-6.5（optimum pH 6.5）, anaerobic to microaerobic conditions （optimum 1.0% 02）. Gene expression pat- terns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profun- dus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.

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.