猪乳铁蛋白在4种重组乳杆菌中的表达及抑菌活性比较

为了获得优化的猪乳铁蛋白乳杆菌表达系统,并比较重组猪乳铁蛋白的抑菌活性,根据乳杆菌使用密码子的偏嗜性优化合成猪乳铁蛋白成熟肽编码序列,将其克隆到乳杆菌表达载体pPG612.1的XhoⅠ/BamHⅠ位点,获得了plf乳杆菌表达载体质粒pPG612.1-plf。将获得的重组质粒分别电转化入干酪乳杆菌ATCC393、戊糖乳杆菌KLDS1.0413、植物乳杆菌KLDS1.0344和副干酪乳杆菌KLDS1.0652细胞内,获得4种表达猪乳铁蛋白的重组乳杆菌。经木糖诱导,通过Western blotting和激光共聚焦检测重组猪乳铁蛋白的表达,用ELISA方法检测和比较4种重组菌上清中表达猪乳铁蛋白的量,并用琼脂孔穴扩散抑菌法检测4种重组乳杆菌表达乳铁蛋白的抑菌活性。结果表明,乳铁蛋白在4种重组乳杆菌中均得到正确表达,其产物分子量约73 kDa,重组干酪乳杆菌、重组戊糖乳杆菌、重组植物乳杆菌和重组副干酪乳杆菌的重组猪乳铁蛋白表达量分别为9.6μg/mL、10.8μg/mL、12.5μg/mL、9.9μg/mL。重组猪乳铁蛋白对大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、巴氏杆菌和李氏杆菌均有一定的抑菌作用,对金黄色葡萄球菌的抑菌作用最强,且4种重组乳杆菌中重组植物乳杆菌表达产物的抑菌效果优于其他重组菌的表达产物。结果表明在4种乳杆菌中重组猪乳铁蛋白的最佳表达系统为植物乳杆菌,该结果为猪乳铁蛋白的乳杆菌表达系统进一步开发与应用奠定了基础。     

细菌重组乳杆菌疫苗的研制现状

细菌是人类感染的常见病原体,研制疫苗防治细菌感染是当前的热点研究领域之一。乳杆菌是一种新型的疫苗载体,我们简要综述了乳杆菌介导的破伤风梭菌、肺炎链球菌、幽门螺杆菌、铜绿假单胞菌、霍乱弧菌和炭疽芽孢杆菌等几种细菌疫苗的构建及其作用机制等方面的研制现状。     

短乳杆菌与植物乳杆菌的发酵特性

【背景】目前对于酸菜发酵的研究主要关注点是植物乳杆菌(Lactobacillus plantarum),有关短乳杆菌(Lactobacillus brevis)在酸菜方面的研究报道很少。【目的】为了挖掘短乳杆菌的发酵性能并开发酸菜发酵剂,将2株短乳杆菌分别与1株植物乳杆菌进行组合并发酵酸菜,分析短乳杆菌对酸菜发酵品质的影响。【方法】分别测定短乳杆菌与植物乳杆菌的单菌株生长产酸性能、耐酸性及亚硝酸盐降解力,并将两菌种组合后发酵酸菜,分析1-7d内酸度、乳酸菌活菌数、亚硝酸盐含量及酸菜质构特性的变化趋势。【结果】相较于短乳杆菌Lb-9-2,短乳杆菌Lb-5-3的生长和产酸速率较慢、酸耐受力较弱,但其亚硝酸盐降解力较强。两株短乳杆菌分别与植物乳杆菌Lp-9-1组合后产酸力显著增强,并在3 d时达到最低pH值(约3.10);植物乳杆菌Lp-9-1的添加使酸菜中总体乳酸菌生长延迟,在5 d时达到最高活菌数;组合菌种的样品中亚硝酸盐含量在1-7 d内变化较为平缓,前5天内两个组合之间差异不显著;接种乳酸菌会降低酸菜硬度和弹性,发酵3d时Lb-5-3/Lp-9-1组合的硬度最大,感官评价得分最高。【...     

四环素抗性重组鼠李糖乳杆菌的构建

将含有鼠李糖乳杆菌同源序列的自杀质粒pUC-ldhD-Ter转化到鼠李糖乳杆菌JCM 1553中,成功获得2株含四环素抗性的重组鼠李糖乳杆菌GL-1和GL-2。采用PCR技术鉴定重组菌株染色体基因组上含有四环素抗性基因Ter;生长曲线说明四环素抗性基因的插入对鼠李糖乳杆菌的生长没有较大影响。抗性菌株GL-1和GL-2在含10%葡萄糖的摇瓶发酵培养基发酵结果显示:37℃,200 r/min发酵40h,菌体密度OD₆₀₀最大达到25.49和24.66,残糖含量为0.60%和0.63%,L-乳酸最高产量为93.956 g/L、93.693 g/L,葡萄糖转化率达到94.82%、94.56%,与原始菌株没有显著区别。     

乳杆菌电转化条件的研究

用Pgk12、Pmg36c等质粒电转化不同的乳杆菌。研究了影响转化效率的多种因素。受体细胞经20μg/ml氨苄青霉素处理1h,可以提高转化效率200倍。同时,发现电击后的细胞必需在高渗培养基中才能存活,电击后2～3h的复苏表达期和用亚抑制抗生素浓度选择转化子,这些对电转化成功以及提高转化效率都是十分关键的。在改进电转化方法中,各种参数为电场强度8.75kV/cm,电阻100Ω或200Ω,电容25μF和2×磷酸缓冲液。     

乳杆菌和双歧杆菌与人类健康的关系

乳杆菌和双歧杆菌与人类健康的关系原解放军３０９医院北京１０００４５何道生１前言为了健康的目的人类利用乳杆菌的历史可以追朔到１９０８年。细菌学的创始人梅奇尼可夫提出,“为了延长人类的生命,应该饮用乳杆菌发酵过的牛奶。”他说,肠道微生物能产生“毒素”,使...     

新型乳杆菌素产生菌的筛选及菌株特性的研究

从酸菜汁中分离出 2 4株乳酸杆菌 ,采用牛津杯定量扩散法筛选出 1株抑菌活性较高的乳酸杆菌 ,经鉴定为戊糖乳杆菌。排除乳酸对指示菌作用的干扰 ,该菌株的离心发酵液不仅对革兰氏阳性细菌有抑制作用 ,而且对大肠杆菌 (E .coli.) ,沙门氏菌 (Salmonellatyphi)等革兰氏阴性细菌也有一定的抑制作用     

干酪乳杆菌12A碳源代谢的比较基因组学分析

【目的】在基因组学水平上,对干酪乳杆菌的碳源代谢特性及调控机制进行研究。【方法】基于BioCyc和MetaCyc数据库,利用Pathway Tools对菌株12A及7株已公布全基因序列的干酪乳杆菌进行全基因组水平的碳源代谢比较分析。【结果】全基因组比较分析结果显示,干酪乳杆菌12A可以将9种糖转运到细胞内代谢利用;可以将多种寡糖和多糖在细胞外水解成半乳糖和葡萄糖;干酪乳杆菌12A可经异型乳酸发酵或混合酸发酵途径生成乙醇及其副产物。【结论】干酪乳杆菌12A可以代谢多种类型碳源,底物选择范围宽泛,而且可以作为工业乙醇发酵的特定菌株;利用比较基因组学方法建立基因结构与细菌代谢能力的联系是可靠的。     

双歧杆菌与乳杆菌原生质体的融合及筛选

目的：通过双歧杆菌与乳杆菌的原生质体融合构建耐氧双岐杆菌,以解决双岐杆菌制剂开发中始终存在的“活菌数低”和存活时间短等问题。方法：采用不同浓度的Mutanolysin分别制备长岐杆菌和保加利亚乳杆菌的原生质体,在电场作用下诱导长双岐杆菌原生质体和经56℃热灭活30min的保加利亚乳杆菌原生质体融合,并在双层再生培养基上筛选融合菌株。结果：成功地构建出几种较为稳定的兼具长双岐杆菌和保加利亚乳杆菌生物学特性的融合菌株;其中融合株F2具有良好的耐氧性;且能在有氧、CO2条件下良好生长。结论：通过原生质体融合技术能改良双歧杆菌的严格厌氧特性,有利于对双岐杆菌的开发和应用。     

运动发酵单胞菌积累聚羟基丁酸提高乙醇产量

运动发酵单胞菌是一种很有潜力的酒精生产菌。PHB是生物合成的一种聚酯,有研究表明,该类物质在微生物体内的积累能够提高宿主菌的抗逆能力。本文对运动发酵单胞菌进行了如下改造：将PHB合成操纵子phbCAB与来源于运动发酵单胞菌的丙酮酸脱羧酶的启动子准确融合,插入广泛宿主载体pBBR1MCS-1中,并利用电转化的方法转入运动发酵单胞菌中。在重组菌中检测到了PhaA和PhaB的酶活;并首次在运动发酵单胞菌中实现了PHB的积累。摇瓶实验表明,前48小时重组菌的乙醇积累量提高了约10%,后续发酵中可能由于葡萄糖耗尽,重组菌与野生菌乙醇积累量差别不大。     

Production of Ethanol from Starch by Respiration-Deficient Recombinant Saccharomyces cerevisiae

A 100%-respiration-deficient nuclear petite amylolytic Saccharomyces cerevisiae NPB-G strain was generated, and its employment for direct fermentation of starch into ethanol was investigated. In a comparison of ethanol fermentation performances with the parental respiration-sufficient WTPB-G strain, the NPB-G strain showed an increase of ca. 48% in both ethanol yield and ethanol productivity.     

两种乳杆菌联用在食蟹猴阴道的定植效率及其毒性分析

研究乳杆菌鼠李糖乳杆菌-格氏乳杆菌联用制剂(LACT)的定植效率, 评估其有效剂量在动物体内的急性和长期毒性。

在食蟹猴中进行2轮阴道定植试验, 通过检测阴道分泌物pH值和清洁度, 并分离鉴定分泌物中的乳杆菌, 确定乳杆菌联用制剂的定植率和定植剂量; 通过单次和多次阴道给药, 检测联用制剂对食蟹猴体质量、食量、生殖器官及生化指标等的影响, 检测联用制剂在体内的急性和长期毒性。

联用制剂在食蟹猴中连续阴道给药5 d后能成功定植, 改善阴道的微生态; 甲硝唑预处理能够提高定植效率和定植时间; 联用制剂对食蟹猴不具有急性和长期的毒性作用。

在动物模型中, 2种乳杆菌联用制剂对细菌性阴道病具有良好的治疗潜质。

     

酿酒酵母L610利用菊糖生产乙醇发酵条件的研究

以1株能够直接利用菊糖产乙醇的酿酒酵母L610为出发菌株,对其利用菊糖生产乙醇的发酵条件进行了一系列研究。结果表明,L610最适乙醇发酵温度为37℃,且40℃高温发酵对其产乙醇能力无显著影响;L610对酸性发酵环境有良好的耐受性,当发酵液p H值降至3.5时,其糖醇转化率及乙醇产量仍保持较高水平;以0.025~0.10 vvm的通气量通气12 h有利于L610发酵菊糖产乙醇;L610对350 g/L的高浓度菊糖有良好的转化率,乙醇浓度和生产强度分别达到129 g/L和1.35 g/(L·h);当直接以300 g/L菊芋粗粉为唯一底物进行发酵时,L610发酵产乙醇浓度达到89.6 g/L,为理论产量的78.1%。本研究所取得的成果为酿酒酵母一步法发酵菊芋生产乙醇的工业化发展提供参考。     

控制发酵过程氧化还原电位优化酿酒酵母乙醇生产

利用氧化还原电极,研究了在厌氧条件下将氧化还原电位值(ORP)控制在不同水平(-50mV、-100mV、-150mV、-230mV)对乙醇发酵过程的影响。试验结果表明,不同的ORP值水平对乙醇得率,甘油形成、有机酸分泌、生物量和菌体死亡率的影响有明显的差异。当ORP为-50mV时的生物量是ORP为-100mV时的1.26倍、ORP为-150mV时的1.86倍、ORP为-230mV时的2.59倍,甘油浓度分别是后三者的1.2倍、1.1倍、1.7倍,而乙醇浓度却分别只有后三者的0.87倍、0.49倍、0.51倍。综合考虑生物量、乙醇浓度、甘油产量、残糖的测定结果,表明将ORP控制在-150mV时对乙醇发酵极为有利。说明可以用ORP电极来精确控制厌氧发酵条件,从而为酵母细胞合理分配代谢流以实现乙醇生产最优化的宏观控制提供了一种有效的手段。     

利用根霉和酿酒酵母转化生玉米粉为高浓度酒精

利用从自然界中新分离得到的生淀粉酶产生菌-根霉W-08,并首次采用固态和液态相结合的培养方法,使生淀粉糖化酶活量达到72IU/mL,然后以生玉米粉为底物,利用根酶W-09和酿酒酵母Z-06采用同步糖化与发酵工艺,30℃,48h,发酵醪酒精浓度达到21%(V/V),转化率为理论转化率的94.5%。     

Production and Purification of Recombinant Hirudin Expressed in the Methylotrophic YeastPichia pastoris

A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leechHirudo medicinalis,was cloned and expressed in the methylotrophic yeastPichia pastoris.The HIR gene was inserted into theP. pastorispPic9K expression vector such that the gene's expression is under alcohol oxidase (AOX1) promoter control and the HIR coding sequence is fused to theSaccharomyces cerevisiaepre-pro α-mating factor signal sequence. ATn903Kan^rdeterminant andHis4⁺gene are also present on pPic9K, affording a method for selecting chromosomal integrants of the HIR gene. Following electroporation of the DNA into theP. pastorisstrain GS115 (his-4), His⁺transformants were recovered and plated on medium containing increasing concentrations of the aminoglycoside antibiotic G418. The resulting His⁺G418-resistant transformants were grown in shake flasks and screened for those that secreted recombinant hirudin (rHIR) to the growth medium. Clones exhibiting rHIR production and secretion were retained for fermentation studies where optimization of growth conditions was found to dramatically increase rHIR expression. One clone that was retained for further characterization secreted rHIR at a level of 1.5 g/liter. Using a straightforward two-step chromatography procedure, the rHIR was purified to >97% with a recovery yield of 63%. The purified rHIR had the predicted N-terminal amino acid sequence and exhibited the same thrombin inhibition kinetics as a variety of HIR isoforms produced in other heterologous systems. Based on these data,P. pastorisoffers an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.     

Production of Recombinant Human Bile Salt Stimulated Lipase and Its Variant inPichia pastoris

hBSSL and its truncated variant hBSSL-C cDNA clones were expressed inPichia pastorisusing two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45–50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized inP. pastorisand was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.     

Ethanol Production from High-Solid SSCF of Alkaline-Pretreated Corncob Using Recombinant Zymomonas mobilis CP4

In this work, Zymomonas mobilis was genetically improved for pentose utilization to increase the final ethanol concentration. It showed good fermentation ability on both soluble sugar mixture and lignocellulose. Nearly all the glucose and xylose in sugar mixture can be consumed, corresponding to 86 % of theoretic ethanol yield. Simultaneous saccharification and co-fermentation (SSCF) of NaOH-pretreated corncob was then carried out in a high dry matter (DM) loading of 15–25?w/v%. At the DM loading of 15 %, the suitable operating conditions were determined, i.e., Z. mobilis loading of 0.30 g dry weight/L at 30 °C (pH?5.5), under which the ethanol concentration reached 49.2 g/L. Higher final ethanol concentrations were obtained when SSCF was operated at the fed-batch mode. Several amounts of substrate (1 % to 10 %) were added, and the highest final ethanol concentration (60.5 g/L) was obtained at 10 % DM addition.