在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量

透明质酸(HA)是一种在医药及化妆品领域具有广泛应用的天然粘多糖。兽疫链球菌(Streptococcuszooepidemicus)是工业上生产透明质酸的菌种之一。透明颤菌血红蛋白(VHb)具有增强细胞摄氧的作用。对生产透明质酸的兽疫链球菌进行了基因改造:将兽疫链球菌HA的合成基因hasABC以及合成透明颤菌血红蛋白的vgb基因(Vitreoscillahemoglobingene,vgb)分别或同时插入阳性菌表达质粒pEU308中,通过电转化导入兽疫链球菌中。通过一氧化碳(CO)差光谱检测到了VHb的表达。在摇瓶实验中,同时带有hasABC和vgb基因的重组菌比野生菌的透明质酸产量提高了30％。而在发酵罐中,带有这2个基因的重组菌的透明质酸产量达到了6．9g／L,高于重组菌5．5g／L的产量。实验结果表明,vgb基因的存在促进了细胞的生长,hasABC操纵子的过表达增强了透明质酸的合成。首次将VHb导入兽疫链球菌中,获得了表达,并证明其对菌体生长及透明质酸合成有促进作用。通过研究,VHb将可以在阳性菌中获得更广泛的应用。     

透明质酸生产菌溶血素S基因缺失突变菌株的构建及其特性北大核心CSCD

【目的】马链球菌兽疫亚种是工业上生产透明质酸的主要菌种,该菌能产生引起宿主细胞溶血的链球菌溶血素S(streptolysin S,SLS)毒素,因而其产品的安全性一直是人们所担心的问题。本实验的目的就是通过基因敲除的方法构建不产SLS的透明质酸生产工程菌,同时探讨溶血素sag A基因缺失对菌株透明质酸合成和其他毒力因子的影响。【方法】利用温度敏感/自杀性质粒p JR700载体系统,构建马链球菌兽疫亚种sag A基因缺失突变株;通过PCR扩增,溶血平板和SLS含量测定等方法确定sag A基因缺失;采用分光光度、SDS-PAGE和细胞毒性试验等分析方法,对野生菌株和sag A基因缺失突变菌株透明质酸含量、透明质酸分子量、溶血素Hylc、透明质酸分解酶、甘油醛-3-磷酸脱氢酶和菌体表面蛋白等相关毒力因子进行对比研究。【结果】获得了透明质酸产量提高30%而溶血活性极低的马链球菌兽疫亚种sag A基因缺失突变株。该突变株与野生菌株相比较,透明质酸分解酶活性增加而透明质酸相对分子量降低,此外,与毒力相关的表面蛋白含量、溶血素Hylc和甘油醛-3-磷酸脱氢酶活性也显著降低。细胞毒性实验结果表明,野生菌株与sag A基因缺失突变菌株的培养物上清液,对细胞活性的影响存在显著差异。【结论】在马链球菌兽疫亚种中sag A不仅是表达溶血素SLS的基因,同时sag A基因对菌株透明质酸合成、透明质酸分解酶、菌体表面蛋白、溶血素Hylc和甘油醛-3-磷酸脱氢酶等都具有调节作用。     

响应面法优化氧化胁迫提高透明质酸分子量

本文研究了氧化剂NaClO胁迫培养兽疫链球菌Streptococcuszooepidemicus对透明质酸(HA)分子量的影响。运用响应面分析法优化发酵工艺条件,在单因素实验基础上,采用中心复合法研究了NaClO的加入时间和加入浓度对HA分子量的影响。利用Mintab 16软件分析确定最佳提取工艺,根据实际条件,得到HA最佳的发酵工艺参数为:NaClO加入时间7 h,NaClO加入浓度为0.415 mL/L。HA分子量由178万Da提高到219万Da。     

透明质酸酶的研究进展

透明质酸酶是能降解透明质酸及部分糖胺聚糖的一类糖苷酶,可应用于医疗和美容等领域。透明质酸酶也可用于制备小分子糖胺寡糖,许多研究发现小分子糖胺寡糖具有比大分子糖胺聚糖更高的生物免疫活性。为便于研究人员对透明质酸酶进行进一步的基础研究及应用研究,本文介绍了透明质酸和透明质酸酶,梳理了透明质酸酶的分类、结构和催化机理,归纳总结了透明质酸酶的酶活力测定方法、重组表达、酶学性质和应用,展望了透明质酸酶的研究方向。     

温度对发酵生产透明质酸的影响

为进一步提高兽疫链球菌透明质酸产量,本实验采用分阶段控制温度工艺,当温度由36℃培养到28 h转入38℃培养至发酵结束,其透明质酸产量有很大提高。经发酵罐验证温度优化结果,其粗品产量由684 g提高到735 g。     

链球菌生物合成透明质酸的分子机理与基因工程菌构建进展

透明质酸（Hyaluronan, HA）是由葡萄糖醛酸和N-乙酰氨基葡萄糖为双糖单位交替连接而成的粘多糖物质。目前构建基因工程菌成为提高产量和改善品质的重要手段。本文从发酵菌种、操纵子、关键酶和工程菌构建等方面,综述了链球菌HA生物合成的分子机理,分析了当前生产中存在的问题,并提出了解决问题的方法。     

兽疫链球菌原生质体激光诱变及高产菌株筛选

探索了透明质酸(Hyaluronic acid)产生菌一兽疫链球菌(Streptococcus zooepidemicus)原生质体制备与再生的最佳条件。研究了酶浓度、酶解时间、高渗液的选择、预处理及高渗预培养等因素的影响。确定了最佳条件为：经1．2％甘氨酸预处理及高渗预培养共同作用2h后,用50U／mL溶菌酶,在NaCl高渗体系中,于39℃作用60min。在此条件下,原生质体形成率可达94．6％,再生率可达18．5％。用不同功率的He-Ne激光照射不同时间诱变原生质体,当功率密度为40mW／cm^2,照射时间为300s时,其致死率可达99．88％。从存活变异株中筛选出一株高产透明质酸菌株,其产量(2．21g／L)达原始菌株(0．49g／L)的4．5倍。     

浅议透明质酸发酵条件的优化

现阶段透明质酸已广泛应用于化妆品、美容行业、保健食品、医药等很多领域。国内市场及国际市场得扩展速度都非常快,随之而来的透明质酸的市场竞争也日趋激烈。因此,为了满足国内日益增长的市场需求,我国应大力发展微生物发酵法制备透明质酸的技术,选育高产菌种,完善发酵工艺,以提高工业化的产量与质量。为进一步提高兽疫链球菌透明质酸的产量,优化其发酵条件,采用15L-30L×3的发酵系统进行试验。     

构建无抗性标记双拷贝透明质酸合成酶基因工程菌

【目的】探讨一种构建无抗生素选择标记链球菌透明质酸合成酶基因工程菌的方法。【方法】PCR扩增链球菌透明质酸合成酶操纵子hasABC基因,用温度敏感载体pJR700构建携带hasABC基因重组质粒pXL32;电转化pXL32质粒到马链球菌兽疫亚种感受态细胞,卡那霉素(Kanamycin,kan)平板37℃培养筛选重组子,在不含kan液体培养基中30℃传代,37℃平板分离挑取抗生素敏感菌落,RT-PCR检测工程菌染色体hasABC基因表达,Bitter-Muir法测定菌体透明质酸含量。【结果】在无抗生素选择压力条件下,获得透明质酸产率提高34%的马链球菌兽疫亚种透明质酸合成酶基因工程菌。【结论】用pJR700温度敏感载体系统,构建能提高透明质酸产量的无抗生素选择标记的基因工程菌是可行的。     

谷氨酸棒杆菌一步法发酵糖质原料生产γ-聚谷氨酸

γ-聚谷氨酸在食品、化妆品、生物医药等领域具有广泛的应用,目前主要的生产菌株是谷氨酸依赖型菌株,在生产过程中需要添加谷氨酸作为前体,因而生产γ-聚谷氨酸的成本较高。文中主要研究从糖质原料一步法发酵合成γ-聚谷氨酸的生产工艺。首先,从产γ-聚谷氨酸的菌株枯草芽孢杆菌中克隆γ-聚谷氨酸合成酶的基因簇pgs BCA,在谷氨酸棒杆菌模式菌株ATCC13032中进行诱导型和组成型表达,结果显示,仅诱导型表达菌株可以积累γ-聚谷氨酸,产量为1.43 g/L。进一步对诱导条件进行优化,确定诱导时间为2 h,IPTG浓度为0.8 mmol/L,γ-聚谷氨酸产量为1.98g/L。在此基础上,在一株高产谷氨酸的谷氨酸棒杆菌F343中外源表达pgs BCA,对重组菌进行发酵,结果表明,在摇瓶发酵中γ-聚谷氨酸产量达到10.23g/L,在5L发酵罐中产量达到20.08g/L;继而对γ-聚谷氨酸进行分子量测定,结果显示,产自F343重组菌的γ-聚谷氨酸的重均分子量比产自枯草芽孢杆菌的提高34.77%。文中构建了一步法发酵糖质原料生产γ-聚谷氨酸的新途径,同时为开发其潜在应用奠定了基础。     

Microbial production of low molecular weight hyaluronic acid by adding hydrogen peroxide and ascorbate in batch culture of Streptococcus zooepidemicus

Microbial production of low molecular weight hyaluronic acid (HA) by the addition of hydrogen peroxide and ascorbate during the batch culture of Streptococcus zooepidemicus was investigated. Hydrogen peroxide (1.0 mmol/g HA) and ascorbate (0.5 mmol/g HA) were added at 8h and 12h to degrade HA. With the redox depolymerization of HA, the HA molecular weight decreased from 1,300 kDa for the control to 80 kDa, and the average broth viscosity during 8-16 h decreased from 360 mPa s for the control to 290 mPa s. The average oxygen mass transfer coefficient K(L)a increased from 10h(-1) for the control to 35 h(-1) and the average dissolved oxygen level increased from 1% of air saturation in the control to 10%. HA production increased from 5.0 g/L for the control to 6.5 g/L, and contributed to the increased redox potential and energy charge. This novel process not only significantly enhanced production of low molecular weight HA, but also improved purification efficiency due to a decreased broth viscosity. Low molecular weight HA finds applications in biomedical and healthcare fields.     

营养条件对兽疫链球菌发酵生产透明质酸的影响

透明质酸 (Ｈｙａｌｕｒｏｎｉｃａｃｉｄ ,简称ＨＡ)是Ｎ 乙酰氨基葡萄糖胺和葡萄糖醛酸以 β 1 3糖苷键和 β 1 4糖苷键连接而成的二糖单体重复构建而成的杂多糖 ,广泛存在于高等动物的结缔组织内。由于结构上的特点 ,ＨＡ具有很高的粘弹性和极强的保水性等特征 ,已被大量用于医学医药、化妆品工业[1,2 ] 。1937年Ｋｅｎｄａｌｌ[3 ] 等发现用溶血性链球菌 (Ｓｔｒｅｐｔｏｃｏｃｃｕｓｈａｅｍｏｌｙｔｉｃｕｓ)可以产生ＨＡ。其后 ,陆续发现许多能产生ＨＡ的微生物菌种 ,逐渐开发出一条可替代传统的动物组织提取法[4 ] 生产ＨＡ的新途径…     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

Enhanced hyaluronic acid production by a two-stage culture strategy based on the modeling of batch and fed-batch cultivation of Streptococcus zooepidemicus

This study aimed to enhance hyaluronic acid (HA) production by a two-stage culture strategy based on the modeling of batch and fed-batch culture of Streptococcus zooepidemicus. Batch culture had higher specific HA synthesis rate while fed-batch culture had higher specific cell growth rate. The lower specific HA synthesis rate in fed-batch culture resulted from the competition of cell growth for the common precursors at a low substrate concentration. Based on the modeling of batch and fed-batch culture of S. zooepidemicus, a two-stage culture strategy was proposed to enhance HA production. S. zooepidemicus were cultured in a fed-batch mode with sucrose concentration maintained at 1.0+/-0.2g/L during 0-8h and then batch culture was performed during 8-20h with an initial sucrose concentration of 15g/L. With the proposed two-stage culture strategy, HA production was increased to 6.6g/L compared with 5.0g/L in batch culture with the same total sucrose. The enhanced HA production by the proposed two-stage culture strategy resulted from the decreased inhibition of cell growth and the increased transformation rate of sucrose to HA.     

海豚链球菌诱变发酵法制备透明质酸及其在动物皮肤修复中的应用

透明质酸(HA)是一种非常重要的生物材料,是体内广泛存在的细胞外基质成分之一。为了获得产量、分子量及纯度较高的透明质酸,并研究透明质酸水凝胶在动物皮肤修复中的潜在作用。通过紫外诱变的方法对海豚链球菌进行诱变,并对此突变菌发酵后产物的蛋白含量及HA分子量进行了测定,通过CTAB法对发酵产物进行提纯,运用物理冻融法将透明质酸制成水凝胶后,用于兔背部全层皮肤修复的初探。结果表明通过诱变海豚链球菌产透明质酸的能力从(82.3±3.3)mg/L增加到(120±10.6)mg/L,增加了46.4%;产物经纯化后蛋白含量从(0.178±0.011)mg/L减少到(0.032±0.017)mg/L,减少了82.02%;所制得透明质酸的分子量约为3.0×105 Da;透明质酸水凝胶对兔全层皮肤缺损的修复有较明显的促进作用,能减轻炎症和伤口瘢痕的形成。     

Modeling and optimization of microbial hyaluronic acid production by Streptococcus zooepidemicus using radial basis function neural network coupling quantum‐behaved particle swarm optimization algorithm

Hyaluronic acid (HA) is a natural biopolymer with unique physiochemical and biological properties and finds a wide range of applications in biomedical and cosmetic fields. It is important to increase HA production to meet the increasing HA market demand. This work is aimed to model and optimize the amino acids addition to enhance HA production of Streptococcus zooepidemicus with radial basis function (RBF) neural network coupling quantum‐behaved particle swarm optimization (QPSO) algorithm. In the RBF‐QPSO approach, RBF neural network is used as a bioprocess modeling tool and QPSO algorithm is applied to conduct the optimization with the established RBF neural network black model as the objective function. The predicted maximum HA yield was 6.92 g/L under the following conditions: arginine 0.062 g/L, cysteine 0.036 g/L, and lysine 0.043 g/L. The optimal amino acids addition allowed HA yield increased from 5.0 g/L of the control to 6.7 g/L in the validation experiments. Moreover, the modeling and optimization capacity of the RBF‐QPSO approach was compared with that of response surface methodology (RSM). It was indicated that the RBF‐QPSO approach gave a slightly better modeling and optimization result compared with RSM. The developed RBF‐QPSO approach in this work may be helpful for the modeling and optimization of the other multivariable, nonlinear, time‐variant bioprocesses. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Use of induction promoters to regulate hyaluronan synthase and UDP-glucose-6-dehydrogenase of Streptococcus zooepidemicus expression in Lactococcus lactis: a case study of the regulation mechanism of hyaluronic acid polymer

Aims:  To determine the effects of the ratios of hyaluronan synthase expression level to precursor sugar UDP-GlcA biosynthesis ability on the molecular weight (MW) of hyaluronic acid (HA) in recombinant Lactococcus lactis .
Methods and Results:  The genes sz HasA (hyaluronan synthase gene) and sz HasB (UDP-glucose-6-dehydrogenase gene) of Streptococcus zooepidemicus were introduced into L. lactis under the control of nisA promoter and lacA promoter respectively, resulting in a dual-plasmid controlled expression system. The effects of the ratios of hyaluronan synthase expression level to the precursor sugar UDP-GlcA biosynthesis ability under different induction concentration collocations with nisin and lactose on the MW of HA in recombinant L. lactis were determined. The results showed that the final weight-average molecular weight (     ) of HA correlated with the relative ratios of HasA (hyaluronan synthase) expression level to the concentration of UDP-GlcA.
Conclusions:  Regulating the relative ratios of HasA expression level to the precursor sugar biosynthesis ability was an efficient method to control the size of HA.
Significance and Impact of the Study:  This study put forward a guide to establish an efficacious way to control the size of HA in fermentation.