Production of recombinant protein by baculovirus-infected insect cells in immobilized culture using porous biomass support particles

Immobilization of insect cells using porous biomass support particles (BSPs) and production of a recombinant protein by the immobilized cells after infection with a baculovirus were investigated in a shake-flask culture. Sf9 cells were passively immobilized in reticulated polyvinyl formal (PVF) resin BSPs (2 x 2 x 2 mm cubes) with matrices of 60 mum mean pore diameter in situ in shake-flasks. The cell density in the BSPs was over 5 x 10(7) cells/cm3-BSP in cultures with regular replacement of the culture medium, as estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After infection with a recombinant baculovirus carrying the beta-galactosidase gene, immobilized cells within the BSPs showed a high specific productivity, comparable to the maximum productivity in shake-flask cultures of non-immobilized cells, as long as nutrients in the medium were not depleted. Even when immobilized cells at a high density of 5 x 10(7) cells/cm3-BSP were infected with the baculovirus, efficient beta-galactosidase production with a high specific productivity was possible by replacing the medium at appropriate intervals to avoid nutrient depletion.     

Immobilization of Escherichia coli cells using porous support particles coated with cationic polymers

The technique of cell immobilization using porous biomass support particles (BSPs), which is attractive from the point of view of simplicity and convenience, relies on the inherent ability of adhesive cells, as a consequence of their growth, to form films around the support material or the ability of flocculent cells to create flocs within the porous structure. In the present study, the immobilization of Escherichia coli cells using BSPs was investigated in shake-flask culture. The density of the cells immobilized within the BSPs was evaluated by measuring their intracellular lactate dehydrogenase (LDH) activity. Since E. coli K12 cells were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs with matrices of relatively small pores (pore diameter 60 microm), coating the surface of the BSPs with various polymers was examined as a way of promoting cell attachment. When positively charged polyamino acids such as poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly-L-ornithine were adsorbed onto the particle surface, they were found to increase the immobilized cell density, while neutral and negatively charged polyamino acids including poly-L-asparagine and poly-L-glutamic acid were not effective. These results indicate that E. coli cells can be efficiently immobilized in PVF resin BSPs by electrostatic interaction between the negatively charged ions of the cell surface and the positively charged polymers adsorbed onto the BSP surface. A significantly high immobilized cell density was also achieved by coating the surface of the BSPs with the synthetic polymeric amine polyethyleneimine.     

Kefir Immobilized on Corn Grains as Biocatalyst for Lactic Acid Fermentation and Sourdough Bread Making

Abstract: The natural mixed culture kefir was immobilized on boiled corn grains to produce an efficient biocatalyst for lactic acid fermentation with direct applications in food production, such as sourdough bread making. The immobilized biocatalyst was initially evaluated for its efficiency for lactic acid production by fermentation of cheese whey at various temperatures. The immobilized cells increased the fermentation rate and enhanced lactic acid production compared to free kefir cells. Maximum lactic acid yield (68.8 g/100 g) and lactic acid productivity (12.6 g/L per day) were obtained during fermentation by immobilized cells at 37 °C. The immobilized biocatalyst was then assessed as culture for sourdough bread making. The produced sourdough breads had satisfactory specific loaf volumes and good sensory characteristics. Specifically, bread made by addition of 60% w/w sourdough containing kefir immobilized on corn was more resistant regarding mould spoilage (appearance during the 11^th day), probably due to higher lactic acid produced (2.86 g/Kg of bread) compared to the control samples. The sourdough breads made with the immobilized biocatalyst had aroma profiles similar to that of the control samples as shown by headspace SPME GC‐MS analysis.     

某些化学添加剂在酵母发酵中的作用

<正> 在酒精发酵过程中,对发酵系统加入少量的不饱和脂肪酸或固醇类物质,可以提高发酵效率,这在过去已有报道,1983年,日本长岛实等研究了某些化学添加剂在固定化酵母酒精发酵过程中对发酵效率的影响。以此为基础,本研究试图找到对提高发酵率较有效的化学添加剂配方—添加剂种类、用量及组成比例。研究了一些来源较广,对发酵有效的     

固定化耶氏酵母提高产γ-癸内酯能力

通过添加凹凸棒石粘土增加了固定化细胞凝胶珠的机械强度,同时疏松了凝胶网格,增加了传质性能,提高了固定化细胞发酵生产γ-癸内酯的能力。其固定细胞制备工艺为:将2%海藻酸钠复合2%凹凸棒石粘土,与9g/100mL耶氏酵母悬液等体积混匀,氯化钙浓度为2%,温度30℃,固化pH为7,时间为4h,制得固定化酵母细胞。发酵条件为:在250mL的三角瓶中装30mL发酵培养基,摇床转速为150r/min,于26℃发酵48h。以此方法生产GDL产量可达4.17g/L,是游离细胞的2.5倍。该固定化细胞重复使用3次后,GDL产量仍可达3.87g/L。     

酿酒酵母发酵生产谷胱甘肽的研究

以诱变获得的酿酒酵母突变株YF(ZnCl2r,Ethr)为试验菌株,通过摇瓶发酵、发酵罐补料分批发酵,对突变株发酵生产谷胱甘肽进行研究.确定发酵罐分批发酵的最佳培养条件为:温度30℃,pH 6.0,接种量为20%,搅拌转速为150 r/min,通气量为250 L/h.在补料分批操作方式下,分别考察了摇瓶培养、发酵罐培养...     

聚氨酯泡沫固定化米根霉发酵L-乳酸的研究

对软质聚氨酯泡沫吸附固定化米根霉发酵生产L-乳酸进行了研究，确定了种子培养方式。与单纯固定化发酵相比，采用游离茵丝方式进行种子培养，然后在发酵培养基中添加适量软质聚氨酯泡沫的固定化发酵方式，发挥了固定化发酵和自聚集形成小茵球发酵的双重优点，产酸量得到提高。     

Calcium alginate-immobilized cultures of lactic Streptococci are protected from bacteriophages

Calcium alginate-immobilized cultures of lactic streptococci were grown in milk and assessed for their sensitivity to homologous bacteriophage, proteolytic activity, and acid production. Immobilized cultures of Streptococcus lactis C2 and Streptococcus cremoris HP were protected from attack by bacteriophage due to the exclusion of phage particles from the gel matrix. These cultures were also functionally proteinase-deficient when immobilized in calcium alginate beads and grown in milk. Acid was produced by immobilized cultures at a lower rate than cells freely suspended in milk due in part to the inability of the immobilized cells to hydrolyze milk proteins. Agitation of immobilized cultures slightly increased acid production, suggesting that diffusional limitations of substrate into the beads contributed to decreased acid production. Use of immobilized cultures of lactic streptococci in certain dairy fermentations may be advantageous due to the protection of these cultures from attack by virulent bacteriophage.     

ZIF-8原位合成法固定化脂肪酶CRL

以ZIF-8为固定化载体,通过原位合成法固定褶皱假丝酵母脂肪酶(CRL),研究了不同固定化条件对固定化酶活力的影响以及固定化酶的稳定性,并对固定化酶进行了结构表征。结果表明:在10 m L 40 mmol/L硝酸锌溶液、10 m L 160 mmol/L 2-甲基咪唑溶液、2-甲基咪唑溶液初始p H8、酶添加量2.75 mg、-48℃冷冻干燥条件下,获得的固定化酶CRL@ZIF-8蛋白回收率为93%,比活力为1.08 U/mg;底物的分子大小对固定化酶的催化性能有明显影响,在以相对小分子的乙酸对硝基苯酯为底物时,重复使用9次后固定化酶仍保持72.8%的相对活力;通过对固定化酶进行场发射扫描电镜和氮气吸附脱附表征,表明脂肪酶CRL在载体表面和内部均有分布,固定化酶颗粒粒径约400 nm。     

高产谷胱甘肽酵母菌株的选育

以BY-14面包酵母（Saccharomyces cerevisiae）为出发菌株,通过紫外线和氮离子注入复合诱变,氯化锌、乙硫氨酸耐性平板筛选,获得一株高产谷胱甘肽的面包酵母优良菌株（S．cerevisiae）BL-23。该菌株经摇瓶发酵谷胱甘肽产量为113．46mg／L,较出发菌株提高62％,每1g干细胞含谷胱甘肽20．86mg,较出发菌株提高56％。传代培养结果表明,该菌株具有稳定的遗传性能。     

纤维素载体固定化β-葡萄糖苷酶及其在大豆异黄酮水解中的应用

建立纤维素载体固定化β-葡萄糖苷酶系统,用于黑豆浆中大豆异黄酮去糖基化反应,并对该催化系统进行了可行性评价。结果表明,在40 cm3纤维素颗粒上可结合超过40 mgβ-葡萄糖苷酶,并且能有效将4-硝基苯-β-D-葡糖苷酸水解成对硝基苯酚,其反应最适温度为50℃。经动力学测试后可得其米氏常数Km值为(1.38±0.20)mmol/L。将该固定化酶系统用于黑豆浆中大豆异黄酮去糖基化,通过高效液相色谱检测其转化效率,表明该系统可以在30 min内利用40 cm~3含酶载体将50 mL黑豆浆中的异黄酮全部转化为去糖基化形式。该固定化酶催化系统连续化催化稳定性实验表明,在经过15次反应以及15 d之后,该固定化酶系统仍然可维持其60%的催化活性。     

仿生硅化固定化葡萄糖氧化酶与辣根过氧化酶研究

采用分步法将仿生硅化与脂质体技术相结合,模拟细胞微结构,以卵磷脂为原料,制备脂质体微囊,以胺类为诱导剂,在其表面硅化形成氧化硅壳层,实现对葡萄糖氧化酶（GOx）与辣根过氧化酶（HRP）双酶固定。研究了超声时间、诱导剂PDADMA用量、硅前驱体水解液（TMOS）用量、Triton X-100浓度等因素对固定化酶活性影响。结果表明,在200μL PDADMA、0.7 m L TMOS、超声50 min制得的固定化酶活性最高,能迅速硅化形成直径200 nm左右的球形微囊。在此优化条件下制备的固定化酶经重复使用7次,依然达到初始催化酶活的61.46%,经一个月冷藏后酶活仍能达到最初的74.1%。可见,经过固定化,双酶系统的稳定性得到了显著提高。      

高产油脂酵母菌株的筛选

将20份土样经平板初筛,摇瓶初筛、复筛,筛选得到了6株产油脂量高的酵母菌株,其中一株的产油脂量已达到4．44g／L。     

水相体系中固定化Acetobacter sp.CCTCC M209061细胞催化(S)-3-氯苯丙醇不对称合成

光学纯的3-氯苯丙醇是合成抗抑郁类药物的重要中间体。本论文报道了水相体系中固定化Acetobacter sp.CCTCC M209061细胞高效、高选择性地催化3-氯苯丙酮不对称还原为(S)-3-氯苯丙醇,采用聚乙烯醇与海藻酸钠的混合载体对醋酸杆菌细胞进行固定化,所得的固定化细胞的稳定性(热稳定性、p H稳定性及操作稳定性)均明显优于游离细胞。同时,固定化后的微生物细胞具有较好的重复利用性,连续反应3个批次后固定化细胞仍保留了80.0%以上的活性,而游离细胞的相对活性则小于20%。在所研究的体系中,葡萄糖为该反应的最佳辅底物,其最适浓度为50.0 mmol/L;该反应体系中的最适缓冲液p H值、反应温度、底物浓度分别为5.5、30℃和3.0 mmol/L。在此条件下,反应的初速度、产率以及产物的e.e.值依次为1.77 m M/h,88.9%和99.0%以上。     

Effect of Lactococcus lactis Immobilized Within Pineapple and Yam Bean Segments, and Jerusalem Artichoke Powder on Its Viability and Quality of Yogurt

Lactococcus lactis cells were immobilized within pineapple segments, yam bean segments, and Jerusalem artichoke (JA) powder, and immobilized cells were used separately as adjuncts in producing probiotic yogurt. In parallel, yogurt with free L. lactis cells and yogurt only from starter cultures were also produced. The resulting yogurt samples were stored at 4 °C. Immobilization of cells increased the viability of L. lactis cells compared to free cells during storage of yogurt within pineapple segments, JA powder, yam bean segments, and free cells (43.77 %, 63.62 %, 80.11 %, and 87.14 %, respectively). The pH values of all yogurt samples decreased during storage; however, the pH values of yogurt supplemented with immobilized cells were higher than samples with free L. lactis cells. The increase in lactic acid content during storage was not different among the yogurt samples with added immobilized cells within segments of pineapple, yam beans, and free cells. However, the lactic acid content increase was greater with samples containing immobilized cells within JA powder. The immobilized cells within pineapple segments resulted in a decrease in b* color values (indicating yellowness) and an increase in a* color values (indicating greenness) whereas immobilized cells within yam bean segments resulted in a decrease in b* color values. Immobilized cells within JA powder resulted in a decrease in L* color values (indicating lightness) and an decrease in a* color values when compared to free cells. During storage, the concentration of γ-aminobutyric acid had a tendency to increase, which was not statistically significant. The sensory test revealed that the overall acceptance scores of yogurt with immobilized cells added were quite similar to those of the samples with free cells and controls throughout the storage period of 28 days.     

无机盐对新型固定化酵母蔗汁酒精发酵的影响

以甘蔗作酒精酵母的固定化载体,以蔗汁为发酵培养基进行酒精发酵,通过电镜观察,酒精酵母不仅能吸附在甘蔗外表面,同时也能较好地吸附在甘蔗内部,比较固定化酵母与游离酵母数,发现固定化酵母数达到1011个/g,而游离细胞数只有107个/mL.通过考察不同浓度的CaCl2、MgSO4和KH2PO4对酵母酒精发酵性能的影响,发现1.0 g/L CaCl2和1.5 g/L KH2PO4可在最短时间内使二氧化碳生成速率达到最大值;而KH2PO4可明显地提高酒精发酵效率.     

添加液态烷烃氧载体对法夫酵母发酵生产虾青素的影响

在法夫酵母培养过程中,氧的供给是细胞生长和虾青素合成的重要因素。试验中测得法夫酵母摇瓶培养时比耗氧速率(qp)为60mg(O2)/(g.h)(cell),临界溶氧浓度10%。试验还考察了多种液态烷烃作为氧载体对法夫酵母生长和类胡萝卜素合成的影响,发现正十六烷作为氧载体效果较好,其最佳条件是在法夫酵母接种同时添加10%(v/v)的正十六烷。在此条件下,细胞干重达到10.1g/L,类胡萝卜素产量13.2mg/L,分别比对照增加22%和43%。同时,单位细胞类胡萝卜素产量从1.11mg/g增加到1.31mg/g,合成能力提高了18%。     

固定化酵母菌和醋酸杆菌发酵食醋工艺研究

用海藻酸钠包埋酵母菌，滴入1％～9％的钙离子液固化，固定时间4h，连续酒精发酵凝胶颗粒的完整性和弹性尚好。用2．5％的海藻酸纳与4％的PVA混合作醋酸杆菌包埋剂，滴入2％～4％的钙离子硼酸液制备凝胶球，固化9h～10h，成型性和机械强度很好。固定化醋酸杆菌分批发酵50h，产醋酸达4．06g／100mL，重复使用25d，固定化细胞保持高的活力。     

培养条件对黑曲霉脂肪酶选择性合成1,3甘油二酯的影响

从前期筛菌工作中得到的一株能高选择性合成1,3-甘油二酯（1,3-DAG）的产胞内脂肪酶的黑曲霉GZUF36菌株出发,分别研究了培养时间、温度、接种量、装液量和转速对该菌摇瓶发酵的影响,得出合适的培养条件为培养时间72 h,接种量3%,装液量40 mL/250 mL,培养温度28 ℃,转速180 r/min,在此条件下1,3-DAG的得率和选择性分别为23.87%和82.96%。同时也比较了摇瓶发酵和5 L发酵罐发酵的异同,相比于摇瓶发酵,5 L发酵罐发酵的1,3-DAG的得率有所提高,但是发酵罐发酵的时间有所延长。     

Fermentation of banana media by using kappa-carrageenan immobilized Lactobacillus acidophilus

Fermentation media were prepared by using banana as the raw material, and cell immobilization of Lactobacillus acidophilus by kappa-carrageenan entrapment was applied to enhance the fermentation efficiency. Gel beads of diameters around 3.0 mm were prepared for the immobilized cells, ripe bananas were used for preparation of media, and both free and immobilized cells were employed to carry out the fermentation for 80 h. Cells leaked out from the gel beads and proliferated in the medium solution during the fermentation of immobilized cells. The final viable cell number reached 10(5) CFU/ml in the medium suspension, over 10(8) CFU/(ml gel) in gel beads for the immobilized cell fermentation and around 10(6) CFU/ml for the free cell fermentation. Immobilized cells withstand the adverse conditions in banana media resulting in better fermentation efficiency compared to free cells. Variation of fructooligosaccharides (FOS) in banana media was not significant in immobilized cell fermentation compared to free cell. Immobilized L. acidophilus fermented banana medium was found to possess synbiotic properties.