源自大黄鱼肠道柠檬酸杆菌的外切几丁质酶的特性分析（英文）

【目的】从饲喂富含几丁质饲料的大黄鱼肠道分离具有几丁质分解功能的菌株并分离鉴定新的几丁质酶。【方法】利用胶体几丁质平板分离饲喂杂鱼的大黄鱼肠道中的几丁质分解菌。对几丁质酶基因chi-X进行了克隆并在大肠杆菌中表达。对CHI-X的酶学性质进行了分析。【结果】从饲喂杂鱼的大黄鱼肠道内容物中分离出1株具有几丁质分解功能的费氏柠檬酸杆菌,其中的几丁质酶基因编码1个含493个氨基酸残基的蛋白,其中包含一个糖苷水解酶18家族催化域。CHI-X对胶体几丁质具有分解功能。最适p H和温度分别是4.0和60°C。CHI-X具有很强的pH稳定性,在pH 3.0–11.0的范围培育1 h仍保留90%左右的活性。Mn~(2+),Li~+和K~+可促进CHI-X酶活,Ag~+对CHI-X有抑制作用。CHI-X对蛋白酶和石斑鱼肠道内容物有较强的抗逆性。CHI-X可分解胶体几丁质为N-乙酰葡萄糖胺和N-乙酰葡萄糖胺二聚体,表明它是一个几丁质外切酶。最后,CHI-X和另一个几丁质酶Chi565表现出酶活性的加和效应。【结论】分离自肠道菌的CHI-X能很好适应海水鱼类的肠道环境,可以作为温水海水养殖鱼类的饲料添加剂使用。     

苏云金芽孢杆菌几丁质酶的研究进展

苏云金芽孢杆菌制剂作为无公害农药已经得到社会的认可,如果再开发其几丁质酶抑制真菌和杀虫增效功能,不仅可充分利用这一农业微生物菌种资源,也将给予传统生物农药以新的生命力。综述了苏云金芽孢杆菌几丁质酶方面研究的国内外最新进展。     

芽胞杆菌菌株产几丁质酶发酵条件的研究

从采自大连地区24份土样中分离筛选到一株产几丁质酶活性较高的芽胞杆菌(Bacillus sp.)B-41,该菌株产几丁质酶最适的发酵条件为：碳源为胶体几丁质,氮源为酵母浸汁,pH7．2,温度42℃,振荡培养4天。     

产几丁质酶的苏云金杆菌菌株筛选及酶合成条件研究

从本室保存的 64株苏云金芽孢杆菌中 ,筛选出一株几丁质酶活力较高的菌株WB 50。产酶条件研究表明 :在pH 7.0的基础培养基中添加 2 .0 %的细粉几丁质 ,1.0 %的酵母膏 ,2 2 0rpm 30℃下培养 72小时 ,几丁质酶的产出最大。     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

苏云金杆菌entomocidus亚种几丁质酶基因的克隆与生物信息学分析

从苏云金杆菌(Bacillus thuringiensis,B t)entom oc idus亚种HD109菌株中克隆了几丁质酶基因chiA74-HD109,其序列全长为2 031 bp,编码676个氨基酸,GenBank登录号为AY455290。其编码产物与蜡状芽孢杆菌几丁质酶CHiB(AB041932)的相似性达97.9%,与B t kenyae亚种LB IT-82菌株几丁质酶chiA74(AF424979)的相似性达98.4%,与B t pak istan i亚种几丁质酶(U89796)的相似性为83.0%,与B t kurstak i亚种几丁质酶kchi(AY189740)的相似性达97.8%,与B t israe lensis亚种几丁质酶(AF526379)的相似性达96.6%,与B t几丁质酶(AY074882)的相似性达98.4%,与B t sotto亚种几丁质酶(AY129671)的相似性为97.3%。功能结构域分析显示其编码区包含信号肽、催化区、Ⅲ型粘蛋白同源区及几丁质结合区4个部分。     

苏云金杆菌几丁质酶新基因的筛选和全长基因的扩增

以煮沸冻融法制备PCR扩增模板,利用苏云金芽孢杆菌（Bacillus thuringiensis,Bt）几丁质酶基因特异引物进行15个Bt血清变种的扩增分析,获得9个几丁质酶全长基因扩增产物。经克隆和序列测定,从Bt serovar.entomocidus HD109、Bt serovar canadensis HD224、Bt serovar、alesti HD16和Bt serovar.toumanoffi HD201等4个菌株中分离了几丁质酶新基因。     

苏云金杆菌以色列亚种几丁质酶基因的克隆及序列分析

从苏云金芽孢杆菌以色列亚种（Bacillus thuringiensis subsp israelensis)中提取基因组DNA,通过合成1对特异性引物,用touchdown PCR的方法扩增几丁质酶ichi基因序列（GenBank登录号：AF526379）。ichi序列全长为2570bp,含有1个2067bp的开放阅读框（ORF）,编码688个氨基酸,推测分子量为75.79kDa,等电点pI=5.90的几丁质酶前体。序列和结构比较分析表明：Ichi氨基酸序列与蜡状芽孢杆菌（Bacillus cereus)28-9几丁质酶CW、蜡状芽孢杆菌CH几丁质酶B及苏云金芽孢杆菌墨西哥亚种几丁质酶的同源性分别为97.24%、97.18%、97.63%,而与苏云金芽孢杆菌巴基斯坦亚种的同源性只有63.07%。Ichi编码区由分泌信号肽（46AA）、催化区（105AA)、粘蛋白Ⅲ型同源区（74AA）及几丁质结合区（40AA）组成。     

豇豆几丁质酶部分酶学特性的研究

该文测定了纯化的豇豆几丁质酶部分酶学特性。结果表明,该酶在pH5-8,温度低于60℃的范围内稳定性较好,酶活力最适pH为65,最适温度为50℃。10mmol/L浓度的Hg2 、Mn2 、Mg2 、Co2 等金属离子对酶活力有一定抑制作用,其中Hg2 离子抑制率最高(6883%)。Km(胶状几丁质)值为1662mg/ml;以SDS-PAGE电泳和SephadexG-100柱层析两种方法分别测得分子量为34kD、325kD;IEF电泳测得等电点为83。     

黄杆菌(Flavobacterium sp.)几丁质酶的纯化和性质

黄杆菌(Flavobacteium sp.)在几丁质的诱导下产生几丁质酶.通过(NH_4)_2SO_4沉淀、DEAE纤维素柱层析、Sephacryl 300柱层析及Sephadex G-75柱层析,从Flavobacterium sp.培养上清液中分离纯化了几丁质酶.SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯度分析表明,纯化后的几丁质酶达到了均一的程度.用SDS-PAGE测得该酶的分子量约45D00道尔顿.该酶水解几丁质的最适pH为 7.0,最适温度为50℃,-20C贮存两年以上仍有活性.水解几丁质的Km值为5.0mg/ml.金属离子对几丁质酶活性影响较大,Ca~(2+) 、Co~(2+)’和Cu~(2+)对酶有激活作用.而NH_4~-、Ba~(2+)、Mg~(2+)、Mn~(2+)对酶有抑制作用.几丁质酶水解几丁质的产物是几丁质二糖.     

海水养殖生境中柠檬酸杆菌降解半胱氨酸产生硫化物的机制研究

【目的】含硫氨基酸的生物降解是造成海水养殖环境中毒性硫化物上升的重要因素,而微生物降解含硫氨基酸的机制和影响因素解析是控制该系统中硫化物浓度的关键环节。【方法】本研究利用稀释涂布-叠皿夹法自本实验室海水养殖环境的沉积物中分离得到一株产生硫化物的厌氧菌株,并通过代谢组学研究其以半胱氨酸为底物产生硫化物的机制和途径。【结果】经鉴定,该菌株为弗氏柠檬酸杆菌(Citrobacterfreundii),能够在厌氧条件下还原硫酸盐,能够降解半胱氨酸产生硫化物,投加L-半胱氨酸可提高其还原硫酸盐的能力。该菌株以1g/L半胱氨酸为底物,在35℃、盐度为10、pH 8.0的条件下,硫化物的最高积累量可达302.4 mg/L。对菌株中硫化物产生具有重要贡献的半胱氨酸脱巯基酶研究表明,该酶最适温度为35℃,在pH6.0–8.0有较高的活性,能够快速降解半胱氨酸产生硫化物。结合代谢组学研究发现,该菌株中含有的3-巯基丙酮酸硫酸转移酶、胱硫醚-γ-裂解酶、半胱氨酸脱巯基酶催化半胱氨酸降解是产生硫化氢的主要途径;亚硫酸盐还原酶还原硫酸盐、亚硫酸盐是其产生硫化氢的次要途径。【结论】通过揭示柠檬酸杆菌降解半胱氨酸产生...     

弗氏柠檬酸杆菌及居泉沙雷菌引起中华穿山甲死亡的案例分析

中华穿山甲（Manis pentadactyla）是目前生活在我国长江以南地区的极危物种,野生穿山甲进入人工圈养环境后可能受到多种胁迫因素影响,容易出现免疫或代谢相关的多种疾病。本报告以1例死亡中华穿山甲为研究对象,对其死亡原因进行分析。经尸体剖检、组织器官微生物检测及组织切片病理分析,最终确定该穿山甲死亡原因为感染弗氏柠檬酸杆菌（Citrobacter freundii）和居泉沙雷菌（Serratia fonticola）导致的消化器官炎症反应,进而出现严重腹泻后死亡。药敏试验结果显示,弗氏柠檬酸杆菌对氟喹诺酮类和氨基糖苷类抗生素较为敏感,对青霉素类、四环素类、大环内酯类及头孢唑啉表现出耐药性;居泉沙雷菌对氨基糖苷类抗生素较为敏感,对氟喹诺酮类中度敏感,对青霉素类、四环素类、大环内酯类及头孢唑啉表现出耐药性。本研究的开展不仅为中华穿山甲的死因提供了更为科学准确的诊断依据,还为应对相关病菌感染制定了有效的治疗方案,以期对今后中华穿山甲救助工作提供参考和指导依据。     

一株人源弗氏柠檬酸杆菌的生物学特性及全基因组测序分析

【背景】 弗氏柠檬酸杆菌(Citrobacter freundii)是一种人兽共患条件致病菌,多重耐药菌株的出现给全球公共健康带来严重威胁。2024年9月,扬州大学附属医院从人手掌软组织感染脓液中分离了一株细菌。【目的】 对分离的细菌进行种类鉴定、生物学特性研究和全基因组测序分析。【方法】 通过革兰氏染色、16S rRNA基因测序和全基因组序列分析鉴定该菌种类。通过生长曲线、药物敏感性和致病性测定探究该菌生物学特性。利用二代Illumina PE150测序和三代PacBio测序获得该菌全基因组序列,并借助多种生物信息学工具进行序列分析。通过实时荧光定量PCR (RT-qPCR)检测铁摄取相关毒力因子在铁限制条件下的表达情况。【结果】 经鉴定,该菌为弗氏柠檬酸杆菌。生长曲线测定结果显示,该菌在第1 h左右进入指数生长期,在第7 h OD₅₉₅值最高。药物敏感性测定结果显示,该菌对头孢拉定、阿米卡星、庆大霉素等8种药物耐药,对羧苄西林、头孢唑林、头孢曲松等11种药物敏感。致病性测定结果显示,该菌在斑马鱼模型中表现出较高致病性。全基因组测序分析结果显示,该菌全基因组大小为4 938 581 bp,G+C含量为51.77%,预测编码4 576个基因,其中耐药基因有24个,毒力因子有44个。该菌基因组中预测有27个移动遗传元件、13种RNA调控元件和27个特有基因。RT-qPCR结果显示,该菌的铁摄取相关毒力因子在铁限制条件下显著上调表达。【结论】 分离自人手掌软组织感染脓液的细菌为弗氏柠檬酸杆菌,该菌表现出较强耐药性和较高致病性,其基因组中携带多个耐药基因和毒力因子,临床上需引起重视。     

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.     

Purification and characterization of chitinase from Alcaligenes xylosoxydans

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecularmass of chitinase was estimated to be 45 kDa and44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 °C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l^–1. Cu²⁺ and Na⁺ at 5 mM inhibited chitinase activity to 25% while Ca²⁺, Mg²⁺ and Ba²⁺ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.     

Molecular Cloning of the Phytase Gene from Citrobacter braakii and its Expression in Saccharomyces cerevisiae

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppA_Ec and rAppA_Sc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppA_Sc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppA_Ec.     

Microsatellite–Centromere Mapping in Large Yellow Croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) Using Gynogenetic Diploid Families

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. Inheritance of 22 heterozygous microsatellite loci was examined in normal crossed diploid families and meio-gynogenetic families in P. crocea. Two gynogenetic families were produced via inhibition of the second polar body in eggs fertilized with UV-irradiated sperm. The ratio of gynogenesis was proven to be 100% and 96.9% in the two families, respectively. Of the 22 examined loci, 4 showed a segregation distortion in both control and gynogenetic families. Microsatellite–centromere (M–C) map distances were examined using 18 loci with normal Mendelian segregation. Estimated recombination rates ranged between 0 and 1.0 under the assumption of complete interference. High recombinant frequencies between heterozygous markers and the centromere were found in large yellow croaker, as in other teleosts. The average recombination frequency was 0.586. Ten loci showed high M–C recombination with frequency greater than 0.67. M–C distances provide useful information for gene mapping in large yellow croaker.