高密度发酵重组大肠杆菌产海藻糖合成酶

酶法催化是目前生产海藻糖的主要手段。本文中,笔者通过高密度发酵重组大肠杆菌产海藻糖合成酶,进而以麦芽糖为底物,催化生产海藻糖。首先根据大肠杆菌高密度发酵条件要求,在揺瓶中对基因工程菌进行了培养基、发酵条件和诱导条件的逐一优化;然后在5 L和50 L发酵罐中进行批次补料发酵的放大实验;最后采用变指数-恒pH法的策略发酵重组大肠杆菌,结果OD_(600)达到97,海藻糖合成酶酶活达到(24 000±350) U/mL,实现了海藻糖合成酶的重组大肠杆菌高密度发酵生产,极大提高了海藻糖的生产效率。     

红酵母RY—98产类胡萝卜素培养基的优选及其发酵生理学研究

对红酵母ＲＹ－９８菌株产生类胡萝卜素的培养基组成和培养条件进行了初步研究,并对发酵过程作了动态分析。结果表明,培养基组成、糖浓度和添加剂以及培养基初始ｐＨ值、摇瓶装量与培养等均对该菌细胞生物量和类胡萝卜素产量有影响,其中ＺＳ添加剂的加入可以明显促进菌体类胡萝卜素的形成,这是研究新发现。在初步优化的培养基组成（葡萄糖４０ｇ／Ｌ、玉米浆１５ｇ／Ｌ、（ＮＨ４）２ＳＯ４２ｇ／Ｌ、ＺＳ添加剂１ｇ／Ｌ）和培养     

组成型表达粪产碱杆菌青霉素G酰化酶重组大肠杆菌发酵条件研究

目的:对重组大肠杆菌组成型表达粪产碱杆菌青霉素G酰化酶(AfPGA)进行了发酵条件研究。方法:在摇瓶和5L发酵罐中研究了(NH4)2SO4和葡萄糖浓度对质粒的分离稳定性及青霉素G酰化酶表达的影响。结果:该工程菌质粒具有分离不稳定性,培养基中无(NH4)2SO4时发酵过程中pH和糊精水解生成葡萄糖的浓度变化较小,细胞前期(0h-12h)的生长速率降低,质粒分离稳定性和青霉素G酰化酶的表达水平提高。发酵过程中维持低葡萄糖水平可以限制细胞的生长速率,提高质粒稳定性和促进青霉素G酰化酶的合成。采用混合碳源发酵,发酵培养基含糊精2g/L,12h后以1g/L.h恒速流加葡萄糖至35h,控制流加过程葡萄糖浓度0.1g/L左右,平均比生长速率为0.06h-1,发酵结束时质粒稳定性为86%,青霉素G酰化酶的表达水平达23 000U/L。结论:重组大肠杆菌组成型表达青霉素G酰化酶的研究对工业生产有一定指导意义。     

海藻糖产生菌的筛选及其发酵条件研究

通过液体发酵筛选,从土壤中分离的241株菌株及保藏的95株菌株中得到15株海藻糖生产菌,对这些菌株进一步筛选得到产海藻糖最多的一株菌Bacillus sp。其发酵最适合条件为：以麦芽糖为碳源,发酵初始pH值7.0,接种量为6%,30℃条件下培养48h,另外该菌株在分别以葡萄糖和蔗糖为碳源的培养基中都能产生海藻糖。     

一株海藻糖合成酶产生菌的鉴定及产酶条件优化

目的对一株海洋来源的产海藻糖合成酶菌株进行鉴定及产酶条件的初步优化。方法通过16SrDNA基因序列的同源性分析,对一株来源于东海海水的海藻糖合成酶产生菌进行鉴定,并通过单因素分析初步研究其培养特性和最佳的发酵条件。结果该菌16SrDNA序列与GenBank中已知序列相比,最高相似度为100％,鉴定为假单胞菌属（Pseudomonas）,命名为Pseudomonassp．A50。其最佳碳源和氮源分别为2％麦芽糖和0．5％酵母膏,最佳NaCl浓度为2．5％,在初始pH7．8,接种量1％,装液量125mL／250mL,28℃,130r／min发酵48h,海藻糖合成酶活力达到最高。结论此产海藻糖合成酶菌株为假单胞菌属,优化后,海藻糖合成酶活力达到14．16U／mL。     

限磷对产甘油假丝酵母甘油合成与胞内磷积累的影响

研究了磷酸盐限量对产甘油假丝酵母甘油合成与胞内磷积累的影响。结果表明, 当酵母细胞从适磷或富磷培养基转接入低磷培养基时, 发酵过程中胞内积累的磷逐渐减少; 而当菌体从低磷培养基转接入适磷或富磷培养基时, 发酵过程中胞内聚磷酸盐的积累量迅速增加。当细胞在第14小时和第38小时从适磷培养基转接入低磷培养基时甘油得率分别高达60.9%和61.4%, 而甘油产率则分别为2.03 g/(L·h)和2.23 g/(L·h)。这些现象说明限制发酵培养基中的磷浓度是产甘油假丝酵母高产甘油的必要条件, 并为其反复分批发酵法生产甘油提供了重要依据。     

阿拉伯胶酶产生菌株的筛选及其发酵培养基的优化

从土壤中筛选产阿拉伯胶酶的微生物菌株,并通过紫外诱变选育后得到高产突变菌株ZHB05F,依据菌落和孢子形态特征初步鉴定为镰刀茵(Fusarium sp.).通过单因素试验,优化了产酶培养基的主要组分的浓度和pH值,得到最佳的产酶发酵培养基组成为:阿拉伯胶30 g/L,(NH4)2SO4 8 g/L,K2HPO4 1g/...     

玉米浆在产甘油假丝酵母甘油发酵中的作用机理

以复合培养基和合成培养基进行比较发酵,研究了玉米浆在产甘油假丝酵母甘油发酵过程中的作用机理。结果表明:玉米浆中的磷、氮和微量元素是影响产甘油假丝酵母甘油发酵的3个关键因素。当玉米浆磷浓度为121·75mg/L(玉米浆浓度为14g/L),最大甘油转化率达到53·44%。玉米浆磷可以调节EMP途径与HMP途径之间碳架代谢流的分布,随着玉米浆浓度进一步增加,过量磷能抑制HMP途径而激活EMP途径,因而复合培养基各项发酵参数的变化非常显著。玉米浆氮对磷的调节功能有协同作用,但并不是产甘油假丝酵母甘油发酵的理想氮源。玉米浆中的微量元素能够显著提高葡萄糖的消耗速率、促进菌体的生长和增加甘油的产量。     

假单胞菌产脂肪酶条件的初步探索

对假单胞菌（Pseudomonassp）2106菌株产脂肪酶条件的初步探索表明,该菌株脂肪酶为组成型,不受油脂类底物的诱导。碳源的种类（单糖、双糖、多糖）和浓度对产酶影响不大,氮源以豆饼粉和玉米浆混合添加最好。最适发酵温度为32℃,摇瓶转速140r／min。实验中还通过正交试验优化了产酶培养基组成。     

发酵性丝孢酵母(Trichosporon fermentans)发酵产油脂的研究

通过摇瓶发酵,考察了碳源浓度、氮源种类和浓度对发酵性丝孢酵母(Trichosporonfermentans)发酵产油脂的影响,对发酵产油脂条件的初步优化结果为:在葡萄糖100 g/L、蛋白胨1.8 g/L、初始pH 7.0的培养基中,以10%的接种量,于33℃、190 r/min的摇床上发酵120 h,可得菌体生物量为18.2 g/L,干细胞的油脂含量为68.5%。     

海洋中海藻糖产生菌的筛选及发酵条件优化

从180余份海水、海泥样品中筛选得到60株产海藻糖较高的菌株,编号为2-14的菌株海藻糖产量最高,为127.9mg/g cell。对2-14菌株进行形态特征、培养特征及生理生化试验,鉴定该菌株为红酵母属(Rhodotorula sp.)。研究摇瓶发酵条件对红酵母海藻糖产量的影响,结果为:初始pH5.5,发酵温度28℃,装液量75mL(250mL三角瓶中)。采用优化后发酵条件红酵母海藻糖产量为193.3mg/g cell,优化前对照值为132.1mg/g cell,优化后的结果是优化前的1.46倍。在5L发酵罐中培养得到最佳发酵时间为54h,发酵罐培养发酵液中海藻糖含量最高达2.5g/L,为摇瓶培养的1.6倍。     

胶样菌CB39产海藻糖的研究

从长白山天池水中筛选到一株低温条件下产糖的细菌。通过薄层层析、成脎反应、毛细管等速电泳以及红外光谱法确定该糖为海藻糖;经鉴定此菌株是胶样菌属中的一个新种(ColloidesSp.)定名为CB39;与已报道的产海藻糖菌株不同的是,菌株CB39能将产生的海藻糖分泌到细胞外,18℃时其培养液中海藻糖含量为2562mg/g干菌体;采用紫外诱变法筛选到一株在25℃条件下产海藻糖量为4167mg/g干菌体的高产突变株5,产糖量是同温度下野生菌的8倍。     

Physiological role of beta-phosphoglucomutase in Lactococcus lactis

A beta-phosphoglucomutase (beta-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of beta-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h(-1), while the deletion of beta-PGM resulted in a maximum specific growth rate of 0.05 h(-1) on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as beta-glucose 1-phosphate in the medium. Furthermore, the beta-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of alpha-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the beta-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded beta-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Trehalose-6-phosphate-mediated phenotypic change in Acinetobacter baumannii

The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD-glucose and glucose-6-phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose-6-phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose-6-phosphate phosphatase activity accumulated trehalose-6-phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose-6-phosphate. Our results demonstrate that trehalose-6-phosphate affects multiple physiological activities in A. baumannii ATCC 19606.     

Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification

In this study, corn starch was used as the substrate for cell growth and trehalose accumulation by Saccharomycopsis fibuligera A11. Effect of different aeration rates, agitation speeds, and concentrations of corn starch on direct conversion of corn starch to trehalose by S. fibuligera A11 were examined using a Biostat B2 2-l fermentor. We found that the optimal conditions for direct conversion of corn starch to trehalose by this yeast strain were that agitation speed was 200 rpm, aeration rate was 4.0 l/min, concentration of corn starch was 2.0% (w/v), initial pH was 5.5, fermentation temperature was 30°C. Under these conditions, over 22.9 g of trehalose per 100 g of cell dry weight was accumulated in the yeast cells, cell mass was 15.2 g/l of the fermentation medium, 0.12% (w/v) of reducing sugar, and 0.21% (w/v) of total sugar were left in the fermented medium within 48 h of the fermentation. It was found that trehalose in the yeast cells could be efficiently extracted by the hot distilled water (80°C). After isolation and purification, the crystal trehalose was obtained from the extract of the cells.     

Regulation of energy metabolism in Saccharomyces cerevisiae. Relationships between catabolite repression, trehalose synthesis, and mitochondrial development.

Changes in trehalose accumulation and in cytochromes during diauxic growth in glucose medium were examined in a normal Saccharomyces cerevisiae strain. While no appreciable disaccharide accumulation occurred during most of the logarithmic phase, a rapid synthesis took place during the final stages. The intrinsic capacity of cells to accumulate trehalose was also determined under nonproliferating conditions, in glucose medium lacking a nitrogen source. Cells harvested at an early growth stage had a much lower trehalose accumulation capacity than cells taken after glucose was exhausted from the culture medium. A high trehalose accumulation capacity could also be obtained at any growth stage by using maltose or galactose as carbon source. Since cells grown under various conditions exhibit a correlated change in cytochrome development and in trehalose accumulation capacity, it was concluded that the level of glucose repression determines the concentration and/or state of activation of the trehalose synthetase-trehalase complex. Independent control of trehalose accumulation capacity and mitochondrial biogenesis by the level of glucose repression was shown in two ways: by demonstrating derepression of trehalose accumulation without development of cytochromes a and c in microaerobic cells, and by showing repression-dependent changes in a cytoplasmic respiration-deficient (ρ^?) mutant, which lacked functional mitochondria. Therefore, the capacity of a cell to accumulate trehalose is not regulated solely by the supply of ATP generated by oxidative phosphorylation.     

Trehalose and glycogen accumulation is related to the duration of the G1 phase of Saccharomyces cerevisiae

Several factors may control trehalose and glycogen synthesis, like the glucose flux, the growth rate, the intracellular glucose-6-phosphate level and the glucose concentration in the medium. Here, the possible relation of these putative inducers to reserve carbohydrate accumulation was studied under well-defined growth conditions in nitrogen-limited continuous cultures. We showed that the amounts of accumulated trehalose and glycogen were regulated by the growth rate imposed on the culture, whereas other implicated inducers did not exhibit a correlation with reserve carbohydrate accumulation. Trehalose accumulation was induced at a dilution rate (D)相似文献   

The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture.

Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0.5 M-NaCl. Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant. The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis. Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic. The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption. Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.     

不同渗透压调节剂对Candida krusei生理代谢的影响

比较了氯化钠、氯化钾、甘露醇存在的高渗环境下克鲁氏假丝酵母(Candida kru-sei)的生理代谢。3种渗透压调节剂对C.krusei生理代谢影响有显著差异。与甘露醇相比,氯化钠和氯化钾对细胞生长的影响更为显著,而氯化钾对细胞的毒性则又小于氯化钠。细胞对糖的消耗速率依次为甘露醇>氯化钾>氯化钠。甘油和海藻糖是C.krusei在高渗环境下的主要相容性溶质。氯化钠和氯化钾对甘油合成的促进作用明显高于甘露醇。在0.6mol/L氯化钠、氯化钾、甘露醇存在时,细胞甘油浓度较对照提高了74%、63%、57%;胞内甘油最大含量也分别达到对照的3.1,2.4和1.8倍。高渗环境下胞内海藻糖含量在发酵前期均有所降低,但发酵后期在0.6mol/L氯化钾和甘露醇存在时海藻糖迅速积累,其含量分别达对照的1.6和1.4倍。     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.