Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization

Fibrinolytic enzymes are important in treatment of cardiovascular diseases. The present work reports isolation, screening and identification of marine cultures for production of fibrinolytic enzymes. A potent fibrinolytic enzyme-producing bacterium was isolated from marine niches and identified as Bacillus subtilis ICTF-1 on the basis of the 16S rRNA gene sequencing and biochemical properties. Further, media optimization using L(18)-orthogonal array method resulted in enhanced production of fibrinolytic enzyme (8814 U/mL) which was 2.6 fold higher than in unoptimized medium (3420 U/mL). In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent. A fibrinolytic enzyme was purified from the culture supernatant to homogeneity by three step procedures with a 34.42-fold increase in specific activity and 7.5% recovery. This purified fibrinolytic enzyme had molecular mass of 28 kDa, optimal temperature and pH at 50 °C and 9, respectively. It was stable at pH 5.0-11.0 and temperature of 25-37 °C. The enzyme activity was activated by Ca(2+) and obviously inhibited by Zn(2+), Fe(3)(+), Hg(2+) and PMSF. The purified fibrinolytic enzyme showed high stability towards various surfactants and was relatively stable towards oxidizing agent. Considering these properties purified fibrinolytic enzyme also finds potential application in laundry detergents in addition to thrombolytic agent. The gene encoding fibrinolytic enzyme was isolated and its DNA sequence was determined. Compared the full DNA sequence with those in NCBI, it was considered to be a subtilisin like serine-protease.     

Purification,characterisation, and quantification of the soy allergen profilin (Gly m 3) in soy products

Profilins are pan-allergen proteins present in various plant foods and pollens. The objective was to develop a method for purification and characterisation of profilin from soy protein isolate. Furthermore, profilin was quantified in soy products and the effect of processing evaluated. Profilin was purified using poly-l-proline affinity chromatography, dialysis and ultrafiltration, and its quantification was implemented by indirect ELISA. Profilin in soymilks ranged from 4.37 ± 0.14 to 7.24 ± 0.30 mg/g protein, while in fermented products profilin ranged from 1.67 ± 0.02 to 5.47 ± 0.02 mg/g protein. Pasteurisation of soymilk was an ineffective method to completely eliminate profilin. Food matrix influenced thermal stability; at 100 °C, β-sheet and random coil structures were altered, while the α-helices remained intact. Induced fermentation of soybean meal by Bifidobacterium lactic, Lactobacillus plantarum and Saccharomyces cerevisiae resulted in 68.3% to 72.7% reduction of soy profilin. Heat treatment, fermentation and hydrolysis effectively reduced soy profilin.     

Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga)

Pepsinogen (PG) from the stomach of albacore tuna (Thunnus alalunga) was purified to homogeneity by using a series of chromatographies involving Sephacryl S-200HR, Sephadex G-50 and DEAE-cellulose with a 658-fold increase in purity. Based on the native-PAGE and zymography, PG showed a single band with pepsin activity. Molecular weights (MW) of PG and active pepsin were estimated to be 39.9 and 32.7 kDa as determined by SDS–PAGE, respectively. PG was converted to the corresponding pepsin through an intermediate form (MW ≈ 36.8 kDa) and the complete activation was observed after 30–60 min. The N-terminal amino acid sequence of the first 15 amino acids of activation segment of pepsinogen was FHKLPLIKGKTAREE. The optimal pH and temperature for pepsin activity were 2.0 and 50 °C, respectively. The activity was stable in the pH range of 2–5. Residual activity more than 85% was found after heating at temperatures up to 50 °C for 30 min. Pepsin activity was strongly inhibited by pepstatin A, whilst E-64, ethylenediaminetetraacetic acid (EDTA) and soybean trypsin inhibitor exhibited the negligible effect. SDS and cysteine also showed inhibitory effects, whilst ATP, molybdate, NaCl and CaCl₂ had no impact on pepsin activity.     

Expanded bed adsorption as a fast technique for the large-scale purification of the complete isoform pool of Ber e 1, the major allergen from Brazil nuts

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Ber e 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1.     

Bovine muscle 20S proteasome: I. Simple purification procedure and enzymatic characterization in relation with postmortem conditions

Over the last decade, several sets of evidence support a possible contribution of the 20S proteasome to the meat tenderizing process. This assumption was emphasized by recent investigations demonstrating that the 20S proteasome was active in the absence of activators and exhibited endo- and exoproteolytic activities, a status often strongly debated before. In the present work, we developed a new rapid and simple purification procedure for muscle 20S proteasome and revisited the physicochemical properties of this complex in relation with the postmortem muscle environmental conditions, i.e. temperature, pH, osmolarity, etc. From a crude extract obtained from freshly excised muscle tissue, reasonable amounts of highly pure proteasome were prepared within a maximum of 4 days using only three chromatography steps. This purified proteasome was used to investigate the effect of pH, temperature, ionic strength and sodium dodecyl sulphate (SDS) on the major hydrolytic activities of this complex, i.e. trypsin-like (TL), chymotrypsin-like (CL) and peptidylglutamyl peptide hydrolase (PGPH) activities. Taken together, the data obtained suggest that the 20S proteasome constitutes a high hydrolytic potential in postmortem muscle conditions. To attest this finding, the 20S proteasome was further quantified by ELISA in at death and postmortem muscles including Longissimus, Rectus abdominis, Diaphragma pedialis and Tensor fascia latae bovine muscles. The primary conclusion was that time course changes in proteasome concentrations were not dependent on the kinetics of the pH fall. Secondly, the proteasome concentration in conditioned meat was in good agreement with previously reported proteolytic activity. Furthermore, the decrease in the muscle proteasome concentration can be considered as slow and this is particularly true in type 1 muscles for which the decrease in the amount of this complex did not exceed 7% during the first three days postmortem. This would suggest that the 20S proteasome was relatively stable during meat conditioning, a feature supporting a potential role in the meat tenderizing process.     

机械离心复合静电技术去除油烟的实验研究

首先,研究了单独使用机械离心油烟净化器情况下,机械离心净化器转速对自身阻力的影响。其次,研究了机械离心净化器和静电式油烟净化器联合使用情况下,机械离心净化器转速对静电式油烟净化器清洗周期、油烟净化效率的影响。最后,为了优化设备效率,将机械离心净化器的金属网转盘进行覆膜来延长机械离心净化器的清洗周期,研究了镀膜后机械离心净化器的摩擦系数对自身净化效率和整个系统净化效率的影响。结果表明,机械式离心净化器的使用可延长静电式油烟净化器的清洗周期,静电式油烟净化器的使用可提高机械式离心净化器的净化效率,机械离心净化器和静电式油烟净化器联合使用油烟净化效果最佳。     

层析法分离纳豆激酶的研究

选高产酶的纳豆杆菌发酵,发酵液离心除菌后加入饱和度为30%硫酸铵去杂,然后加入65%的硫酸铵析出纳豆激酶粗提物,然后将粗提物分别过阴阳离子交换柱和疏水层析柱进行提纯酶。比较其提纯倍数和回收率,得出较好得分离纯化方案为:样品依次经过DEAE-SepharoseFastFlow阴离子层析、CM-SepharoseFastFlow阳离子层析和Penpyl-SepharoseCl-4B疏水层析柱,纳豆激酶最终纯化倍数达到32.2,回收率为13.2%。     

超滤法精制L—异亮氨酸

国内传统的氨基酸粗品精制基本上全部采用活性炭脱色。活性炭色黑质轻,污染环境,且其产品质量差。在氨基酸生产工艺中采用无相变、低能耗、环境友好的膜分离技术,是氨基酸工业技术进步中一大突破。采用超滤技术代替现有的活性炭脱色工艺,实现氨基酸的高效提纯与精制。研究了不同切割相对分子质量的膜,操作压力和温度对膜通量的影响及膜的清洗方法。结果表明,在操作压力为0.15～0.20MPa,操作温度为45℃时经杭州水处理中心生产截留相对分子质量为30000的聚丙烯腈超滤膜超滤浓缩精制制得的异亮氨酸产品得率高,质量好,为超滤技术应用于异亮氨酸精制提供了依据。另外,采用超滤膜过滤除去热原也很有效,同时还能达到无菌的要求。     

Mixed Surfactant-Based Reverse Micellar Extraction Studies of Bovine Lactoperoxidase

The suitability of reverse micellar extraction for recovery of bovine lactoperoxidase (LP) from aqueous solution was evaluated using systems formed by ionic and nonionic surfactant mixtures. The influence of ionic surfactant concentration, organic solvent, and pH on the extraction of LP into the reverse micellar phase was studied. The Tween® series surfactants with Aerosol-OT (bis-(2-ethylhexyl) sulfosuccinate) showed better extraction of LP in the reverse micelles (RM) compared to the Triton® and Span® series of surfactants. Complete extraction of LP from an aqueous phase of initial concentration 25 mg L⁻¹ occurred with the RM formed by 90 mM Aerosol-OT/8 mM Tween® 80 in isooctane. The optimal pH, ionic strength, and positively charged ionic surfactant concentration for back extraction were also studied and a maximum of 95.5% back extraction efficiency and 66% LP activity recovery was obtained for a pH of 10.5,1 M KCl and 60 mM cetyltrimethylammonium bromide system.     

海带多糖的提取及组份LI的分离与鉴定

采用热水提取法考查了海带多糖的提取条件对多糖提取率的影响。实验结果表明,海带多糖的最佳提取温度为１００℃,提取时间为６ｈ,加水量为９０ｍＬ·ｇ－１。海带粗多糖经ＤＥＡＥ－纤维素（ＤＥ－５２,Ｂ４Ｏ２－７型）柱层析分离,以０．０５ｍｏｌ／Ｌ硼砂溶液为洗脱液,得到一种水溶性多糖（ＬＩ）,通过纸层析、ＳｅｐｈａｄｅｘＧ－２００柱层析检查为单一组成,其酸性水解物经纸层析检查,表明其单糖组成为木糖、葡萄糖、岩藻糖,并含有硫酸基。葡聚糖凝胶过滤层析法测得其相对分子质量近似为１．１×１０５。ＬＩ结构鉴定有待于进一步研究。