瑞替普酶在乳酸乳球菌中的表达

为建立溶栓药物瑞替普酶(r PA)的乳酸菌表达系统,从实验室前期构建的大肠杆菌表达质粒p ET22b-rpa上扩增出目的基因rpa,将该基因与乳酸菌表达质粒pCYT连接,构建了pCYT-rpa质粒.此外,为提高r PA在乳酸乳球菌NZ9000中的稳定性,同时构建了rpa位于葡萄球菌(Staphylococcal)耐热核酸酶基因nuc下游融合表达的重组质粒pCYT-nuc-rpa.将质粒p CYT-rpa和p CYT-nuc-rpa分别电转化至乳酸乳球菌NZ9000中,经Nisin诱导表达,Western blot结果显示Nuc可提高r PA在乳酸乳球菌中的稳定性,从而增加了rPA在乳酸菌中的表达量.重组r PA及Nuc-rPA在复性后均具有溶栓活性,活性分别为800,U/L和1,000,U/L.     

Microencapsulated Engineered <Emphasis Type="Italic">Lactococcus lactis Cells</Emphasis> for Heterologous Protein Delivery: Preparation and In Vitro Analysis

This article demonstrates the potential of encapsulated, engineered Lactococcus lactis as a vehicle for the oral delivery of therapeutic proteins. Using alginate-poly-l-lysine-alginate membrane-encapsulated L. lactis engineered to secrete the reporter protein Staphylococcal aureus nuclease, we show comparable viability and protein secretion between free and immobilized cells. After 12 h, microcapsules with a cell density of 4.8 × 10⁵ colony forming unit (CFU) ml⁻¹ grew to 2.2 × 10⁸ CFU ml⁻¹ and released 0.24 arbitrary unit (AU) ml⁻¹ of nuclease, producing similar results as free cells, which grew from 3.4 × 10⁵ to 1.9 × 10⁸ CFU ml⁻¹ and secreted 0.21 AU ml⁻¹ of nuclease. Moreover, encapsulated cells at a density of 4.4 × 10⁷ CFU ml⁻¹ grew to 2.2 × 10¹⁰ CFU ml⁻¹ in 12 h and secreted 15.3 AU ml⁻¹ of nuclease although 3.1 × 10⁷ CFU ml⁻¹ of free cells reached only 2.3 × 10⁹ CFU ml⁻¹ and released 5.6 AU ml⁻¹ of nuclease. We also show the sustained stability of the microcapsules during storage at 4°C over 8 weeks.     

玉米酒糟为主要氮源的Nisin生产菌株的诱变选育

为使乳酸乳球菌适应玉米酒糟作为发酵生产乳酸链球菌素(Nisin)的主要氮源,首先对培养基进行了初步优化,发现在酒糟清液中加入蔗糖、酵母膏和磷酸氢二钾明显促进了乳酸乳球菌的生长.在此基础上,使用等离子体对乳酸乳球菌进行诱变处理,利用24方孔深孔板技术可实现对高产Nisin突变菌株的选择性筛选.结果表明,诱变菌株在只有5.0%存活率时,得到的正突变菌株可达26.2%.借助摇瓶发酵实验对其生产Nisin的发酵水平进行研究,发现有1株诱变选育菌株发酵Nisin的活性高达6 520 U/mL,并且其发酵能力比诱变起始菌株能力更强.     

酸浆中作用菌的研究Ⅰ．乳链球菌的来源分析

对酸浆法生产甘薯淀粉中的加工用水、加工场地空气、鲜薯的分析测试表明：酸浆中的乳链球菌主要来源于甘薯表面和空气，精洗甘薯表面乳链球菌为０．８个／ｃｍ２；甘薯加工地区空气中该菌含量是非加工区的５倍以上。     

响应面法优化乳酸乳球菌电转化效率研究

乳酸乳球菌NZ9000是乳酸菌食品级高效蛋白表达系统中使用最广泛的宿主菌株。为获得高效的电转化效率,首先采用单因素试验,对影响NZ9000电转化效率的各因素:生长阶段、甘氨酸浓度、质粒浓度、电场强度、复苏培养和复苏时间进行优化;然后利用响应面法对其中的关键因素进一步优化,确定最佳电转化条件为吸光值OD600 0.2,甘氨酸浓度8 g/L和电场强度12.5 kV/cm,电转化效率达到3.76×10~7 CFU/μg DNA,比优化前提高了250%。本研究通过优化乳酸乳球菌NZ9000感受态的制备方法,提高了电转化效率,为深入挖掘乳酸乳球菌感受态潜力奠定了基础。     

Identifying ligand-binding specificity of the oligopeptide receptor OppA from Bifidobacterium longum KACC91563 by structure-based molecular modeling

As a ATP-binding cassette (ABC) transporter the OppA receptor plays key roles in protecting the host organism and transporting nutrients across the intestine by the oligopeptide transporter from symbiotic bacteria and directs maturation of the host immune system. Among lactic acid bacteria, Bifidobacterium longum KACC91563, isolated from fecal samples of healthy Korean neonates, has the capability to alleviate food allergy effects. Operating as a peptide importer, the extracellular OppA receptor from gram-positive B. longum KACC91563 translocates nutrients, specifically peptides, from the outside environment of the intestinal tract to the inside of symbiotic cells. In the present study we attempt to explicate the relationship between the substrate’s specificity from the OppA importer and the probiotic effects of B. longum KACC91563 in the host intestine. It was first identified in this study the specialized structure–function relationship from the OppA importer of B. longum KACC91563 with its structural and functional determinants. This could provide insights into substrate specificity of unique immunological properties and a key switch for the substrate’s metabolism to reprogramming immune responses in the host intestine by structure-based molecular modeling. The probiotic effects of oligopeptide substrate (such as a proline-rich peptide containing at least one branched residue of leucine, isoleucine, and valine) and its metabolism for the OppA from B. longum KACC91563 are attributed to enhancement of the epithelial barrier by several different strain specific pathways to prevent the strong adhesion of pathogens.     

Detection of nisin expression by Lactococcus lactis using two susceptible bacteria to associate the effects of nisin with EDTA

Nisin, a bacteriocin produced during the exponential growth phase of Lactococcus lactis ATCC 11454, inhibits the growth of a broad range of Grampositive bacteria. Gram-negative bacteria can also be inhibited by nisin with EDTA. In this study, nisin production was assayed by the agar diffusion method using Lactobacillus sake ATCC 15521 and a recombinant Escherichia coli DH5-α expressing the recombinant green fluorescent protein as the nisin-susceptible test organisms. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/mL of medium) and converted to standard nisin concentration (Nisaplin^®, 25 mg of pure nisin with an activity of 1×10⁶ AU/mL). The expression and release of nisin by L. lactis in skimmed milk (9.09% total solids) with Man Rugosa Shepeer-Bacto Lactobacilli broth (1∶1) was monitored in a 5 L New Brunswick fermentor. Combining EDTA with nisin increased the bactericidal effect of nisin on the bacteria examined. The presence of EDTA was necessary to inhibit E. coli growth with nisin. L. sake was shown to be a good indicator for the evaluation of nisin release in the culture media, including with the addition of EDTA.     

乳链菌肽产生菌P-99的摇瓶发酵条件

文章对天然生物防腐剂乳链菌肽产生菌——乳链球菌P-99的摇瓶细胞生长和发酵条件进行了研究,发现该菌株细胞的生长与发酵是同步的,最适种龄为8～18h,产乳链菌肽表现出初级代谢动力学特征;在M17培养基、30℃、起始pH6.5条件下发酵良好,通气量和Ca2+、Zn2+、Fe2+等金属离子对其细胞生长与乳链菌肽的合成无明显影响,但Mn2+对乳链菌肽的合成有促进作用,而Cu2+则有较强的抑制作用。     

乳酸链球菌发酵的系统计算

以乳酸链球菌代谢途径为基础,自动化生成了代谢物的标准化反应速率微分方程组,并建立了系统模型.在设定参数的情况下,计算得到了乳酸链球菌代谢物的浓度变化,量化了乳酸链球菌代谢过程.结果表明: 1)当菌量足够时,葡萄糖初值不同,不影响反应达到平衡所需时间和代谢物极值出现时刻;2)模型经改写,对突变菌株也适用,可以为菌株筛选提供指导,减少实验的盲目性与繁杂性.     

嗜温乳酸菌与白地霉混合发酵酸乳的研究

与高温发酵酸乳相比,嗜温乳酸菌与霉菌联合发酵酸乳有更丰富的风味物质,且嗜温发酵酸乳粘稠、细腻,香气浓郁、纯正,酸甜适口.采用编号为3-7和3-9的嗜温乳酸菌与1株产香白地霉联合发酵生产酸乳,通过混料试验设计,得到酸乳发酵剂的优化菌种体积配比(乳酸菌3-7∶乳酸菌3-9∶白地霉)为50∶5∶45时,其风味、质地和气味较佳.该酸乳具有很高的有效营养成分,其胞外多糖质量浓度高达643.2 mg/L,具有较高的生理功能.其中白地霉不仅能与乳酸菌互利共生产生真菌胞外多糖,而且其产生的香气成分还能有效地提高呈香型物质的种类和含量,改善酸乳的风味质量.