Objective To clone and express a new PL25 family ulvan lyase,TsUly25A,and to characterise its properties and explore its application for the preparation of ulvan oligosaccharides.Methods The gene encoding ulvan lyase ...Objective To clone and express a new PL25 family ulvan lyase,TsUly25A,and to characterise its properties and explore its application for the preparation of ulvan oligosaccharides.Methods The gene encoding ulvan lyase of the PL25 family,tsuly25A,was cloned from Thalassomonas sp.LD5 and then expressed in Escherichia coli BL21(DE3).The recombinant enzyme TsUly25A was subsequently purified and its enzymatic properties were investigated.Results Its enzyme activity was highest at 50℃and pH 8.0,with a specific activity of 0.45 U/mg.NaCl exerted marginal effects on the activity and thermal stability of TsUly25A.TsUly25A could still degrade ulvan polysaccharides in pure water and retained about 60%of its activity.Conclusions Ulvan lyase TsUly25A could degrade ulvan polysaccharides in pure water,laying the foundation for the industrial application of ulvan lyase in the production of ulvan oligosaccharides.展开更多
Blocking the development of edible mushrooms will affect the production cycle and yield of fruiting bodies.Phenylalanine ammonia lyase(PAL,EC 4.3.1.24.)is an enzyme that catalyzes the deamination of phenylalanine to f...Blocking the development of edible mushrooms will affect the production cycle and yield of fruiting bodies.Phenylalanine ammonia lyase(PAL,EC 4.3.1.24.)is an enzyme that catalyzes the deamination of phenylalanine to form trans-cinnamic acid.Previous studies have shown that a decrease in pal1 gene transcription delays fruiting body development in Pleurotus ostreatus.Herein,we used wild type(WT)and RNA interference(RNAi)strains to study the molecular regulation of pal1 by RNA sequencing and Agrobacterium-mediated genetic transformation.Our results showed that interference with the pal1 gene resulted in reductions in the total PAL enzyme activity and the total phenol content,as well as an increase in the intracellular H_(2)O_(2)content.RNA-Seq data demonstrated that the significantly enriched KEGG terms were mainly related to the peroxisome pathway,MAPK signaling pathway-yeast and three other pathways,and the catalase(CAT)gene cat1 is also involved in multiple pathways that were enriched above.Exogenous H_(2)O_(2)significantly enhanced the transcription of the cat1 gene and elevated total CAT enzymatic activity.Moreover,the levels of cat1 gene transcription and the total CAT enzymatic activity in the RNAi-pal1 strains gradually become closer to those in the WT strain through the removal of H_(2)O_(2),which indicated that pal1 regulated the expression of cat1 by affecting the intracellular H_(2)O_(2)content.Finally,the overexpression of the cat1 gene in P.ostreatus caused growth retardation,especially during the process of primordia formation.In conclusion,this study demonstrated that PAL1 affects cat1 gene expression through the signaling molecule H_(2)O_(2)and regulates the development of P.ostreatus.The findings of this study enhance our understanding of the molecular developmental mechanism of edible mushrooms.展开更多
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 info...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.展开更多
Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-B...Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P.pastoris.Furthermore,diverse strategies,encompassing the optimization of expression cassette components,elevation of gene dosage,and co-expression of chaperone factors,were employed to augment PMGL-Ba production in P.pastoris.The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements.By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup,a strain yielding high PMGL-Ba production was achieved.In shake flask fermentation lasting 144 h,the total protein concentration reached 1.81 g/L,and the enzyme activity reached 1821.36 U/mL.For further scale up production,high-density fermentation transpired in a 5 L fermenter for 72 h.Remarkably,the total protein concentration increased to 12.49 g/L,and the enzyme activity reached an impressive 12668.12 U/mL.The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.展开更多
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 lya...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.展开更多
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), ...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.展开更多
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...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.展开更多
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 culturi...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.展开更多
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 populati...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.展开更多
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 amin...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.展开更多
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 s...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.展开更多
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 molecul...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.展开更多
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 st...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.展开更多
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. 51...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.展开更多
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 r...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 bacteria展开更多
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. c...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.展开更多
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 manifest...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.展开更多
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...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.展开更多
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...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.展开更多
文摘Objective To clone and express a new PL25 family ulvan lyase,TsUly25A,and to characterise its properties and explore its application for the preparation of ulvan oligosaccharides.Methods The gene encoding ulvan lyase of the PL25 family,tsuly25A,was cloned from Thalassomonas sp.LD5 and then expressed in Escherichia coli BL21(DE3).The recombinant enzyme TsUly25A was subsequently purified and its enzymatic properties were investigated.Results Its enzyme activity was highest at 50℃and pH 8.0,with a specific activity of 0.45 U/mg.NaCl exerted marginal effects on the activity and thermal stability of TsUly25A.TsUly25A could still degrade ulvan polysaccharides in pure water and retained about 60%of its activity.Conclusions Ulvan lyase TsUly25A could degrade ulvan polysaccharides in pure water,laying the foundation for the industrial application of ulvan lyase in the production of ulvan oligosaccharides.
基金supported by the National Key R&D Program of China(2022YFD1200600)the National Natural Science Foundation of China(32002110)the earmarked fund for China Agriculture Research System(CARS-20)。
文摘Blocking the development of edible mushrooms will affect the production cycle and yield of fruiting bodies.Phenylalanine ammonia lyase(PAL,EC 4.3.1.24.)is an enzyme that catalyzes the deamination of phenylalanine to form trans-cinnamic acid.Previous studies have shown that a decrease in pal1 gene transcription delays fruiting body development in Pleurotus ostreatus.Herein,we used wild type(WT)and RNA interference(RNAi)strains to study the molecular regulation of pal1 by RNA sequencing and Agrobacterium-mediated genetic transformation.Our results showed that interference with the pal1 gene resulted in reductions in the total PAL enzyme activity and the total phenol content,as well as an increase in the intracellular H_(2)O_(2)content.RNA-Seq data demonstrated that the significantly enriched KEGG terms were mainly related to the peroxisome pathway,MAPK signaling pathway-yeast and three other pathways,and the catalase(CAT)gene cat1 is also involved in multiple pathways that were enriched above.Exogenous H_(2)O_(2)significantly enhanced the transcription of the cat1 gene and elevated total CAT enzymatic activity.Moreover,the levels of cat1 gene transcription and the total CAT enzymatic activity in the RNAi-pal1 strains gradually become closer to those in the WT strain through the removal of H_(2)O_(2),which indicated that pal1 regulated the expression of cat1 by affecting the intracellular H_(2)O_(2)content.Finally,the overexpression of the cat1 gene in P.ostreatus caused growth retardation,especially during the process of primordia formation.In conclusion,this study demonstrated that PAL1 affects cat1 gene expression through the signaling molecule H_(2)O_(2)and regulates the development of P.ostreatus.The findings of this study enhance our understanding of the molecular developmental mechanism of edible mushrooms.
基金supported by the Ministry of Agriculture and Rural Affairs,China(2023ZD04039-01)the National Natural Science Foundation of China(32172008)the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang,China(2019R01002)。
文摘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.
基金supported by the National Key R&D Program of China(NO.2021YFC2100400).
文摘Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P.pastoris.Furthermore,diverse strategies,encompassing the optimization of expression cassette components,elevation of gene dosage,and co-expression of chaperone factors,were employed to augment PMGL-Ba production in P.pastoris.The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements.By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup,a strain yielding high PMGL-Ba production was achieved.In shake flask fermentation lasting 144 h,the total protein concentration reached 1.81 g/L,and the enzyme activity reached 1821.36 U/mL.For further scale up production,high-density fermentation transpired in a 5 L fermenter for 72 h.Remarkably,the total protein concentration increased to 12.49 g/L,and the enzyme activity reached an impressive 12668.12 U/mL.The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.
基金supported by the Pro-gramme for Changjiang Scholars and the Innovative Research Team in Universities of China (PCSIRT0607)by the National Natural Science Foundation of China (30871727+2 种基金 30872037)the National Key Project of Scientific and Technical Supporting Programmes Funded by the Ministry of Science & Technology of China (2006BAD01A15 2006BAD24B04)
文摘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.
基金supported by grants from the National Natu-ral Science Foundation of China (81972779)Ministry of Education (MOE) Key Laboratory on signaling Regulation and Targeting Therapy of Liver Cancer,and Shanghai Key Laboratory of Hepato-biliary Tumor Biology,Chinese National Key Project (2018ZX10723204-006-003)。
文摘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.
文摘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.
文摘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.
基金supported by the Special Fund for Agro-scientific Research in the Public Interest in China (201503114)
文摘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.
基金Supported by the Public Science and Technology Research Funds Project of Ocean(No.201505026-4)the Chinese Polar Environment Comprehensive Investigation&Assessment Programs(No.CHINARE2016-01-05)+1 种基金the Basic Scientific Research Funds of First Institute of Oceanography,State Oceanic Administration(SOA)(No.2014T09)the Qingdao Applied Basic Research Project(No.14-2-4-14-jch)
文摘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.
基金The Marine Public Welfare Project of SOA under contract No.201505032the Scientific and Technological Innovation Project financially of Qingdao National Laboratory for Marine Science and Technology under contract No.2016ASKJ14
文摘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.
基金supported by the National Science and Technology Major Project of China (2008ZX08001-004)
文摘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.
基金Supported by the Technology Development Funds of Education Department of Jilin Province,China(No.2008110)
文摘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.
文摘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.
基金supported by the Natural Science Foundation of China(30571637)
文摘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 bacteria
基金Supported by the National Natural Science Foundation of China (30870519 and 30870541)
文摘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.
基金Supported by the Natural Science Foundation of Shandong Province,No.ZR2019MH060。
文摘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.
基金Supported by the Programfor Changjiang Scholors and Innovative Research Teamin University(No.IRT0526)
文摘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.
文摘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.
