不同细菌刺激后仿刺参体腔液中免疫相关酶的应答变化

为了解不同细菌刺激后仿刺参(Apostichopus japonicus)体腔液中免疫因子的应答变化,分别用灿烂弧菌(Vibrio splendidus)、哈维氏弧菌(V.harveyi)、假交替单胞菌(Pseudoalteromonas nigrifacien)、溶壁微球菌(Micrococcus lysodeikticus)和停乳链球菌(Streptococcus dysgadysgalactiae)注射刺激仿刺参,然后分别采用对硝基苯基磷酸酯(p NPP)底物法、氯化硝基四氮唑蓝(NBT)法、溶壁微球菌粉法和多巴络合物生成法对体腔液上清中的酸性磷酸酶(ACP)与碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)、溶菌酶(LYZ)和酚氧化酶(PO)的活力进行了测定。结果显示,灿烂弧菌刺激后,酸性磷酸酶和碱性磷酸酶活力显著升高,而超氧化物歧化酶、溶菌酶和酚氧化酶活力显著降低;哈维氏弧菌刺激后,酸性磷酸酶、超氧化物歧化酶、溶菌酶和酚氧化酶活力显著升高,碱性磷酸酶活力变化不规律;假交替单胞菌刺激后,酸性磷酸酶、溶菌酶和酚氧化酶活力显著升高,超氧化物歧化酶活力先升高后降低,碱性磷酸酶活力变化不规律;溶壁微球菌刺激后,酸性磷酸酶和酚氧化酶活力显著升高,超氧化物歧化酶活力先升高后降低,溶菌酶活力先升高后降低,而后在72 h恢复至对照水平,碱性磷酸酶活力变化不规律;停乳链球菌刺激后,除碱性磷酸酶活力在4 h有所下降外,其余免疫相关酶活力均显著升高。研究结果表明,酚氧化酶是仿刺参非特异性免疫系统中最敏感、高效的免疫因子之一;革兰氏阳性细菌与革兰氏阴性细菌之间在诱导仿刺参免疫因子应答变化上无明显规律性差异;溶壁微球菌诱导溶菌酶的应答变化与灿烂弧菌、哈维氏弧菌、假交替单胞菌和停乳链球菌存在明显差异,溶菌酶可能是仿刺参清除入侵溶壁微球菌的主要免疫因子;灿烂弧菌诱导仿刺参免疫因子应答变化显著不同于其他4株细菌,显示出本研究选取的5个免疫指标在预警灿烂弧菌病害上具有潜在应用价值;停乳链球菌在仿刺参中具有作为免疫增强剂的潜在应用价值。     

Purification and characterization of lysozyme produced by Streptomyces erythraeus.

A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18,500 as determined by ultracentrifugation. Its isoelectric point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetyl-muramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcbeta(1 leads to 4) MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.     

日本对虾c型溶菌酶的高效重组表达及产物分析

从日本对虾(Marsupenaeus japonicus)血液中提取总RNA,根据GenBank已登录的该cDNA序列(AB080238),通过RT-PCR技术扩增出日本对虾溶菌酶(MjLys)成熟肽基因。该基因完整的开放阅读框为477 bp,编码158个氨基酸(aa),前18 aa为信号肽,成熟肽由140 aa组成,分子量为16.4 kD,理论等电点(pI)为8.80。经分析表明,该基因含有一个完整的c型溶菌酶结构域(1-130 aa),包括c型溶菌酶特有的两个活性中心Glu33和Asp50,以及8个保守结构Cys残基。将MjLys成熟肽基因亚克隆至原核表达载体pET-32a(+),在大肠杆菌细胞BL21(DE3)pLysS中诱导发酵,实现了重组MjLys蛋白的高效表达,并测定了该重组蛋白对几种细菌的抑菌活性。结果表明,重组日本对虾溶菌酶对革兰氏阳性菌金黄色葡萄球菌和溶壁微球菌均有显著的溶菌活性。     

Affinity chromatographic purification of human lysozyme, with special reference to human leukemia lysozyme.

Lysozyme [EC 3.2.1.17] was purified from human tears, serum, and urine of acute monocytic leukemia patients, renal disease patients, and residents in cadmium-polluted areas of Tsushima Island using an affinity adsorbent containing lysozyme-lysate of Micrococcus lysodeikticus cell walls as the ligand. By means of this procedure, leukemia lysozyme was purified 100- to 200-fold with an activity recovery of 80%. It was crystallized at pH 10. This purified preparation appeared homogeneous in disc electrophoresis and showed a specific activity 2.5-fold higher than that of crystalline lysozyme from hen egg-white. Tear lysozyme was also purified to a nearly homogeneous state while the enzymes from normal serum and urine of a nephrosis patient and of residents in cadmium-polluted area were still disc electrophoretically heterogeneous and showed low specific activity as compared with purified leukemia lysozyme.     

Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.     

Purification of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose.

Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.     

Preparation of bioactive and surface functional oligomannosyl neoglycoprotein using extracellular pH-sensitive glycosylation of mutant lysozyme having N-linked signal sequence in yeast

Bioactive oligomannosyl lysozyme with improved surface functionalities was successfully prepared by using an extracellular pH-sensitive glycosylation system for heterogeneous protein in yeast cell. A recombinant Saccharomyces cerevisiae carrying a mutant lysozyme gene encoding the signal sequence of an N-linked glycosylation site at position 49 was cultivated in various pH conditions to investigate the effects of extracellular pH on the glycosylation patterns and the expression of the protein. A large polymannose (Man(310)GlcNAc(2)) chain-linked lysozyme was predominantly expressed accompanied by small amounts of a core-type oligomannose chain (Man(14)GlcNAc(2))-linked lysozyme in the yeast medium where the extracellular pH was kept at 3.5 or above, while an oligomannose chain lysozyme was preferentially expressed in the yeast medium where the pH was less than 3. The lytic activities of the oligomannosyl and the polymannosyl lysozymes were found to be 70.4 and 5.1%, respectively, of the wild-type lysozyme when Micrococcus lysodeikticus cells were used as the substrate. The enzymatic activity of the oligomannosyl lysozyme was totally conserved for the glycolysis assay with a soluble substrate, glycol chitin, whereas that of the polymannosyl lysozyme was not. After heating the sample up to 95 degrees C at pH 7.0 where no visible protein coagulation was observed, thermostability of the enzymatic activity of the oligomannosyl lysozyme was drastically improved with more than 60% of residual lytic activity. Emulsifying properties of the protein also were highly improved by the oligomannosylation, in which the emulsifying activity was 3.2 times higher than that of the wild-type protein. Corresponding to the increase of the surface functionalities, the surface tension of the oligomannosyl protein exhibited a significantly (p < 0.05) lower value compared to that of the wild-type. By using the lower pH medium at 3.0, it was revealed that a substantial amount (0.31 mg/L) of the oligomannosyl lysozyme was successfully obtained in the culture medium. Therefore, the extracellular pH-sensitive glycosylation system can be used to obtain bioactive and surface functional neoglycoproteins.     

Purification of a lysozyme from skin secretions of Bufo andrewsi

A novel toad lysozyme (named BA-lysozyme) was purified from skin secretions of Bufo andrewsi by a three-step chromatography procedure. BA-lysozyme is a single chain protein and the apparent molecular weight is about 15 kDa as judged by SDS-PAGE. The specific lytic activity against Micrococcus lysodeikticus of BA-lysozyme is 2.7 x 10(5) units/mg, indicating that it is a potent lysozyme. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli with minimal inhibitory concentrations (MIC) of 1 and 8 microM, respectively. The deduced primary structure of BA-lysozyme from cloned cDNA was confirmed by N-terminal sequencing and peptide mass fingerprinting. Its amino acid sequence shares 56.5% identity with that of chicken egg-white lysozyme. Phylogenetic analysis indicates that B. andrewsi lysozyme is closely related to that of turtle. This is the first report on the isolation and primary structure determination of amphibian lysozyme.     

Substrate specificity and antifungal activity of recombinant tobacco class I chitinases

Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.     

凡纳滨对虾溶菌酶基因在毕赤酵母中的分泌表达和活性检测

原核重组表达的凡纳滨对虾(Litopenaeus vannamei)溶菌酶蛋白主要以包涵体形式存在, 经变性和复性处理后活性仍较差。研究将凡纳滨对虾溶菌酶基因(Lvlyz基因)克隆至毕赤酵母分泌型表达载体pPIC9K中, 电击转化毕赤酵母GS115细胞, 经组氨酸营养缺陷培养基筛选和PCR检测获得转化子。对其进行连续甲醇诱导表达, 利用SDS-PAGE和C端携带的6×His标签,对发酵液上清进行Western blot检测, 结果表明19.3 kD左右的条带即是重组表达的溶菌酶蛋白。用溶壁微球菌平板抑菌法鉴定表达产物具有较强的抑菌能力。研究首次利用毕赤酵母真核表达系统实现对虾溶菌酶基因的可溶性表达, 并且表达产物的活性良好。       

七鳃鳗g型溶菌酶的分子特征和抗菌活性

溶菌酶是先天免疫系统中对抗细菌病原体感染的一种关键蛋白．本研究从七鳃鳗中克隆g型溶菌酶基因. 其酶基因cDNA为701 bp（GenBank 序列号KP204854）,开放阅读框为555 bp,编码由184个氨基酸组成的多肽,理论分子质量为20.24 kD,等电点为5.48,含有1个半胱氨酸残基,无信号肽．实时荧光定量PCR分析表明,七鳃鳗g型溶菌酶基因在各组织中广泛表达,其中在肠中表达量最高．脂多糖（LPS）体内刺激七鳃鳗后发现,溶菌酶在口腔腺和头肾表达量显著升高．以溶壁微球菌和哈维弧菌为底物检测重组g型溶菌酶的活性时,均表现出抗菌活性,最适pH为7.5,最适温度为35℃．扫描电镜分析表明,重组酶能够使溶壁微球菌破裂．以上结果均表明,g型溶菌酶在七鳃鳗的先天免疫系统防御病菌感染中起到重要作用．     

菜心溶菌酶的提纯及酶学性质

菜心（ＢｒａｓｓｉｃａｃａｍｐｅｓｔｒｉｓＬ．ｓｓｐ．ｃｈｉｎｅｎｓｉｓｖａｒ．ｕｔｉｌｉｓ）叶子高速捣碎后,滤液经酸碱处理,硫酸铵分步沉淀,凝胶柱层析等步骤分离纯化溶菌酶,酶比活力达３４１４．６Ｕ／ｍｇ,纯化倍数为１９７．４。菜心溶菌酶在较宽的温度或ｐＨ值范围均有活性,最适温度为６０℃,最适ｐＨ值为５．８,底物Ｋｍ值为８７μｇ／ｍＬ。该酶对热和酸碱的稳定性较高,巯基和酪氨酸残基不是该酶活性中心的必需基团。     

Characteristics of a Lytic Enzyme Induced by Bacteriophage Infection of Micrococcus lysodeikticus

A lytic enzyme induced in Micrococcus lysodeikticus strain 1 by infection with N1 bacteriophage was purified 45- to 50-fold by ammonium sulfate precipitation, acid precipitation, and selective adsorption of contaminating proteins with calcium phosphate gel. The optimal pH for activity of the enzyme was 6.5 to 7.0. Maximal activity occurred at 45 to 50 C and at an ionic strength of 0.06. The enzyme had a limited specificity and lysed cell walls of M. lysodeikticus with the release of dinitrofluorobenzene reactive groups. Living cells were lysed in the absence of phage; however, the rate of lysis increased when phage was present in excess of 10 particles per bacterial cell. Young cells were most sensitive, and the sensitivity decreased to a minimum with stationary-phase cells. Acting synergistically, lysozyme and the N1-induced lysin caused lysis of cells which were resistant to either enzyme acting independently. The N1 lysin did not exhibit proteolytic activity.     

Differential Patterns of Bacteriolytic Activities in Potato in Comparison to Bacteriophage T4 and Hen Egg White Lysozymes

A comparison of the enzymatic activities of endogenous potato bacteriolytic enzymes with bacteriophage T4 and hen egg white lysozyme has been performed. Using Erwinia carotovora atroseptica and Pseudomonas solanacearum as substrates in, comparison to Micrococcus lysodeikticus a differential pattern of bacteriolytic activities could be detected. The expression pattern of endogenous potato lysozymes suggests that their functional activity against phytopathogenic bacteria in planta is unlikely. Antibacterial activities in transformed, T4 lysozyme expressing and non–transformed potato plants are evaluated.     

Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101.

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.     

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 degrees C and cold stress at 5 degrees C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in the muscle sinus where injection took place. Injection of V. anguillarum or M. lysodeikticus induced significant up-regulation of lysozyme gene expression, but only 2-3days post-injection, with no change in the total hemocyte counts but an increased percentage of hemocytes expressing lysozyme mRNA. Neither the control injection of PBS-NaCl nor temperature stress modified the lysozyme expression pattern. Consequently, the hemocyte population appears to be capable of discriminating between stress factors, and even between 2 Vibrio species.     

人LYC5溶菌酶基因在毕赤酵母中的重组表达

LYC5是一种c型人溶菌酶蛋白。根据毕赤酵母密码子的偏爱性,对LYC5的mRNA编码序列进行优化设计,将优化后的基因序列克隆至毕赤酵母分泌型表达载体pPIC9K中,构建重组酵母表达质粒pPIC9K- LYC5 。重组质粒经线性化处理后转化毕赤酵母GS115,应用G418抗性筛选出高拷贝转化子,并对其进行摇瓶诱导表达,产物经SDS-PAGE电泳检测,发现在约15 kDa的位置出现了一条特异蛋白条带,此条带经LTQ Orbitra pelite MS鉴定,证明此蛋白即LYC5溶菌酶蛋白,表达量约为20 mg/L。对表达上清液进行活性分析,发现表达上清对溶壁微球菌具有较好的溶菌活性,活性约为40 000 U/mg,最适酶活反应温度为45℃,最适pH为5.0。采用基因工程方法,首次表达出了有生物学活性的人源LYC5溶菌酶蛋白,为深入探讨人溶菌酶家族成员的抗菌谱及其应用前景的研究奠定了基础。