以壳聚糖为载体亲和层析胰蛋白酶

用活化的壳聚糖为载体,鸡卵粘蛋白(CHOM)为配基,制备了胰蛋白酶的亲和吸附剂。采用该吸附剂亲和层析胰酶,所得产物经SDS-PAGE电泳检测,带中只有一条带颜色较深,且与标准胰蛋白酶带位置几乎相同。实验结果表明1 g壳聚糖可以固定60 mg鸡卵粘蛋白,制成的亲和吸附剂可吸附胰蛋白酶的最大量为118 U/g。以壳聚糖为载体的亲和吸附剂制备过程简单、安全。     

用高碘酸盐活化葡聚糖凝胶制备亲和吸附剂的研究

 <正> 用高碘酸盐活化的Sephadex与鸡卵类粘蛋白偶合制备分离纯化胰蛋白酶的亲和吸附剂。该方法比CNBr活化Sepharose 4B制备亲和吸附剂的方法具有操作安全,价格低廉等优点。活化载体在4℃保存较长时间不失去键合能力。该亲和吸附剂可制备得比活力为11228.8u/mgBAEE单位电泳单带纯胰蛋白酶,并能反复使用十余次,仍具有较强亲和吸附能力。酶     

果胶酶固定化方法的研究

用海藻酸钠、二醋酸纤维、明胶分别作为固定化果胶酶的载体,研究比较载体的用量、交联剂的浓度、用酶量、制备的ｐＨ。结果表明,选择１５％浓度的明胶为载体,５％浓度的戊二醛为交联剂,用酶量５％,在ｐＨ３．５条件下制备固定化果胶酶,其酶活力回收率可达６７．７４％,重复回收使用１０次后,酶活力还可保留８０％以上,是果胶酶固定化的一种较好的方法。     

魔芋葡苷聚糖(KGM)凝胶为亲和层析载体与 Sepharose 4B的比较研究Ⅱ.KGM染料亲和层析分离人血清白蛋白

将KGM和Sepharose 4B凝胶在相同条件下活化、偶联接上染料Cibacron Blue F3GA制成了KGM和Sepharose 4B染料亲和吸附剂,并用来与牛血清白蛋白(BSA)作用,每毫升KGM亲和吸附剂可吸附BSA 28 mg,用NaSCN洗脱时回收为84.5%,而Sepharose 4B梁料亲和吸附剂每毫升可吸附BSA 15.3 mg,用NaSCN洗脱时回收为81.7%,并对两种凝胶的染料亲和吸附性能进行了比较.用KGM染料亲和吸附剂分离的人血清白蛋白与人血清白蛋白标准品有同样的纯度,都达到了电泳纯.     

果胶酶固定化方法的研究

用海藻酸钠、二醋酸纤维、明胶分别作为固定化果胶酶的载体,研究比较载体的用量、交联剂的浓度、用酶量、制备的ｐＨ。结果表明,选择１５％浓度的明胶为载体,５％浓度的戊二醛为交联剂,用酶量５％,在ｐＨ３．５条件下制备固定化果胶酶,其酶活力回收率可达６７．７４％,重复回收使用１０次后,酶活力还可保留８０％以上,是果胶酶固定化的一种较好的方法。     

明胶/海藻酸钠/沙蒿胶复合水凝胶的制备及表征

为探究明胶（G）、海藻酸钠（SA）,沙蒿胶（ASKG）对复合水凝胶的力学性能、溶胀和保湿性能的影响,采用共混-离子交联法制备海藻酸钠/明胶/沙蒿胶复合水凝胶,并对制得的水凝胶进行结构表征和溶血率测试。结果表明:当G质量分数为2.5%,SA为1.5%,ASKG为0.7%时,复合水凝胶压缩强度达到427.2 kPa,拉伸强度达到563.449 kPa,断裂伸长率为117%,溶胀率为744%,且具有较好的保湿性能。红外光谱表明,由于沙蒿胶中存在大量羟基,因此加入沙蒿胶后在3 300 cm^-1～3 600 cm^-1羟基峰形变宽。G/SA/ASKG复合水凝胶溶血率低于5%,具有较好的网络孔结构和血液相容性,为复合水凝胶在医用敷料方面的应用提供一定的参考价值。     

草酸青霉BZH-2002产果胶酶特性研究

对草酸青霉菌（Penixillium oxalicum）BZH-2002菌株固体发酵果胶酶的特性及浸提条件进行了研究,确定其最佳产酶时期为72—92h,利用干物质量最快的时期为24—72h发酵产酶期间培养基介质中pH值呈“V”字形变化。该菌株以甜菜渣、葵花盘为碳源,以（NH）2SO4为氮源时产酶率最高,分别高达4．33万和4．56万U／g干物质,（NH4）2SO4最佳用量0．9g/10g甜菜渣。此外,浸提方法对果胶酶产率也有一定影响,1％NaCl作为浸提液的效果最好,最佳浸提时间1h,浸提温度25—40℃。     

草酸青霉BZH-2002果胶酶固体发酵条件研究

对草酸青霉菌(Penixillium oxalicum)BZH-2002菌株固体发酵果胶酶的主要影响因子温度、初始pH值、含水量及接种量进行了实验探讨,确定了最佳培养条件：温度为30℃,初始pH值为4．8,固体培养基含水量控制在30～35ml/10g甜菜渣,接种量3～4%。同时对该菌株固体发酵提取液中果胶酶的酶学特性进行了初步研究,结果表明,该酶最适反应温度和pH分别为55℃和pH4．8,在40℃温度下和pH3．5～5．5范围内,酶活性较稳定。     

高产碱性果胶酶菌株的育种及其发酵条件的研究

以克劳氏芽孢杆茵(Bacillus clausii)S-4为出发菌株,经紫外诱变育种和固态发酵条件的选育,得到产碱性果胶酶较高的新茵株N-10,并研究了其固态发酵条件和部分酶学性质.结果表明,以甜菜渣为碳源和酶的诱导物以及棉粕作为氮源较适宜.较优的固态发酵条件为:甜菜渣5g,棉粕0.125 g,麦芽糖0.1 g,KH2PO40.0075 g,Na2CO30.15 g,水12.5 mL,种龄24 h,接种量2mL,发酵温度40℃,发酵时间84 h;酶产率可达4780 u/g(干甜菜渣),较菌株S-4提高108%.该酶的最适pH 10,最适温度55℃;分别在pH 7.5～9.5和30～40℃范围内较稳定;Ca2 、Mg2 、Fe2 对酶活有明显激活作用,Cu2 、Zn2 对酶活有强烈抑制活作用.     

免疫亲和层析法纯化苦瓜几丁酶

用扁豆几丁酶免疫家兔,获得抗扁豆几丁酶的抗体,将此抗体与Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联,制备免疫亲和吸附剂,用以纯化苦瓜几丁酶．苦瓜叶片的粗提液经过免疫亲和吸附柱后,可获得电泳纯的几丁酶,其分子量为３５ ｋＤ,与用几丁质凝胶为亲和吸附剂的纯化结果一致．表明利用植物几丁酶在结构上的保守性,用免疫亲和法可纯化不同植物的同类几丁酶．与几丁质凝胶亲和柱相比,免疫亲和法纯化植物几丁酶具有快速、亲和柱可重复使用等的优点．利用免疫亲和层析获得的纯化样品,研究了苦瓜几丁酶对真菌的抑制试验,研究结果表明,苦瓜几丁酶能分解棉花枯萎病菌的菌丝体细胞壁制备物,并对其孢子芽管的伸长有一定抑制作用．     

Isolation of the lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis by affinity chromatography.

The lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis was isolated from solutions obtained after cell disintegration by a novel affinity chromatographic method using an adsorbent composed of human haptoglobin covalently attached to a Sepharose 4B matrix. The haemagglutinin was bound to the adsorbent at pH 6.5 and eluted by a stepwise change to a pH 10 buffer. A 300--600-fold purification of the haemagglutinin was achieved by this one-step process. The chemical and biological properties of the haemagglutinin isolated by affinity chromatography were found to be similar to those of the protein isolated by other workers from culture supernatants. The affinity chromatographic method was found to be specific for the purification of the lymphocytosis promoting factor-haemagglutinin and no purification of the fimbrial-haemagglutinin of Bordetella pertussis was achieved by the method.     

Fractionation of plant aminoacyl-tRNA synthetases on tRNA-Sepharose columns.

It has been shown that tRNA-Sepharose, a chromatographic adsorbent containing unfractionated tRNA bound to a Sepharose matrix, is a useful, group-specific adsorbent for fractionation of the plant aminoacyl-tRNA synthetases. Conditions are described in which Val-, Trp-, Phe-, Leu- and Ile-tRNA synthetases from yellow lupin seeds can be separated from each other on the tRNA-Sepharose columns. Factors affecting affinity chromatography on the t-RNA-Sepharose columns are discussed. The affinity chromatography procedure for the purification of lupin Ser-tRNA synthetase to homogenity is described.     

Synthesis and characterization of pseudo-affinity ligand for penicillin acylase purification

The aim of this work was to test a chromatographic affinity support containing methacryloyl antipyrine (MAAP) for penicillin acylase (PA) purification by using pure penicillin acylase and crude extract. First, MAAP as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Polymer beads (average size diameter: 40–120 μm) were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAAP. This approach for the preparation of adsorbent has several advantages over conventional preparation protocols. An expensive and time consuming step in the preparation of adsorbent is immobilization of a ligand to the adsorption matrix. In this procedure, affinity ligand MAAP acts as comonomer without further modification steps. Poly(EGDMA-MAAP) beads were characterized by FTIR, NMR and screen analysis. Elemental analysis of MAAP for nitrogen was estimated as 89.3 μmol/g. The prepared adsorbent was then used for the capture of penicillin acylase in batch system. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) beads was found to be 82.2 mg/g at pH 5.0. Chromatography with crude feedstock resulted in 23.2-fold purification and 93% recovery with 1.0 M NaOH.     

Rational combinatorial chemistry-based selection,synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca(2+)-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (approximately 1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.     

Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5+/-3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.     

Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 . 10(5). The molecule does not contain sugars. The dissociation constant of the protein . sulfobromophthalein complex has been found to be 4. 10(-6) M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.     

Validation of leaf and microbial pectinases: commercial launching of a new platform technology

Almost all current genetically modified plant commercial products are derived from seeds. The first protein product made in leaves for commercial use is reported here. Leaf pectinases are validated here with eight liquid commercial microbial enzyme products for textile or juice industry applications. Leaf pectinases are functional in broad pH/temperature ranges as crude leaf extracts, while most commercial enzyme products showed significant loss at alkaline pH or higher temperature, essential for various textile applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, leaf pectinase powder was stored up to 16 months at ambient temperature without loss of enzyme activity. Commercial pectinase products showed much higher enzyme protein PAGE than crude leaf extracts with comparable enzyme activity without protease inhibitors. Natural cotton fibre does not absorb water due to hydrophobic nature of waxes and pectins. After bioscouring with pectinase, measurement of contact‐angle water droplet absorption by the FAMAS videos showed 33 or 63 (leaf pectinase), 61 or 64 (commercial pectinase) milliseconds , well below the 10‐second industry requirements. First marker‐free lettuce plants expressing pectinases were also created by removal of the antibiotic resistance aadA gene. Leaf pectinase powder efficiently clarified orange juice pulp similar to several microbial enzyme products. Commercial pilot scale biomass production of tobacco leaves expressing different pectinases showed that hydroponic growth at Fraunhofer yielded 10 times lower leaf biomass per plant than soil‐grown plants in the greenhouse. Pectinase enzyme yield from the greenhouse plants was double that of Fraunhofer. Thus, this leaf‐production platform offers a novel, low‐cost approach for enzyme production by elimination of fermentation, purification, concentration, formulation and cold chain.     

Separation and purification of enzymes by continuous pH-parametric pumping

Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. epsilon-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes.     

Recent trends and some applications of isothermal titration calorimetry in biotechnology

Isothermal titration calorimeters (ITCs) are thermodynamic instruments used for the determination of enthalpy changes in any physical/chemical reaction. This can be applied in various fields of biotechnology. This review explains ITC applications, especially in bioseparation, drug development and cell metabolism. In liquid chromatography, the separation/purification of specific proteins or polypeptides in a mixture is usually achieved by varying the adsorption affinities of the different proteins/polypeptides for the adsorbent under different mobile-phase conditions and temperatures. Using ITC analysis, the binding mechanism of proteins with adsorbent solid material is derived by elucidating enthalpy and entropy changes, which offer valuable guidelines for designing experimental conditions in chromatographic separation. The binding affinity of a drug with its target is studied by deriving binding enthalpy and binding entropy. To improve the binding affinity, suitable lead compounds for a drug can be identified and their affinity tested by ITC. Recently ITC has also been used in studying cell metabolism. The heat produced by animal cells in culture can be used as a primary indicator of the kinetics of cell metabolism, which provides key information for drug bioactivity and operation parameters for process cell culture.     

Magnetic porous corn starch for the affinity purification of cyclodextrin glucanotransferase produced by Bacillus circulans

Magnetic porous corn starch was prepared as an affinity adsorbent for the efficient and simple scale-up procedure for one-step purification of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans. Magnetic affinity separation enabled isolation of CGTase from cultivation media (volumes between 10 and 400 mL) with ca 60–70% recovery after elution with alkaline buffers containing soluble starch; the enzyme purification factor was 19–25 in different batches. The majority of ballast proteins were removed during the purification process, which shows high selectivity of the affinity material used.