耐有机溶剂脂肪酶产生菌的筛选及其酶学性质

以体积分数10%的甲苯等有机溶剂为筛选压力,从油污和污水等样品中筛选到一株产耐有机溶剂脂肪酶的有机溶剂耐受茵LX1,经16 S rDNA序列分析,该菌株鉴定为Pseudomonas aeruginosa.研究发现菌株LX1所产脂肪酶最适反应pH和温度分别为7.0和40℃,在较宽的pH范围内(6.5～10.5)具有较高的稳定性;金属离子鏊合剂EDTA和Ba~(2+)、Ca~(2+)对LX1脂肪酶活力均有明显的激活作用,推测该脂肪酶为金属激活酶.LX1脂肪酶在体积分数25%的疏水性和亲水性有机溶剂中均具有较好的耐受性,在十六烷、十四烷、十二烷、正庚醇、正辛醇和正己醇体系中半衰期显著延长到10 d以上,在DMF和DMSO体系中的半袁期为5 d以上,均高于在无溶剂体系中的半衰期.展现了LX1脂肪酶在有机相生物催化中的良好应用前景.     

纳豆激酶的分离纯化及其特性研究

从实验室保存的1株高产纳豆激酶菌株出发,进行发酵产酶,确立具有纤溶活力的纳豆激酶的分离纯化工艺,主要通过硫酸铵分级盐析法和Phenyl Sepharose疏水柱层析进行分离纯化,用纤维蛋白平板法测定酶活力,用SDS-PAGE电泳验证为电泳纯,分子质量为28 ku。试验了不同作用温度、pH值和金属离子对NK活力的影响。结果表明,NK的适宜温度为25~50℃,适宜pH值为7.0,Cu2+,Mn2+,Hg2+是其抑制剂,而Mg2+,Ca2+为激活剂。     

Influence of ion accumulation on the emulsion stability and performance of semi-synthetic metalworking fluids

The metalworking industry is one of the largest in the United States. Although metalworking fluids (MWFs) are ubiquitous in manufacturing as coolants and lubricants, these emulsified fluids have a significant environmental impact over their life cycle. Accordingly, it has become necessary to better understand emulsion destabilization mechanisms that lead to MWF deterioration and disposal so that MWF formulations can be designed for increased longevity. This paper investigates the impact of pH and a wide range of hard water salts on MWF emulsion stability. While expected trends from the emulsion science literature are observed, it is shown that MWF destabilization can lead to an increase in the microbial load that the MWF can sustain while only slightly improving manufacturing performance as measured by the tapping torque test. Experimental observations also indicate that these trends are strongly correlated with increased emulsion particle size, regardless of whether increased particle size is achieved by aging, by reductions in pH, or by the addition of hard water salts. In MWF systems, these conditions typically result from the accumulation of divalent and trivalent cations over time due to hard water additions and exposure to metal workpieces and tools. While MWFs are formulated with EDTA to avoid emulsion destabilization due to cation accumulation, it is shown that EDTA can be ineffective or highly inefficient for this purpose due to direct interactions between EDTA and the MWF emulsifier system. Given the ineffectiveness of EDTA and commonly utilized MWF emulsifier systems to maintain stable emulsion size in the presence of high concentrations of hard water salts, a more effective and environmentally preferable technological change to the MWF formulation design is proposed and successfully demonstrated.     

Enzymatic characterization of transglutaminase from Streptomyces mobaraensis DSM 40587 in high salt and effect of enzymatic cross-linking of yak milk proteins on functional properties of stirred yogurt

Streptomyces transglutaminase (TGase) purified from high-salt medium was characterized and applied into yak yogurts. The purified enzyme presented a Michaelis constant of 40.47 mmol and a maximum velocity of 44.44 U/mg of protein for N-carboxybenzoyl-l-glutaminyl-glycine in the hydroxamate procedure. The purified TGase exhibited optimum activity at 55°C and pH 6.0. The enzyme was not stable above 50°C and was stable within a pH range of 5.0 to 10.0 at 4°C for 12h and pH 5.0 to 9.0 at 37°C for 30 min. The TGase activity was not affected by Ca(2+), K(+), Ba(2+), or Na(+), but slightly inhibited by Fe(2+), Mg(2+), and Mn(2+), and strongly by Cu(2+) and Zn(2+). To explore yak milk products, it was used to produce yogurt and TGase was used. It was found that TGase-catalyzed cross-linking was effective in improving functional properties of stirred yak yogurt. Treated yogurt produced a strong acid gel, higher consistency, cohesiveness, index of viscosity, and creamier mouth feel than the untreated product. Furthermore, yak yogurt treated with TGase presented lower wet yak hair or sweat odor, or both. Therefore, TGase can be used to pave the way for exploration of novel yak products to overcome the issues of peculiar wet yak hair or sweat odor, or both.     

假单胞菌H3壳聚糖酶的纯化及部分酶学性质

对 Pseudmona sp.H3产生的壳聚糖酶粗酶液采用(NH_4)_2SO_4盐析、Sephadex G-25脱盐、Sepharose Q-XL阴离子交换层析和Superdex G-75分子筛层析进行纯化,经SDS-PAGE鉴定为单蛋白带,分子质量约为33.8ku,酶反应最适温度为40℃,最适pH为5.0,降解壳聚精(D.A.90.14％)Km值为3.59g/L,V_(max)值为 3.80mmol/(L·min);Ba~(2+)、K~+、Co~(2+)对该酶有激活作用,而Zn~(2+)、Mn~(2+)、Al~(3+)、Cu~(2+)则对酶有抑制作用;此外,该酶除了能降解壳聚糖以外,还具有CMCase活性。     

黑曲霉酸性β-甘露聚糖酶曲盘发酵与粗酶性质研究

用黑曲霉菌株(Aspergillus niger)LW—1进行固态发酵生产酸性β-甘露聚糖酶,其曲盘(大小φ20 cm×4 cm)发酵的最优工艺为:培养基最初料水比为1:1.8,起始pH自然,接种量10%(相对于干物料),曲盘中装料150g(以干料计),通过中间补水和翻曲,32℃培养72 h,最高酶活可达22 250 IU/g(干曲)。对粗酶性质进行研究,结果表明,酶的最适反应温度和pH分别为70℃和3.5,低于60℃、pH5.0～8.0酶稳定,Zn~(2+)、Ca~(2+)、ED- TA对酶有明显的激活作用;Mn~(2+)、Fe~(2+)、pb~(2+)、Sn~(2+)、Fe~(3+)、Cu~(2+)、Al~(3+)、Ag~+对酶有一定的抑制作用。     

Purification and properties of an arginyl aminopeptidase from Debaryomyces hansenii

A metallo arginyl aminopeptidase (EC 3.4.11.6) activated by Co(2+) was isolated from Debaryomyces hansenii CECT 12487. The enzyme was purified after precipitation with protamine sulphate, followed by a weak anion exchange chromatography, gel filtration chromatography and a strong anion exchange chromatography. The arginyl aminopeptidase (AAP) was purified 337 folds, with a 18% recovery. The AAP appeared to be a dimer with a molecular mass of 101 kDa. The enzyme was active in the pH range from 6 to 9. The optimal activity was detected at pH 7.0 and at 37 degrees C. AAP activity was inhibited by typical aminopeptidase inhibitors (puromycin and bestatin), reducing agents (DTT), chelating agents (EDTA, EGTA and phenantroline) and sulphydryl groups reagents (iodoacetate). Ca(2+), Mn(2+) and Co(2+) activated the enzyme, while Cu(2+), Cd(2+), Hg(2+) and Mg(2+) inhibited it. The K(m) values calculated for Arg-AMC (7-amido-4-methylcoumarin) and Leu-AMC were 0.071 and 0.094 mM, respectively. The enzyme showed maximum specificity for basic amino acids (Arg and Lys), but was also able to hydrolyze non-charged amino acids (Leu, Met and Ala) and, at a minor rate, aromatic amino acids (Phe and Tyr). AAP showed higher activity when an acid residue was located at the C-terminal position of dipeptides.The described purification of an arginyl aminopeptidase from the yeast D. hansenii can contribute to the lack of knowledge about the exopeptidase activity in one of the yeasts more frequently isolated in sausage and to understand its role during the ripening of a fermented sausage.     

Purification and characterization of a novel serine protease from the mushroom Pholiota nameko

A novel serine protease, with a molecular mass of 19 kDa and the N-terminal sequence of ARTPEAPAEV, was isolated from dried fruiting bodies of the mushroom Pholiota nameko. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, Q-Sepharose and SP-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on DEAE-cellulose and Q-Sepharose but adsorbed on SP-Sepharose. It exhibited an optimum temperature at 50°C, an optimum pH at pH 8.8, a Km of 5.64 mg/mL and a Vmax of 0.98 μmol/min/mL against substrate casein. A number of metal ions inhibited the enzyme including Pb(2+), Mn(2+), Ca(2+), Hg(2+), Zn(2+), Cu(2+), Co(2+), Fe(3+) and Al(3+), with the inhibition of the last two cations being the most potent. K(+) and Mg(2+) slightly enhanced, while Li(+) moderately potentiated the activity of the protease. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease.     

一种耐热性植酸酶的分离纯化及其酶学性质研究

采用半固态发酵方式培养泡盛曲霉(Aspergillus awamori)AS3.324,通过有机膜超滤、阴离子交换层析、凝胶层析后,得到纯化的植酸酶。酶学性质表明,其反应最适温度为50-55℃,最适pH为5.5,在37℃下以植酸钠为底物的Km值为1.03 nmol/L,Vmax为2.13μmol/(L.min)。EDTA基本不影响植酸酶活性;Ca2+,Mg2+,Mn2+对植酸酶活性有轻微的抑制作用;Fe2+,Zn2+对酶促反应有显著的抑制作用。对该酶的耐热性研究表明,经较高温度条件处理后,仍有较高残余酶活性,与当今商品化的植酸酶相比,有较强的耐热性。泡盛曲霉植酸酶作为动物饲料添加剂具有广泛的应用前景。     

木霉LE02 β-1，3-葡聚糖酶酶学特性的研究

对木霉菌株LE02所产β-1,3-葡聚糖酶的酶学特性进行了研究。结果表明,该酶最适反应温度为55℃、最适反应pH值为5.0。Co~(2+)、K+、Zn~(2+)、Li~+、Ba~(2+)、Cu~(2+)以及1.0mmol/L的Fe~(2+)对该酶没有影响,Cd~(2+)和10.0mmol/L的Mg~(2+)对酶具有部分抑制作用,而低浓度的Hg~(2+)、5.0mmol/L以上的Mn~(2+)和10.0 mmol/L的Fe~(3+)能强烈抑制该酶的活性。该酶只能作用于β-1,3-糖苷键,以Larinami为底物时其米氏常数K_m值为128.34μg/mL,最大反应速度V_m为23.01μg/(min·mL)。经过SDS-PAGE测定的分子质量近似为80.137ku。     

榆干离褶伞溶栓酶的纯化及酶学性质研究

采用离子交换层析、凝胶过滤层析及快速蛋白液相色谱等蛋白质分离纯化技术,从榆干离褶伞菌丝体中纯化一种溶栓酶,并探讨各种因子对榆干离褶伞溶栓酶活性的影响,观察溶栓酶对纤维蛋白及纤维蛋白原的水解特征和酰胺水解活性。实验结果表明,其分子质量约为50 ku,该酶的最适pH和最适温度为pH 6.0,35℃,并对tPA底物S-2288最敏感。Mn~(2+)和Mg~(2+)激活酶活性,而Cu~(2+),EDTA对酶活有抑制作用。该酶不仅直接水解纤维蛋白,还可以水解纤维蛋白原。榆干离褶伞很有潜力成为治疗血栓的新的药物来源。     

Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil

The Pseudomonas aeruginosa san-ai strain was isolated from water-soluble cutting oil used for cooling and lubrication during industrial metal-working processes. This strain, which is grown in a high alkaline (pH 10) mixture of surfactants and mineral oil, produces an extracellular proteolytic enzyme. We have purified and characterized this 18 kDa protease. The P. aeruginosa san-ai protease functions optimally at pH 9.0 and 60 degrees C. Additionally, it is a Zn-containing metalloenzyme, and its monomeric structure contains at least one disulfide bond. Because the enzyme is stable in the presence of organic solvents, it is suitable for peptide synthesis. Furthermore, the P. aeruginosa san-ai protease could be used in an intelligent drug delivery system (DDS) designed for applications in the metal industry for prevention of putrefaction of cutting oil.     

Purification and characterization of organic solvent-stable lipase from organic solvent-tolerant Pseudomonas aeruginosa LST-03

An organic solvent-stable lipase (LST-03 lipase) secreted into the culture broth of the organic solvent-tolerant Pseudomonas aeruginosa LST-03 was purified by ion-exchange and hydrophobic interaction chromatography in the presence of 2-propanol. The purified enzyme was homogeneous as determined by SDS-PAGE. The molecular mass of the lipase was estimated to be 27.1 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum pH and temperature were 6.0 and 37 degrees C. LST-03 lipase was stable at pH 5-8 and below 40 degrees C. Its hydrolytic activity was highest against tricaproin (C6), methyl octanoate (C8), and coconut oil respectively among the triacylglycerols, fatty acid methyl esters, and natural oils investigated. The enzyme cleaved not only the 1,3-positioned ester bonds, but also the 2-positioned ester bond of triolein. It exhibited high levels of activity in the presence of n-decane, n-octane, DMSO, and DMF as well as in the absence of an organic solvent. In addition, LST-03 lipase was stabler in the presence of n-decane, ethyleneglycol, DMSO, n-octane, n-heptane, isooctane, and cyclohexane than in the absence of an organic solvent.     

Extracellular polymeric substances from Bacillus subtilis associated with minerals modify the extent and rate of heavy metal sorption

Extracellular polymeric substances (EPS) are an important source of organic matter in soil. Once released by microorganisms, a portion may be sorbed to mineral surfaces, thereby altering the mineral?s ability to immobilize heavy metals. EPS from Bacillus subtilis were reacted with Ca-saturated bentonite and ferrihydrite in 0.01 M KCl at pH 5.0 to follow the preferential uptake of EPS-C, -N, and -P. The sorption kinetics of Pb(2+), Cu(2+), and Zn(2+) to the resulting EPS-mineral composites was studied in single and binary metal batch experiments ([metal](total) = 50 μM, pH 5.0). Bentonite sorbed much more EPS-C (18.5 mg g(-1)) than ferrihydrite (7.9 mg g(-1)). During sorption, EPS were chemically and size fractionated with bentonite favoring the uptake of low-molecular weight components and EPS-N, and ferrihydrite selectively retaining high-molecular weight and P-rich components. Surface area and pore size measurements by N(2) gas adsorption at 77 K indicated that EPS altered the structure of mineral-EPS associations by inducing partial disaggregation of bentonite and aggregation of ferrihydrite. Whereas mineral-bound EPS increased the extent and rate of Pb(2+), Cu(2+), and Zn(2+) sorption for bentonite, either no effect or a decrease in metal uptake was observed for ferrihydrite. The extent of sorption always followed the order Pb(2+) > Cu(2+) > Zn(2+), which also prevailed in binary Pb(2+)/Cu(2+) systems. In consequence, sorption of EPS to different minerals may have contrasting consequences for the immobilization of heavy metals in natural environments by inducing mineral-specific alterations of the pore size distribution and, thus, of available sorption sites.     

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.     

Partial purification and characterisation of dipeptidyl peptidase II from porcine skeletal muscle

The purification and study of biochemical properties of dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from porcine skeletal muscle have been carried out in the present work. The purification included ammonium sulphate fractionation and two HPLC chromatographic separations using a Resource-Q anion exchange column. The enzyme was purified 1270 fold, with a 1.6% recovery and was completely separated from DPP IV activity. The pure enzyme displayed one main protein band with a Mr of 58 kDa on SDS-PAGE. Maximum activity was reached at pH 5.5 and 65°C. Those synthetic substrates containing Pro in N-penultimate position were the most efficiently hydrolysed, whereas in the case of peptides, DPP II efficiently hydrolysed both X-Pro- and X-Ala- peptides. The serine peptidase inhibitors PMSF and Pefabloc SC suppressed DPP II activity in a high degree, whereas 3, 4-DCI and cysteine peptidase inhibitors exerted little effect. Alkaline metal salts inhibited the enzyme activity according to the size of the cation, and among the assayed divalent cations, only Cu(2+), Fe(2+) and Hg(2+) showed significant inhibition of the activity. This is the first time that porcine muscle DPP II has been purified and its biochemical characteristics studied. So, these results contribute to improve the knowledge in relation with the proteolytic chain and the generation of flavour characteristics in meat products.     

微生物产大豆异黄酮糖苷酶分离纯化的研究

利用离子交换层析法分离纯化了由菌种Absidia sp.SI39d发酵所产的1种高活性大豆异黄酮糖苷酶,经SDS-聚丙烯酰胺凝胶电泳检验得到电泳单点的纯酶,并对其酶性质进行了研究。提纯酶的活力达173U/ mL,酶蛋白的分子量为61ku,最适反应温度为40℃,最适pH值为5.0,在20～60℃,pH4.0～7.0之间酶活力相对稳定。Co~(2+)、Ca~(2+)、Zn~(2+)对酶活性无影响;Ag~+、Cu~(2+)对酶活性有抑制作用;C_([Fe~(3+)])<50mmol/L时,对酶活性无影响;当C_([Fe~(3+)])>50mmol/L时,有抑制作用。该酶的酶反应动力学方程为V=2.07[s]/(1.45+[s])。     

植酸与金属离子络合的研究

植酸络合金属离子可用于抑制酶促褐变和非酶褐变的发生，因而在食品工业表现出潜在的应用前景。应用量子化学EHT方法研究了植酸对钙、镁、铜、钴、镍金属离子的络合性能。络合结构分析表明，植酸与金属离子形成络合物的稳定性和配体分子轨道与金属轨道耦合程度有关；植酸对金属离子络合能力来自于其分子中磷酸基团的富电子性。     

Effective self-purification of polynary metal electroplating wastewaters through formation of layered double hydroxides

Heavy metal ions (Ni(2+), Zn(2+), and Cr(3+)) can be effectively removed from real polynary metal ions-bearing electroplating wastewaters by a carbonation process, with ～99% of metal ions removed in most cases. The synchronous formation of layered double hydroxide (LDH) precipitates containing these metal ions was responsible for the self-purification of wastewaters. The constituents of formed polynary metals-LDHs mainly depended on the Ni(2+):Zn(2+):Cr(3+) molar ratio in wastewaters. LDH was formed at pH of 6.0-8.0 when the Ni(2+)/Zn(2+) molar ratio ≥ 1 where molar fraction of trivalent metal in the wastewaters was 0.2-0.4, otherwise ZnO, hydrozincite, or amorphous precipitate was observed. In the case of LDH formation, the residual concentration of Ni(2+), Zn(2+), and Cr(3+) in the treated wastewaters was very low, about 2-3, ～2, and ～1 mg/L, respectively, at 20-80 °C and pH of 6.0-8.0, indicating the effective incorporation of heavy metal ions into the LDH matrix. Furthermore, the obtained LDH materials were used to adsorb azoic dye GR, with the maximum adsorption amount of 129-134 mg/g. We also found that the obtained LDHs catalyzed more than 65% toluene to decompose at 350 °C under ambient pressure. Thus the current research has not only shown effective recovery of heavy metal ions from the electroplating wastewaters in an environmentally friendly process but also demonstrated the potential utilization of recovered materials.     

米黑根毛霉脂肪酶基因在毕赤酵母中的高效表达

脂肪酶是主要工业用酶制剂之一,在食品、洗涤剂和制药等领域广泛应用。将编码米黑根毛霉(Rhizo-mucor miehei)脂肪酶RML的基因克隆到pPIC9K载体中,构建了分泌型表达载体pPIC9K-RML,载体经线性化后转化Pichia pastorisGS115,G418梯度筛选获得了分泌表达RML的重组毕赤酵母工程菌,SDS-PAGE分析显示表达的脂肪酶分子量大小与预期一致。初步研究表明,重组脂肪酶最适温度为40℃,最适pH值为8.0,以橄榄油为底物时,发酵上清液酶活最大可达102 U/mL,表明构建的重组毕赤酵母工程菌具有较好的工业化生产潜力。