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。     

玉米水解糖液体培养灵芝发酵条件的优化

目的：优化液体培养灵芝的发酵条件,提高多糖产量。方法：采用玉米水解糖为主要成分的培养基,通过单因素和正交实验,对赤芝G22菌株液体培养过程中影响多糖产量的发酵温度、摇床转速等工艺条件进行了研究。结果：经极差分析和方差分析确定了多糖高产的最佳发酵条件为：发酵温度27℃、摇床转速170r/min、培养基初始pH值6.5、发酵时间144 h。结论：通过优化液体发酵条件,可显著提高灵芝多糖的产量。在最佳发酵条件下液体培养G22菌株,灵芝总多糖产量由1.851g/L提高到2.439g/L,提高了31.0%。     

栖热菌属高温菌RH99-02菌株产类胡萝卜素的研究

从腾冲热海分离到最高生长温度达80℃的一株嗜热非芽孢菌RH99-02菌株,经鉴定属Thermus属,对其所产类胡萝卜素进行了吸收光谱扫描及薄层层析分析。发酵研究表明振荡培养时的菌体生物量和色素产量上均高于静置培养,培养至50h菌体生物量和色素产量达到最大值,菌体色素产量为762μg/g干重。     

栖热菌属高温菌RH99-02菌株产类胡萝卜素的研究*

从腾冲热海分离到最高生长温度达 80℃的一株嗜热非芽孢菌RH99- 0 2菌株 ,经鉴定属Thermus属。对其所产类胡萝卜素进行了吸收光谱扫描及薄层层析分析。发酵研究表明振荡培养时的菌体生物量和色素产量上均高于静置培养 ,培养至 50h菌体生物量和色素产量达到最大值 ,菌体色素产量为 762 μg/g干重。     

玫瑰亚栖热菌RH9901菌株产红色色素的研究

从腾冲热海分离到最高生长温度达 70℃的 1株嗜热非芽孢菌RH990 1菌株 ,鉴定为玫瑰亚栖热菌新种 (Meiothermusrosaceus)。对其所产色素进行吸收光谱扫描及薄层层析分析表明为类胡萝卜素。振荡培养在菌体生物量和色素产量上均高于静置培养 ,培养至 5 0h菌体生长量和色素产量达到最大值 ,菌体色素产量为 1.14 7mg/ g干重。     

温度对青蒿毛状根生长和青蒿素生物合成的影响

本实验研究了不同温度(15℃～35℃)对青蒿毛状根生长和青蒿素生物合成的影响,发现25℃有利于毛状根生长,30℃促进了青蒿素生物合成。通过温度改变的二步培养技术(培养前20d温度控制在25℃,后10d温度提高到30℃),青蒿素的产量得到明显提高,高于在恒温培养时(25℃或30℃)的结果。     

两种UDP-葡萄糖脱氢酶对透明质酸生物转化的影响

分别从大肠杆菌和化脓链球菌中扩增出编码UDP-葡萄糖脱氢酶基因ecohas B和spyhas B,并将其插入T7表达载体p RX2构建重组质粒p RXEB和p RXSB。在大肠杆菌BL21(DE3)中重组表达,并对经镍柱纯化后的UDP-葡萄糖脱氢酶的酶学性质进行分析。酶学性质研究表明:spy Has B的最适反应温度是30℃,最适p H 10,最适条件下的比活力是12.2 U/mg;eco Has B的最适反应温度是30℃,最适p H 9,最适条件下的比活力是5.55 U/mg。从多杀巴氏杆菌扩增出的透明质酸合成酶基因pmuhas A分别与ecohas B和spyhas B构建共表达载体p BPAEB和p BPASB。将其转化到大肠杆菌BW25113中,经生物转化生产透明质酸(HA),并对转化条件进行了优化。结果表明:重组菌株进行透明质酸转化时,UDP-葡萄糖脱氢酶酶活力越高,稳定性越好,HA产量越高;转化条件优化后,p BPAEB/BW25113和p BPASB/BW25113在摇瓶中的产量分别是1.52和1.70 g/L,比之前报道的提高了2-3倍。     

透明质酸产生菌的选育及发酵培养基的优化

将实验室保存UVD-34透明质酸产生菌再进行超声波处理,挑选出了一株透明质酸产量较高的菌,并对其发酵培养基进行初步优化。结果表明当可溶性淀粉质量分数为2%,复合氮源为3%,碳酸氢钠为0.1%时,其透明质酸的产量可达4.59 g.L-1,比优化前提高了1.29倍。该突变株值得进一步研究。     

4,4,4-三氟乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

对实验室保藏菌种进行筛选,得到一株葡萄汁酵母Saccharomyces uvarum SW-58,对其产酶条件进行优化,其发酵培养基组成:醋酸钠60 g/L,玉米浆30 g/L,KH2PO46 g/L,MgSO4.7H2O 1 g/L;培养条件为:发酵温度30℃,初始pH 6.0,发酵周期36 h。4,4,4-三氟乙酰乙酸乙酯羰基还原酶酶活最高可达388.1 U/L,产物的浓度由优化前的3.5 g/L提高到4.6 g/L,所得产物的光学纯度由优化前的60.8%e.e.提高到85.0%e.e。     

赤霉素A9检定方法的建立和发酵的初步研究

通过对50株野生型藤仓赤霉菌的筛选,获得一株GA9组分较高而GA3、GA+7组分较低的菌株农大201(ND-201),然后通过多次紫外诱变,使其产量从原来的34μg／ml提高到260μg／ml。当发酵条件采用变温培养(培养72h后由28℃转到34℃、调节pH值(72h后pH由4．5调到6．2)．产量可达300μg／ml。在培养基接最佳组分配制后,GA9的产量可达350μg／ml。发酵产物经提取、层析并经气相色谱-质谱联机(GC-MS)鉴定确证为GA9。硅胶G薄层层析和荧光光度法可简便地对GA9进行定量测定。HPLC法可测得GA9组分的比例含量。     

Optimization of hyaluronic acid production and its cytotoxicity and degradability characteristics

Abstract

In the present study, culture conditions of Streptococcus equi was optimized through Box–Behnken experimental design for hyaluronic acid production. About 0.87?gL^?1 of hyaluronic acid was produced under the determined conditions and optimal conditions were found as 38.42?°C, 24?hr and 250?rpm. The validity and practicability of this statistical optimization strategy were confirmed relation between predicted and experimental values. The hyaluronic acid obtained under optimal conditions was characterized. The effects of different conditions such as ultraviolet light, temperature and enzymatic degradation on hyaluronic acid produced under optimal conditions were determined. 118?°C for 32?min of autoclaved HA sample included 63.09 µg mL^?1 of d-glucuronic acid, which is about two-fold of enzymatic effect. Cytotoxicity of hyaluronic acid on human dermal cells (HUVEC, HaCaT), L929 and THP-1 cells was studied. In vitro effect on pro or anti-inflammatory cytokine release of THP-1 cells was determined. Although it varies depending on the concentration, cytotoxicity of hyaluronic acid is between 5 and 30%. However, it varies depending on the concentration of hyaluronic acid, TNF-α release was not much increased compared to control study. Consequently, purification procedure is necessary to develop and it is worth developing the bacterial hyaluronic acid.     

L-谷氨酸补料分批发酵的研究

对谷氨酸棒杆菌(Corynebacteriuin glutamicum)HCJ46产L-谷氨酸的补料分批发酵条件进行研究.结果表明:最适初糖质量浓度和最佳残糖维持质量浓度分别为100和(10～20)g/L;对发酵控温方式进行研究,确定了最佳温度控制策略为0～8h维持32℃,8～16h维持34℃、16～32h维持36℃,同时发现相对溶氧控制在30%左右时产酸最高.在以上的优化条件下,L-谷氨酸产量从72g/L提高到95g/L,提高了31.9%.     

Effect of trimethylcolchicinic acid on the synthesis and excretion of proteoglycans in tissue culture.

The action of trimethylcolchicinic acid on the synthesis and excretion of proteoglycans has been studied on the L cell strain. The incorporation of precursors has been measured, and proteoglycans produced in the culture medium have been extracted and their concentration determined. The mucopolysaccharide components have been studied by electrophoresis. Control cultures produce hyaluronic acid, dermatan sulfate and very low concentrations of chondroitin 4-sulphate or 6-sulphate. Cultures treated with trimethycolchicinic acid (4 mu g/ml) produce hyaluronic acid, very high concentrations of chondroitin 4-sulphate or 6-sulphate and only traces of dermatan sulphate. So, trimethylcolchicinic acid does not modify the synthesis of hyaluronic acid: it considerably increases the production of chondroitin 4-sulphate or 6-sulphate and inhibits the production of dermatan sulphate. Protein fraction of the proteoglycans is proportionally increased in treated cultures, but there is no marked difference between amino acid concentrations of proteoglycans extracted from control and treated cultures. A slight fall in the cystine concentrations was the only change in the amino acid content of proteoglycans extracted from treated cultures. A hypothesis to explain these results is discussed.     

Hormonal control of hyaluronic acid production in fibroblasts and its relation to nucleic acid and protein synthesis.

Whole serum and elevated pH previously had been found to stimulate both cell multiplication and hyaluronic acid production by chick embryo fibroblasts in culture. In a study to determine whether cell multiplication and hyaluronic acid production both respond to a single well-defined substance, insulin was found to stimulate, and cortisol to inhibit both processes coordinately. It appears, therefore, that multiplication and differentiated function in fibroblasts respond to a common underlying regulatory signal. Inhibition of ribosomal RNA synthesis by actinomycin D does not prevent serum stimulation of hyaluronic acid production, but inhibition of total RNA synthesis does. If total RNA synthesis is inhibited only after the hyaluronic acid production has reached a new high level, it continues at that level for the next five hours. The stimulatory treatment causes an increase in the activity of the enzyme hyaluronate synthetase. Inhibition of protein synthesis prevents any increase in hyaluronic acid production, and reduces the basal level of production. Reduction of the availability of Mg2+ in the medium coordinately inhibits DNA synthesis and hyaluronic acid production. The results are discussed in the light of a model for coordinate control growth and metabolism based on the availability of Mg2+.     

Effects of adenosine 3'':5''-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts.

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.     

Change of hyaluronic acid synthesis during differentiation of myogenic cells and its relation to transformation of myoblasts by Rous sarcoma virus

Hyaluronic acid synthesis was examined in cultures of differentiating chick embryo muscle cells before, during and after fusion. Prior to fusion, hyaluronic acid was synthesized and secreted into the medium, but once fusion began this synthesis was reduced significantly. Synthesis then increased again after completion of fusion. Thus, production of hyaluronic acid was lowest at the time of or right before cell fusion. When myoblasts were transformed by Rous sarcoma virus (RSV), a higher amount of hyaluronic acid was synthesized, and cells were not able to fuse. The turnover rate of hyaluronic acid might be different between myotubes and RSV-transformed myoblasts. The addition of exogenous hyaluronic acid to myoblast cultures resulted in the partial inhibition of fusion. The effect was reversible because fusion took place after removal of the exogenous hyaluronic acid. These observations suggest that hyaluronic acid plays an important role in the differentiation of myogenic cells, and that elevated hyaluronic acid synthesis may partly be the reason for inhibition of myotube formation upon transformation by Rous sarcoma virus.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substance by cultured fibroblasts (3) induction of hyaluronic acid synthetase by prostaglandin F2α

The mechanism of the stimulatory effect of prostaglandin(PG) F_2α on the production of hexosamine-containing substance by cultured fibroblasts was studied. Treatment of the cells with 1 μg/ml of PGF_2α resulted in a doubled net synthesis of acidic glycosaminoglycans during 20 hrs measured with uronic acid as index, and also resulted in 300 per cent increase of ³H-glucosamine incorporation into hexosamine-containing substances during the first 6 hrs. Fractionation of the PGF_2α-stimulated hexosamine-containing substances with double isotope technique revealed that hyaluronic acid was the most stimulated component. Prior to the increase of hyaluronic acid, hyaluronic acid synthetase activity was found to be augmented by PGF_2α as high as 4 times over the control. The augmentation of hyaluronic acid synthetase activity by PGF_2α did not take place if actinomycin D was simultaneously present in the culture medium, suggesting that PGF_2α induced the enzyme.     

Stable production of hyaluronic acid in Streptococcus zooepidemicus chemostats operated at high dilution rate

Hyaluronic acid is routinely produced through fermentation of both Group A and C streptococci. Despite significant production costs associated with short fermentations and removal of contaminating proteins released during entry into stationary phase, hyaluronic acid is typically produced in batch rather than continuous culture. The main reason is that hyaluronic acid synthesis has been found to be unstable in continuous culture except at very low dilution rates. Here, we investigated the mechanisms underlying this instability and developed a stable, high dilution rate (0.4 h-1) chemostat process for both chemically defined and complex media operating for more than 150 h of production. In chemically defined medium, the product yield was 25% higher in chemostat cultures than in conventional batch culture when arginine or glucose was the limiting substrate. In contrast, glutamine limitation resulted in higher ATP requirements and a yield similar to that observed in batch culture. In complex, glucose-limited medium, ATP requirements were greatly reduced but biomass synthesis was favored over hyaluronic acid and no improvement in hyaluronic acid yield was observed. The successful establishment of continuous culture at high dilution rate enables both commercial production at reduced cost and a more rational characterization and optimization of hyaluronic acid production in streptococci.     

高温胁迫下西伯利亚蝗体内抗逆物质含量变化

【目的】新疆气候变暖是导致西伯利亚蝗Gomphocerus sibiricus (L.)持续严重发生的重要原因之一,前期研究表明近40年来西伯利亚蝗严重发生与新疆同期气候变暖有显著相关性,本研究进一步探讨了温度升高条件下西伯利亚蝗的生理生化适应机理。【方法】采用生理生化研究方法,研究了24, 27, 30, 33, 36, 39和42℃下西伯利亚蝗体内海藻糖、甘油、油酸、亚油酸和亚麻酸5种抗逆物质的积累与变化过程。【结果】在24~42℃范围内,西伯利亚蝗体内5种物质的积累量随温度的升高呈现出先增后减的变化趋势。海藻糖、甘油、油酸、亚油酸和亚麻酸含量在30℃时含量最高,分别为18.691 μg/g, 261.432 μg/g, 79.063 mg/g, 78.664 mg/g和227.593 mg/g;42℃高温时为最低,含量依次为18.218 μg/g, 104.588 μg/g, 4.343 mg/g, 3.039 mg/g和11.067 mg/g。5种抗逆物质积累的速率不同,其中随温度升高亚油酸含量增、减速率最快,分别为832.189%和63.988%,海藻糖含量增、减速率最慢,分别为 0.893%和0.224%。【结论】在24~30℃之间,随着温度升高,西伯利亚蝗可以通过积累体内抗逆物质,尤其通过快速积累不饱和脂肪酸以提高自身对阶段性高温的耐受能力;超过30℃,蝗虫体内抗逆物质积累下降,死亡率增加,虫体对高温胁迫失去耐受力。这预示着在气候变暖趋势下,西伯利亚蝗仍将是新疆草原最重要的生物灾害之一。