pH对毕赤酵母表达重组人复合a干扰素的降解影响

使用Pichia pastoris表达重组人复合a干扰素(cIFN)会发生降解、聚合等不均一表达的现象。在5 L发酵罐中考察了不同诱导pH对cIFN表达产生降解的影响, 结果发现在适合酵母生长的pH 3.0~7.0范围内, 当诱导pH为4.0~5.0时, cIFN不均一表达现象最少, 生物活性达到2.5×108 IU/mL。通过测定发酵液中总蛋白酶活和细胞活性寻找了cIFN降解出现的原因：发现低诱导pH下细胞死亡率升高释放更多酶系, 高诱导pH下蛋白酶活性明显增大, 两者都使蛋白酶作用加强, 加剧cIFN的降解; 特别是诱导pH为7.0时, 适宜的pH使蛋白酶酶活陡升, 将cIFN完全降解。     

pH对毕赤酵母表达重组人复合α干扰素的降解影响

使用Pichiapastoris表达重组人复合α干扰素(cIFN)会发生降解、聚合等不均一表达的现象.在5 L发酵罐中考察了不同诱导pH对cIFN表达产生降解的影响,结果发现在适合酵母生长的pH 3.0～7.0范围内,当诱导pH为4.0～5.0时,cIFN不均一表达现象最少,生物活性达到2.5×108 IU/mL.通过测定发酵液中总蛋白酶活和细胞活性寻找了cIFN降解出现的原因:发现低诱导pH下细胞死亡率升高释放更多酶系,高诱导pH下蛋白酶活性明显增大,两者都使蛋白酶作用加强,加剧cIFN的降解;特别是诱导pH为7.0时,适宜的pH使蛋白酶酶活陡升,将cIFN完全降解.     

温度对巴斯德毕赤酵母表达瑞替普酶(reteplase)的影响

研究了温度对巴斯德毕赤酵母表达瑞替普酶(reteplase,rPA)的影响。结果发现,在BMMY摇瓶培养条件下,诱导温度在20℃、25℃,rPA表达量较高,分别是30℃诱导条件下(18.2mg/L)的1.4倍和1.34倍。在高密度发酵过程中,降低诱导温度(20℃、25℃),rPA的表达量在诱导表达84h时达到最高,分别为207.9mg/L和199.5mg/L,比30℃诱导条件下分别提高了35%和30%。通过对细胞活性、蛋白酶和AOX酶活性分析发现,降低温度不仅提高了发酵过程中酵母活细胞率,降低了蛋白酶的活性,减少了rPA的降解,而且提高了AOX酶活,增强了rPA的表达。     

褐藻胶裂解酶基因的克隆表达与酶学性质

随着大型褐藻生产燃料乙醇以及褐藻寡糖重大药用价值的发现,褐藻胶裂解酶成为国内外多个领域的研究重点。文中对解藻酸弧菌上与褐藻胶降解相关的5个基因分别进行克隆表达,通过SDS-PAGE和酶活性定量测定,发现该基因簇中的4个基因有降解褐藻胶活性。对酶活最高的rAlgV3进行了诱导条件的优化、酶蛋白纯化及酶性质研究,发现优化诱导条件后重组酶rAlgV3的酶活由2.34×10~4 U/L上升为1.68×10~5 U/L,比优化前提高了7.3倍;对酶性质进行表征发现该酶在4–70℃均有活性,最适反应温度为40℃,在4–20℃酶相对稳定;该酶在pH 6.5-9.0环境下均有较高的酶活,最适pH为8.0;pH稳定性好,在pH 4.5–9.5环境下可以稳定存在;适量的NaCl浓度和Fe~(2+)、Fe~(3+)等离子具有促进酶活的作用,SDS和Cu~(2+)离子可明显抑制酶活力。对该酶的底物特性的研究发现,该酶不仅可以降解褐藻胶中的Poly-M片段,也能降解Poly-G片段,具有广泛底物特性;其降解海藻酸钠主要释放二糖和三糖,是一种内切酶。该酶对于第三代燃料乙醇的发展及褐藻寡糖的生产具有重要作用。     

诱导相温度对毕赤酵母表达重组人复合α干扰素聚合的影响

研究了Pichia pastoris表达重组人复合α干扰素(cIFN)时诱导期温度对cIFN形成聚合体的影响。在5L罐上考察毕赤酵母在不同温度(30、25、20℃)下诱导时菌体生长和cIFN表达的差异,发现毕赤酵母在20℃下菌体生长不受影响,总蛋白表达量有所降低,相当于30℃发酵时胞外总蛋白的67.8%。但是通过非还原性电泳、Native-PAGE及Western blotting分析发现较高温度(30℃)诱导表达时,分泌到胞外的cIFN容易形成聚合体,单体含量很少。诱导相控制温度为20℃时,明显降低了cIFN聚合体的形成,cIFN单体量为570mg/L,发酵上清液抗病毒活性为1.05×109IU/mL。与控制诱导相温度为30℃相比,cIFN单体量提高了7.2倍,发酵上清液中单位体积的cIFN抗病毒活性提高了38.7倍。     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

邻单胞菌(Plesiomonas sp.90-1)降解直链烷基苯磺酸钠酶的诱导

在Plesiomonas sp.90-1中,降解直链烷基苯环酸钠(LAS)相关的酶为诱导酶.使用正交多因子法研究了细胞降解LAS酶活诱导的最佳条件为:细胞培养温度30℃,LAS诱导浓度10ppm,酵母膏0.008%,pH8.0,通气.在此条件下,LAS酶活比未经诱导者提高1.4倍.碳源的加入会阻遏LAS降解酶的活力形成.在所试氮源中,以(NH_4)H_2PO_4对LAS降解酶的形成最有利.低浓度的磷酸盐对LAS降解酶活形成无影响,但高浓度的磷酸盐对LAS降解酶活形成不利.利用微量检压技术发现经此条件诱导的细胞耗氧量上升2—3倍.     

芽孢杆菌α-淀粉酶基因的克隆、表达和酶学性质分析

在仔猪结肠内容物中分离出一株能利用淀粉的芽孢杆菌Bacillussp.WS06,构建了全基因组DNA文库,从中筛选出α_淀粉酶基因amyF,分析测定了其核苷酸序列并进行了表达;其中amyF编码的蛋白有526个氨基酸、分子量为58.6kD;它与已报道的Bacillusmegaterium的α_淀粉酶序列有93%的同源性。经过氨基酸序列比较分析还发现,AmyF含有淀粉酶家族中4个高度保守的酶催化活性区。经多步纯化,重组酶的比活共提高了22.2倍,获得凝胶电泳均一的蛋白样品;经SDS_PAGE检测,AmyF酶分子量为57kD。该酶的最适反应温度为55℃～60℃,酶的最适反应pH为7.0,在温度不超过55℃时,酶活较稳定;AmyF能迅速降解淀粉生成麦芽寡糖,属于内切糖苷酶。     

甲醇浓度对毕赤酵母表达重组人复合α干扰素分离纯化得率的影响

研究了毕赤酵母Pichia pastoris表达的重组人复合α干扰素(cIFN)时不同诱导甲醇浓度对cIFN分离纯化得率的影响,并分析了原因.在5L罐中采用0.25、0.50和0.75％(W/V)三个甲醇浓度诱导时,在0.75％高甲醇浓度诱导下cIFN表达水平最高,达到2.06 g/L,是0.25％低甲醇浓度诱导的1.24倍,但是低甲醇浓度诱导下cIFN分离纯化得率却高于高甲醇诱导浓度下3.75倍.另外,低甲醇浓度下发酵上清液cIFN抗病毒活性为2.85×108IU/mL,较高甲醇浓度提高了4.48倍.进一步采用SDS-PAGE和Native-PAGE免疫印迹分析不同条件下发酵液中cIFN存在状态,发现在高甲醇浓度下cIFN容易形成大量的聚合体,分别为共价聚合和非共价聚合,而cIFN单体含量较少,但是低甲醇浓度诱导下情况完全相反.最终在0.25％甲醇诱导下分离纯化1L发酵上清液可得0.73 g单体cIFN,是0.75％甲醇诱导下的3.84倍.     

巨大芽孢杆菌β-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆(Bacillus megaterium)的全基因组DNA文库中筛选出一个β-淀粉酶基因amyG,分析测定了其核苷酸序列并进行了诱导表达;其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD,与已报道的巨大芽孢杆菌DSM319的β-淀粉酶序列有着94.5%的同源性.经氨基酸序列比较分析发现,AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成.其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区.经多步纯化,重组酶的比活共提高了7.4倍,获得凝胶电泳均一的蛋白样品;经SDS-PAGE电泳测定,酶AmyG的分子量为57 kD.该酶的最适反应温度为60℃,最适反应pH为7.0;在温度不超过60℃时,酶活较稳定:AmyG能迅速降解淀粉生成麦芽糖,属于外切β-糖苷酶.     

Bioprocessing strategies for improving hen egg-white lysozyme (HEWL) production by recombinant Aspergillus niger HEWL WT-13-16

Hen egg-white lysozyme (HEWL) production by recombinant Aspergillus niger HEWL WT-13-16 from a cDNA under the control of the A. niger glucoamylase promoter was used as a model system. The fungal mycelium was either immobilized on porous Celite 560 micro-carrier or grown in suspension as pelleted and dispersed forms. The objective was to reduce the protease activity that adversely affects the expressed HEWL. Free suspension culture at uncontrolled pH served as the benchmark. The control of pH during growth at pH 4.0 gave rise to a greater than five-fold reduction of protease activity in suspension culture. An additional 38.5% decrease in protease activity was achieved in mycelial-pellet cultures in comparison to a 40.9% decrease in protease activity obtained with Celite 560 beads in an airlift vessel at controlled pH. The specific HEWL yields were 5.8, 5.0 and 4.1 mg/g dry wt. for the free suspension, mycelial-pellet, and Celite-560-immobilized cultures, respectively.     

The inhibition of aggregation of recombinant human consensus interferon-α mutant during <Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentation

Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation of recombinant human consensus interferon-α mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20°C, the cIFN secreted into the medium was in the form of monomers instead of aggregates. The maximum specific activity at 20°C was 4.04 times as high as that at 30°C. There was no obvious effect on the cell growth at 20°C, but the total protein level was decreased. Similar inhibition effect on cIFN aggregation was observed when 0.2 g l⁻¹ Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20°C was 19.9-fold higher than that without Tween-20 at 30°C.     

Incomplete formation of intramolecular disulfide bond triggers degradation and aggregation of human consensus interferon-α mutant by Pichia pastoris

Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-α mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of β-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.     

Polygalacturonase-Mediated Solubilization and Depolymerization of Pectic Polymers in Tomato Fruit Cell Walls : Regulation by pH and Ionic Conditions

The hydrolysis of cell wall pectins by tomato (Lycopersicon esculentum) polygalacturonase (PG) in vitro is more extensive than the degradation affecting these polymers during ripening. We examined the hydrolysis of polygalacturonic acid and cell walls by PG isozyme 2 (PG2) under conditions widely adopted in the literature (pH 4.5 and containing Na⁺) and under conditions approximating the apoplastic environment of tomato fruit (pH 6.0 and K⁺ as the predominate cation). The pH optima for PG2 in the presence of K⁺ were 1.5 and 0.5 units higher for the hydrolysis of polygalacturonic acid and cell walls, respectively, compared with activity in the presence of Na⁺. Increasing K⁺ concentration stimulated pectin solubilization at pH 4.5 but had little influence at pH 6.0. Pectin depolymerization by PG2 was extensive at pH values from 4.0 to 5.0 and was further enhanced at high K⁺ levels. Oligomers were abundant products in in vitro reactions at pH 4.0 to 5.0, decreased sharply at pH 5.5, and were negligible at pH 6.0. EDTA stimulated PG-mediated pectin solubilization at pH 6.0 but did not promote oligomer production. Ca²⁺ suppressed PG-mediated pectin release at pH 4.5 yet had minimal influence on the proportional recovery of oligomers. Extensive pectin breakdown in processed tomato might be explained in part by cation- and low-pH-induced stimulation of PG and other wall-associated enzymes.     

Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris

Previous report has shown that the expression of recombinant human consensus interferon-α mutant (cIFN) in Pichia pastoris in bioreactor is limited with respect to the incorrectly folded cIFN with incomplete disulfide bond, which lead to the degradation and aggregation of cIFN. In this study, the origin of incorrectly folded cIFN is firstly studied. Fed-batch fermentation in bioreactor shows that the incorrectly folded cIFN is formed intramolecularly and secreted to the extracellular environment. Further chemostat cultures indicate that the specific growth rate is the critical factor for the production of incorrect cIFN. In addition, cell shows reduced expression level of cIFN at high specific growth rate. We also demonstrate that the incorrectly folded cIFN could form aggregates intracellularly and these aggregates are non-covalent forms. Taken together, these results suggest that the efficient heterologous expression of cIFN is limited by high cell growth that is unique from expression limitations seen for soluble proteins. A balance has to be found between the increase for high efficient expression of heterologous proteins and requirement of the high cell growth during the expression of recombinant proteins in P. pastoris.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

Yarrowia (Candida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica, designated CL1 and CL2, with casein as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50 degrees C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

R.E. LEVIN AND R. WITKOWSKI. 1991. Yarrowia (Canadida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica , designated CL1 and CL2, with casin as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50.C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0     

Degradation of Zein during Germination of Corn

The degradation of zein during germination of corn was investigated in special relevance to protease activity, Polyacrylamide gel electrophoresis demonstrated that two main subunits of zein were rapidly degraded as the germination proceeded. It was observed that the zein degradation was followed by the formation of free amino acids, especially phenylalanine and tyrosine. A protease activity increased remarkably during the germination. A crude extract was obtained which could degrade zein in vitro to form relatively large amounts of free phenylalanine and tyrosine. The crude extract showed a maximum activity for casein at pH 3 and for zein at pH 4.5. The activity was enhanced when determined in the presence of 2-mercapto- ethanol. A sulfhydryl protease was estimated to occur which might be responsible for the degradation of zein during germination.