argE基因在大肠杆菌中的表达优化

N-乙酰鸟氨酸脱酰基酶可在重组菌BL21（DE3）-pET22b-argE中表达。首先确定了该酶的细胞表达定位,再研究了诱导温度、诱导剂种类及浓度、诱导起始菌体密度、诱导时间等因素对重组菌生长及目的蛋白表达活性的影响。结果表明,IPTG和乳糖皆可诱导目的蛋白表达,乳糖的诱导效果优于IPTG。在诱导起始0D600为0．46时加入15g／L乳糖,20℃诱导18h最适于目的蛋白的活性表达。表达条件优化后,酶活从1．68U／mL提高至282．99U／mL,约为原来的168倍。     

海藻糖生产过程中产酶发酵条件的研究

研究了产酶的培养基组分和比例以及最佳培养条件对微球菌生产麦芽寡糖基海藻糖合成酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶(MTHase)的影响,得到最优培养基组成为：葡萄糖2．0％,酵母膏2．0％,蛋白胨1．0％,磷酸氢二钾0．1％,硫酸镁0．05％;优化后的培养条件为：以15％的接种量接种至250mL的锥形瓶中,装液量为50mL,初始pH值7．5～8．5,培养温度为30℃,摇床培养4d。经优化后菌体干重由原来的1．938g／L增加到18．5g／L,生物量几乎增长了10倍;而酶活也由原来的30．64U／g增加到206．11U／g,酶活提高了接近7倍。     

2,5-DKG还原酶Ⅰ的分离纯化与性质研究

欧文氏菌ER97高效表达了从棒状杆菌SCB3058克隆的2,5-二酮基-D-葡萄糖酸（2,5-DKG）还原酶Ⅰ基因,5L罐发酵后,收集菌体破碎,将胞内可溶性的蛋白通过硫酸铵分级沉淀、DEAE—Sepharose CL-6B离子交换柱层析和Phenyl Sepharose CL-4B疏水柱层析后分离纯化到了2,5-DKG还原酶Ⅰ,纯化了5倍,得率27％,比活力为3,418U／mg。测定了该酶的一些特性参数：分子量为34kD,等电点为6．0,它以NADPH为辅酶,将2,5-DKG还原为2-酮基-L-古龙酸（2-KLG）,对NADPH和2,5-DKG底物的Km值分别是0．29mmol／L和14．7mmol／L,1mmol／L Cu^2＋、Zn^2＋等有强烈抑制作用,EDTA和巯基乙醇对该酶没有抑制作用,酶的最适pH为7．0,最适反应温度为40℃。     

真空微波诱变结合化学诱变选育酸性纤维素酶高产菌株

以酸性纤维素酶产生菌绿色木霉（Trichoderma viride）WL0512作为原始出发菌株,首先经自然分离筛选出一株产酶较稳定的菌株TVN-18,其羧甲基纤维素酶活（CMC酶活）达2765．8U／g,滤纸酶活（FPA酶活）达48．5U／g。再经真空微波和甲基磺酸乙酯（EMS）逐级诱变处理,获得了一株高产、稳产酸性纤维素酶的E6—1菌株,其CMC酶活达4396．6U／g,FPA酶活达126．0U／g,分别是菌株TVN-18的1．59倍和2．60倍。通过对固态发酵培养基麸皮和稻草比例、料水比以及初始pH值的优化,突变株的产酶能力进一步得到提高,其产的CIVIC酶活和FPA酶活分别提高了22．3％和22．4％。     

利用重组大肠杆菌生产α-环糊精葡萄糖基转移酶

将来源于软化类芽孢杆菌（Paenibacillus macerans）的α-环糊精葡萄糖基转移酶（α-CGT）基因插入含pelB信号肽的质粒pET-20b（＋）中,构建了表达载体pET-20b（＋）／cgt,并将其转化表达宿主E.coli BL21（DE3）。对重组菌E．coli BL21／pET-cgt进行摇瓶发酵条件的优化,确定了其胞外表达α-CGT酶的最适条件：葡萄糖8g/L,乳糖0．5g/L,蛋白胨12g/L,酵母膏24g/L,K2HPO472mmol／L,KH2PO417mmol／L,CaCl2 2．5mmol／L;初始pH为7．0,诱导温度为25℃。在该条件下培养90h后最终α-CGT酶的胞外比活达到22．1u／mL,与来源菌Pmacerans所产天然酶比活相比提高了42倍,实现了α-CGT酶的高效生产。将基因工程菌在上述条件下于3L发酵罐中发酵,90h后胞外酶比活达到22．6U／mL,证实了工业化放大的可能性。     

重组人胰激肽原酶复性工艺的研究

目的:研究重组人胰激肽原酶包涵体变性及复性的工艺。方法:对本实验室构建的重组人胰激肽原酶大肠杆菌进行IPTG诱导表达表达成功后,菌体经超声破碎释放包涵体,包涵体经洗涤、变性、稀释和尿素梯度凝胶过滤色谱这两种方法复性后(Sephadex-G75),通过测定酶活检验复性效果。结果:①重组人胰激肽原酶工程菌经过IPTG诱导后能够表达目的蛋白,目的蛋白以包涵体形式存在,将细胞破碎后,包涵体经过3次洗涤,纯度达到71.93%;②变性包涵体经24小时稀释复性后,蛋白浓度达到72.61μg/m L,酶的比活达到13.84 U/mg;③变性包涵体经过2个小时的尿素梯度凝胶过滤复性后,蛋白浓度可达到830.07μg/mL,酶的比活达到48.61 U/mg。结论:两种复性方法均可以使包涵体达到一定的浓度和比活,比较发现尿素梯度凝胶过滤色谱具有复性时间短和比活力高等优点,可作为重组人胰激肽原酶复性的一种有效的手段。     

粪产碱杆菌青霉素G酰化酶的分离提取

比较研究了几种破碎大肠杆菌细胞的方法,如渗透压法、超声波法、玻珠震碎法、玻珠研磨法、有机溶剂法、冻融法以及盐酸胍/EDTA法等,以确定出一种简单、快速、高效的破碎重组大肠杆菌细胞的方法获得粪产碱杆菌青霉素G酰化酶(AfPGA)用于后续试验。结果表明玻珠震碎法、超声波法和渗透压法是较优的细胞破碎方法,活力回收率分别为99.7%、78.4%、60.7%,其他方法均低于22%。而比活力以渗透压法为最高,达到4.40 U/mg。     

刺孢小克银汉霉菌体氨基酰化酶的性质研究

首次选育出有较高氨基酰化酶活性的菌株刺孢小克银汉霉（Cunninghamella echinulata）9980,并进行液体培养,比较了3种不同培养基中菌体细胞氨基酰化酶活性,考察了几种因素对菌体细胞酶活的影响。结果表明：蛋白胨培养基中菌体细胞酶活最高,达680u／g。菌体细胞酶活最适温度55％,最适pH7．0,最佳底物浓度为0．2mol／L,缓冲液中的无机离子对酶活有抑制作用,10^-3-10^-4mol／L的Co^2＋对酶活有激活作用。     

黑曲霉固态发酵生产单宁酶的条件优化

研究采用响应面法优化黑曲霉固态发酵生产单宁酶的培养条件。应用Plackett—Burman试验筛选出重要影响因子：五倍子粉含量、（NH4）2SO4浓度以及接种孢子量,最陡爬坡试验逼近最大响应区域。应用Box．Behnken响应面试验对重要影响因子进一步优化。得到最佳培养条件：每250mL三角瓶中装入1．0g五倍子粉、4．4g稻壳和0．5g麸皮、液固比（mL／g）2：1且营养盐溶液组成为（NH4）2s0421g/L、MgSO4·7H2O1g／L、NaCl1g／L,培养基pH自然,接种5．7×10^7个孢子后在30℃温度下培养4d。在此条件下,单宁酶产量从40U／g提高到114U／g,3次重复验证性试验平均值为115U／g,验证了模型的可靠性。     

产乙醇酸氧化酶的重组毕赤酵母的构建及催化合成乙醛酸的研究

乙醇酸氧化酶（Go）是植物光呼吸途径中的一种关键酶,可以催化乙醇酸生产乙醛酸。从新鲜菠菜叶中提取总RNA,利用RT-PCR技术获得编码GO基因的cDNA片断。通过基因重组将GO基因克隆到载体pA0815中,构建了胞内表达载体pA0815／GO,重组质粒经电转整合至甲醇营养酵母GS115染色体。在混合碳源为10g/L山梨醇和0．5g/L甲醇的培养条件下,细胞的GO酶活达到474IU／g（DCW）。利用该重组毕赤酵母作为催化剂生产乙醛酸,结果表明：在乙醇酸浓度为0．25mol／L,重组酵母湿菌体为10dL,黄素单核苷酸（FMN）浓度为0．01mmol／L,pH8．0,20℃,反应18h后乙醛酸的产率达到51．8％。     

Effect of osmotic shock and shearing forces on ferric enterochelin transport in Escherichia coli K12

The fes mutation in Escherichia coli K12, which inactivates enterochelin esterase, allows the cell to accumulate ferric enterochelin. The ferric complex of enterochelin was released in significant quantities from a fes mutant after osmotic shock. Analysis of the effects of the individual stages of the shock procedure in wild-type cells showed that prior exposure of cells to sucrose and EDTA was not required, careful dilution of cells into a hypo-osmolar medium being sufficient to induce efflux of Fe3+. Prior treatment with EDTA or exposure to shearing forces served either to enhance efflux or to induce efflux in isotonic media. Neither vitamin B12 nor 5'-nucleotidase was released from the periplasm by these procedures. The release observed under mild conditions was stimulated specifically by Co2+, did not occur at 0 degree C, and was inhibited by 2,4-dinitrophenol at 37 degrees C. From these observations, it was concluded that the efflux of Fe3+ represents a physiological response of the cell to exposure to a hypo-osmolar medium. Such changes may enhance survival following physicochemical stressing of the bacterial outer membrane.     

Either periplasmic tethering or protease resistance is sufficient to allow a SodC to protect Salmonella enterica serovar Typhimurium from phagocytic superoxide

Salmonella Typhimurium combats phagocytic superoxide by producing the periplasmic superoxide dismutase, SodCI. The homologous protein, SodCII, is also produced during infection, but does not contribute to virulence. The proteins physically differ in that SodCI is dimeric, protease resistant and non-covalently tethered within the periplasm. Conversely, SodCII is a protease-sensitive monomer that is released normally from the periplasm by osmotic shock. To identify which properties correlate with virulence, we constructed over 20 enzymatically functional hybrid SodC proteins and assayed them for protease susceptibility, release by osmotic shock, multimerization and affinity for metal cofactors. Protease susceptibility maps to the C-terminus of SodCII, while SodCI residues 120-131 are required for tethering. A protease-resistant SodCII hybrid was able to substitute for SodCI during infection. Interestingly, a tethered but protease-sensitive SodCII hybrid was also able to confer protection. Thus, either tethering or protease resistance is sufficient for a SodC to function during infection. These results support our model that in the macrophage, the outer membrane of Salmonella is partially disrupted by antimicrobial peptides. Periplasmic proteins, including SodCII, are released and/or phagocytic proteases gain access. SodCI is both tethered within the periplasm and protease resistant, thereby surviving to detoxify superoxide.     

Relation of R Factor and Chromosomal β-Lactamase with the Periplasmic Space

The release of several R factor and chromosomal beta-lactamases by osmotic shock treatment was studied. It was found that those beta-lactamases with a molecular weight of about 20,000 were released, but those with a molecular weight of about 30,000 to 44,000 were not released during osmotic shock. This differential release did not depend on whether the structural genes were on the chromosome or on the genome of an R factor. The release or retention of the beta-lactamases appeared to be a characteristic of the enzyme rather than the host cell since the same results were obtained when the R factors were harbored by a variety of host bacteria. Studies with bacteria which produced more than one beta-lactamase showed that each enzyme reacted independently to the presence of other beta-lactamases produced by the host bacterium.     

Anchor-chain molecular system for orientation control in enzyme immobilization

An anchor-chain molecular system was constructed for controlled orientation and high activity in enzyme immobilization. A streptavidin recognition peptide (streptag) coding sequence was fused to the 3' end of the phoA gene, which codes for E. coli alkaline phosphatase (EAP). Both the wild-type (WT) and the Asp-101 --> Ser (D1O1S) mutant were modified with the streptag sequence with or without the insertion of a flexible linker peptide [-(Gly-Ser)(5)-] coding sequence. The fused genes were cloned into the vector pASK75 and expressed in the periplasm of the host cell Escherichia coli SM547. The proteins were released by osmotic shock and purified by ion-exchange chromatography. Enzyme activities of all proteins were measured spectrophotometrically with rho-nitrophenyl phosphate as the substrate. Specific activities of D101S-streptag and D101S-linker-streptag enzymes were increased 25- or 34-fold over the WT, respectively. These fusion proteins were then immobilized on microtiter plates through streptag-streptavidin binding reaction. After immobilization, the D101S-linker-streptag enzyme displayed the highest residual activity and the ratio of enzyme activities of the linker to nonlinker enzymes was 8.4. These results show that the addition of a linker peptide provides a spacer so as to minimize steric hindrance between the enzyme and streptavidin. The method provides a solution for controlled enzyme immobilization with high recover activity, which is especially important in construction of biosensors, biochips, or other biodevices.     

Elongation factor Tu and DnaK are transferred from the cytoplasm to the periplasm of Escherichia coli during osmotic downshock presumably via the mechanosensitive channel mscL

Upon osmotic downshock, a few cytoplasmic proteins, including thioredoxin, elongation factor Tu (EF-Tu), and DnaK, are released from Tris-EDTA-treated Escherichia coli cells by an unknown mechanism. We have shown previously that deletion of mscL, the gene coding for the mechanosensitive channel of the plasma membrane with the highest conductance, prevents the release of thioredoxin. We confirm and extend the implication of MscL in this process by showing that the release of EF-Tu and DnaK is severely impaired in MscL-deficient strains. Release of these proteins is not observed in the absence of a Tris-EDTA treatment which disrupts the outer membrane, indicating that, in intact cells, they are transferred to the periplasm upon shock, presumably through the MscL channel.     

Structural properties of periplasmic SodCI that correlate with virulence in Salmonella enterica serovar Typhimurium

Salmonella enterica strains survive and propagate in macrophages by both circumventing and resisting the antibacterial effectors normally delivered to the phagosome. An important aspect of Salmonella resistance is the production of periplasmic superoxide dismutase to combat phagocytic superoxide. S. enterica serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Both enzymes are produced during infection, but only SodCI contributes to virulence in the animal. Although 60% identical to SodCII at the amino acid level with very similar enzymatic properties, SodCI is dimeric, protease resistant, and tethered within the periplasm via a noncovalent interaction. In contrast, SodCII is monomeric and protease sensitive and is released from the periplasm normally by osmotic shock. We have constructed an enzymatically active monomeric SodCI enzyme by site-directed mutagenesis. The resulting protein was released by osmotic shock and sensitive to protease and could not complement the loss of wild-type dimeric SodCI during infection. To distinguish which property is most critical during infection, we cloned and characterized related SodC proteins from a variety of bacteria. Brucella abortus SodC was monomeric and released by osmotic shock but was protease resistant and could complement SodCI in the animal. These data suggest that protease resistance is a critical property that allows SodCI to function in the harsh environment of the phagosome to combat phagocytic superoxide. We propose a model to account for the various properties of SodCI and how they contribute to bacterial survival in the phagosome.     

Periplasmic enzymes in Bdellovibrio bacteriovorus and Bdellovibrio stolpii.

When cells of either Bdellovibrio bacteriovorus 109J or Bdellovibrio stolpii UKi2 were subjected to osmotic shock by treatment with sucrose-EDTA and MgCl2 solutions, only trace amounts of proteins or enzyme activities were released into the shock fluid. In contrast, when nongrowing cells were converted to motile, osmotically stable, peptidoglycan-free spheroplasts by penicillin treatment, numerous proteins were released into the suspending fluid. For both species, this suspending fluid contained substantial levels of 5'-nucleotidase, purine phosphorylase, and deoxyribose-phosphate aldolase. Penicillin treatment also released aminoendopeptidase N from B. bacteriovorus, but not from B. stolpii. Penicillin treatment did not cause release of cytoplasmic enzymes such as malate dehydrogenase. The data indicated that bdellovibrios possess periplasmic enzymes or peripheral enzymes associated with the cell wall complex. During intraperiplasmic bdellovibrio growth, periplasmic and cytoplasmic enzymes of the Escherichia coli substrate cell were not released upon formation of the spherical bdelloplast during bdellovibrio penetration. Most of the E. coli enzymes were retained within the bdelloplast until later in the growth cycle, when they became inactivated or released into the suspending buffer or both.     

Comparative release of alkaline phosphatases from Serratia marcescens by osmotic shock and polymyxin B treatment.

The comparative release of periplasmic enzymes and proteins from two strains of Serratia marcescens by osmotic shock and polymyxin B treatment was studied. There were significant qualitative and quantitative differences in the materials released by these two techniques. The osmotic shock procedure released a higher level of alkaline phosphatase activity and a greater number of protein components than the polymyxin B treatment. The molecular weights of the active components released by the two techniques were shown to be 190,000 +/- 10,000 (A'), 140,000 +/- 10,000 (A) and 110,000 +/- 10,000 (B) daltons. Components released by polymyxin B were also released by osmotic shock. However, the reverse was not true. Component B in the osmotic shock fluids was by far the most active. The differences in the release mechanisms of the two techniques were discussed. It is suggested that polymyxin B treatment is the method of choice because of its selectiveness and mildness, despite the rather low level of activity of alkaline phosphatase released.     

Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization.

We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli. The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions. Specifically, treatment of E. coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host. Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies. Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.     

Polygalacturonic acid trans-eliminase in the osmotic shock fluid of Erwinia rubrifaciens: characterization of the purified enzyme and its effect on plant cells.

An endopolygalacturonic acid trans-eliminase (EC 4.2.2.2), released by osmotic shock of Erwinia rubrifaciens cells, has been purified to near homogeneity (3, 100-fold) by column chromatography on diethylaminoethyl-cellulose, phosphocellulose, and hydroxyapatite-cellulose followed by isoelectric focusing. It has a molecular weight of 41,000, s20,w of 3.09S, an isoelectric point of pH 6.25, pH optimum of 9.5, and a temperature optimum of 37 C and requires Ca2+ with an optimum concentration of 0.5 to 1.0 mM. Mg2+ could not substitute for Ca2+. Tyrosinyl residues seem essential for enzyme catalysis based on rapid inactivation by tetranitromethane. The enzyme prefers unmethylated polygalacturonic acid as the substrate, cleaving alpha-1,4-glycosidic linkages randomly to form unsaturated galacturonides at a Vmax of 1,166 mumol of product/min per mg of protein and a Km of 5 mg of polygalacturonic acid per ml. Over 90% of the enzyme activity is released from osmotically shocked E. rubrifaciens cells. Unlike E. rubrifaciens, trans-eliminase is not released from Erwinia carotovora cells by osmotic shock treatment, but enzyme activity is detected in the culture medium. The release of the enzyme is reduced fivefold by the addition of dibutyryl cyclic adenosine 5'-monophosphate. The hypersensitive reaction in tobacco leaves was induced within 60 min after injection of less than 1 mug of purified E. rubrifaciens trans-eliminase. Single cells of tobacco in suspension culture are readily killed by the enzyme, whereas tobacco protoplasts remain unaffected when treated in the same manner. These results indicate that endopolygalacturonic acid trans-eliminase is a constitutive enzyme possibly located in the periplasmic space of the E. rubrifaciens cell and releases enzyme into the culture medium in the presence of substrate. The release of the enzyme in tobacco tissue and the trans-eliminative cleavage of plant cell wall components may be steps leading to hypersensitivity of the tobacco tissue.