A novel type of lysine oxidase: L-lysine-epsilon-oxidase

The melanogenic marine bacterium M. mediterranea synthesizes marinocine, a protein with antibacterial activity. We cloned the gene coding for this protein and named it lodA [P. Lucas-Elío, P. Hernández, A. Sanchez-Amat, F. Solano, Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea. Biochim. Biophys. Acta 1721 (2005) 193-203; P. Lucas-Elío, D. Gómez, F. Solano, A. Sanchez-Amat, The antimicrobial activity of marinocine, synthesized by M. mediterranea, is due to the hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188 (2006) 2493-2501]. Now, we show that this protein is a new type of lysine oxidase which catalyzes the oxidative deamination of free L-lysine into 6-semialdehyde 2-aminoadipic acid, ammonia and hydrogen peroxide. This new enzyme is compared to other enzymes related to lysine transformation. Two different groups have been used for comparison. Enzymes in the first group lead to 2-aminoadipic acid as a final product. The second one would be enzymes catalyzing the oxidative deamination of lysine releasing H2O2, namely lysine-alpha-oxidase (LalphaO) and lysyl oxidase (Lox). Kinetic properties, substrate specificity and inhibition pattern show clear differences with all above mentioned lysine-related enzymes. Thus, we propose to rename this enzyme lysine-epsilon-oxidase (lod for the gene) instead of marinocine. Lod shows high stereospecificity for free L-lysine, it is inhibited by substrate analogues, such as cadaverine and 6-aminocaproic acid, and also by beta-aminopropionitrile, suggesting the existence of a tyrosine-derived quinone cofactor at its active site.     

Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea

This work describes the purification and partial characterization of a novel antibacterial compound, here named marinocine, produced by Marinomonas mediterranea, a melanogenic marine bacterium with rich secondary metabolism. The antibacterial compound is a protein detected in the medium at death phase of growth. It has been purified to apparent homogeneity from the supernatants of cultures by means of ethanol precipitation followed by column chromatographies on DEAE-Sephadex and Sephacryl HR-200. The protein has an apparent molecular mass of 140-170 kDa according to gel permeation chromatography and non-denaturing SDS-PAGE, although in denaturing SDS-PAGE two mayor bands of 97 and 185 kDa appear. Marinocine is relatively heat-stable and shows a great resistance against many hydrolytic enzymes such as glycosidases, lipase, and proteases. The antibacterial range of the molecule includes Gram-positive and Gram-negative microorganisms, as well as some nosocomial isolates, Staphylococcus aureus and Pseudomonas sp., highly resistant to classical antibiotics. By contrast, marinocine did not show any effect on the eukaryotic microorganisms tested. Regarding eukaryotic CHO cells, the decrease on viability was much lower than the one observed on bacterial cells.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

重组猪乳铁蛋白N端的高效表达及抑菌活性检测

为获得表达猪乳铁蛋白基因的重组菌株,并检测其表达的重组猪乳铁蛋白抑菌活性,应用RT-PCR方法从泌乳3d后母猪乳腺组织中扩增了猪乳铁蛋白N端1077bp的PLF-N基因片段,与GenBank上发表的4株猪乳铁蛋白基因序列相比,核苷酸同源性均达到99%以上。为了得到高表达量的PLF-N基因,以扩增的PLF-N片段为参考模板,经过密码子优化,全基因合成了编码猪乳铁蛋白N端的基因PLF-NS。将其定向插入到原核表达载体pET-30b中,转化大肠杆菌BL21,获得了表达PLF-NS的重组菌pET-PLF-NS/BL21;经IPTG诱导,并对表达条件进行优化,以及通过SDS-PAGE和Western blotting分析均表明猪乳铁蛋白得到了正确表达,其产物分子量约为42kDa,最优表达条件下蛋白表达量占菌体总蛋白的32%,表达产物以包涵体形式存在。包涵体经裂解、纯化、复性处理后纯度达到98%。用琼脂孔穴扩散抑菌法检测表明重组猪乳铁蛋白具有明显的抑菌作用。表明通过基因优化对表达量低的基因进行改造使之高效表达,是一种提高表达效率的有效手段。     

The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity

Antibacterial peptides from various sources express different antibacterial activity. In order to obtain a high activity antibacterial peptide, the sequences of four antimicrobial peptides--Protegrin-1, 4 kDa Scorpion Defensin, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide SMAP-29--were exploited to generate a synthetic antimicrobial peptide cp gene, which was then cloned into the expression vector pPICZalpha-A. The constructed recombinant expression vector pPICZalpha-cp was transformed into Pichia pastoris X-33, in which the synthetic antimicrobial peptide (CP) could be expressed under the control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Results showed that recombinant plasmid is highly stable, and In vitro experiments showed that the recombinant antimicrobial peptide CP is heat and acid-stable, and it has high antibacterial activity against several Gram-positive and -negative bacteria. Only 1 microg of the recombinant antimicrobial peptide CP has an antibacterial activity equivalent to 64 U ampicillin. Thus, this recombinant antimicrobial peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs.     

杂合抗菌肽CecA-mil的改造及在毕赤酵母中的分泌表达

参照毕赤氏巴斯德酵母(Pichia pastorts)偏好密码子,改造并化学合成杂合抗菌肽CecA-mil基因,改造后的CecA-mil基因克隆到pPICZα-A载体中,构建分泌型重组酵母表达载体pPICZα-A-CM,转化Pichia pastoris受体菌X-33。在醇氧化酶(AOX)启动子调控下,分子量约1．9kD的CecA-mil杂合抗菌肽获得表达,经表达条件优化,重组酵母菌的摇瓶发酵产率可达到245μg／mL。抗菌特性研究表明,该表达产物具有广谱抗菌活性,对多数G^-菌及G^ 菌均有较好的抑菌活性,特别是对氨苄青霉素抗性菌和卡那霉素抗性菌抑杀效果更好;具有热稳定性和酸稳定性。这些特点使得重组抗菌肽CecA-mil在食品防腐、疾病防治和动物饲料添加剂等方面显露出很好的应用前景。     

牛抗菌肽Bac7-Bac5融合基因在大肠杆菌中的过表达，纯化及抑菌活性

牛抗菌肽Bac7和Bac5是一种线性阳离子小分子多肽,在机体天然免疫和获得性免疫中都发挥着重要作用。本研究根据Gen bank中公布的牛抗菌肽bac7和bac5成熟肽基因序列,人工合成了融合基因Bac7-Bac5片段,克隆于原核表达载体pET32(a+)中构建了重组表达载体（pET-B7-B5）,将其转化于E coli BL21(DE3) 中实现了重组蛋白B7-B5（rB7-B5）的过表达,表达的rB7-B5以包涵体形式存在,rB7-B5表达量约占细菌总蛋白的36.6％,分子量大小为33kD,与预测大小相符。以经Ni亲和层析柱纯化和多步透析法复性的rB7-B5,对猪胸膜肺炎放线杆菌和耐药性大肠杆菌具有很好的抑菌活性,本研究为新型抗菌制剂的研制和开发奠定了基础。     

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.     

Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine

We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector. This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88. All three constructs were cloned and expressed in Escherichia coli and the proteins purified. The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF. The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays. Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups. This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond. Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration. Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation. These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond.     

Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme

The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.     

棘胸蛙抗菌肽Spinosan-C的串联表达与活性检测

为克服抗菌肽易被蛋白酶降解及对宿主大肠杆菌的杀伤作用,并进一步提高大肠杆菌系统的表达能力,以棘胸蛙Paa spinosa抗菌肽Spinosan-C为研究对象,按照大肠杆菌密码子利用频率进行密码子优化,设计合成8拷贝的串联8×Spinosan-C基因,将合成的串联基因克隆到大肠杆菌表达载体p ET-28a,利用大肠杆菌感受态细胞Rosetta进行原核表达,获得高效表达的串联8×Spinosan-C重组蛋白,用甲酸专一性切割得到抗菌肽Spinosan-C单体。体外抑菌试验表明,切割后的抗菌肽Spinosan-C单体对测试菌生长具有抑制作用,为蛙类抗菌肽的规模化制备提供了参考。     

Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576bp and coded for 191 amino acid residue protein precursors. Each of the deduced elapid cathelicidin has a 22 amino acid residue signal peptide, a conserved cathelin domain of 135 amino acid residues and a mature antimicrobial peptide of 34 amino acid residues. Unlike the highly divergent cathelicidins in mammals, the nucleotide and deduced protein sequences of the three cloned elapid cathelicidins were remarkably conserved. All the elapid mature cathelicidins were predicted to be cleaved at Valine157 by elastase. OH-CATH, the deduced mature cathelicidin from king cobra, was chemically synthesized and it showed strong antibacterial activity against various bacteria with minimal inhibitory concentration of 1-20microg/ml in the presence of 1% NaCl. Meanwhile, the synthetic peptide showed no haemolytic activity toward human red blood cells even at a high dose of 200microg/ml. Phylogenetic analysis of cathelicidins from vertebrate suggested that elapid and viperid cathelicidins were grouped together in the tree. Snake cathelicidins were evolutionary closely related to the neutrophilic granule proteins (NGPs) from mouse, rat and rabbit. Snake cathelicidins also showed a close relationship with avian fowlicidins (1-3) and chicken myeloid antimicrobial peptide 27. Elapid cathelicidins might be used as models for the development of novel therapeutic drugs.     

Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme. Cloning, sequencing, and characterization of the nuclear-encoded gene

The gene COX VII coding for yeast cytochrome c oxidase subunit VII has been cloned by a two-step procedure. Two degenerate oligonucleotides corresponding to amino- and carboxyl-terminal protein segments were used in a polymerase chain reaction for the amplification of a major portion of subunit VII (residues 1-52), which was then used for the cloning of complete COX VII. From the nucleotide sequence, an additional amino-terminal and two additional carboxyl-terminal amino acids are predicted as compared with the described primary sequence (Power, S. D., Lochrie, M. A., and Poyton, R. O. (1986) J. Biol. Chem. 261, 9206-9209). Beside subunit VIIa the subunit described here is the only nuclear encoded subunit of cytochrome c oxidase in yeast without a leader sequence. COX VII exists as a single copy per haploid genome as shown by Southern blot and gene disruption. Null mutants produced by gene disruption at the COX VII locus were respiratory-deficient. No cytochrome c oxidase activity was detectable nor was there an assembly of the oxidase complex.     

通过定点突变提高乳链菌肽对热及pH的稳定性

【目的】通过定点突变技术改变乳链菌肽(nisin)特定位置氨基酸,获得性质改善的nisin突变体,为扩大其应用范围提供依据。【方法】在抑菌谱扩大的nisin单突变体M21K nisinZ的基础上,对M21K nisZ基因第29位丝氨酸密码子进行定点突变;将其克隆至乳酸菌表达载体pMG36e,并在Lactococcus lactis NZ9800中进行表达;双突变体M21K/S29K nisinZ经分离纯化后检测其在抑菌活性、抑菌谱和稳定性等方面的变化。【结果】与单突变体M21K nisinZ及野生型nisinZ(wild-type,WT)相比,双突变体M21K/S29K nisinZ对指示菌的抑菌活性虽有所下降,但其对温度及pH值的稳定性有显著提高。同时其抑菌谱与M21K nisinZ相同,可抑制革兰氏阴性菌,扩大了WT的抑菌谱。【结论】通过改变nisin分子特定位置的氨基酸可以改善nisin分子的理化性质,有可能得到应用范围更广的nisin品种。     

Genome sequence of Rheinheimera sp. strain A13L, isolated from Pangong Lake, India

Rheinheimera sp. strain A13L, which has antimicrobial activity, was isolated from alkaline brackish water of the high-altitude Pangong Lake of Ladakh, India. Here we report the draft genome sequence of Rhienheimera sp. strain A13L (4,523,491 bp with a G+C content of 46.23%). The genome is predicted to contain genes for marinocine and colicin V production, which may be responsible for the antimicrobial activity of the strain.     

β-发卡抗菌肽的全新设计及其生物学活性

通过缬氨酸和精氨酸的交替连接形成β-发卡结构的两条侧链,D-脯氨酸和甘氨酸形成β-转角单元以及侧链末端的两个半胱氨酸连接形成一个二硫键,来设计得到全新的由16残基构成的β-发卡抗菌肽VR。对设计得到的抗菌肽VR的生物学活性进行了检测,主要测定了新型β-发卡抗菌肽VR的最小杀菌浓度、对红细胞的溶血活性、杀菌动力学和盐敏感性。结果发现,VR和蜂毒素具有相似的杀菌活性,而溶血活性远低于蜂毒素,这表明VR比蜂毒素具有更高的细胞选择性。在NaCl的浓度低于100 mmol/L时,VR的杀菌活性没有受到影响;在NaCl的浓度为100 mmol/L时,VR具有50%的杀菌活性。综上可见,VR具有较优异的生物学活性,拥有成为抗生素替代物的发展潜力。     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.