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Characterization of flocculation for cell removal from fermentation broth via polyelectrolyte addition is commonly based on qualitative methods such as physical appearance of the floc. The use of zeta potential as a quantitative measure of floc character was evaluated as an indicator of optimal polymer addition. Zeta potential was found to increase with increasing cationic polyelectrolyte dosage, but never reached zero regardless of the total amount of polymer added, indicating flocculation occurs at least partially through a bridging type mechanism. Experiments were conducted using various polymer concentrations (25-75 g/L) and dosing methods (batch, incremental and continuous addition) that resulted in variable overall polymer requirements to achieve optimum flocculation. Zeta potential was found to be constant at optimal floc character regardless of the total amount of polymer added, polymer concentration, or method of polymer addition. Experiments with two additional types of fermentation broth also showed characteristic zeta potentials at optimal flocculation. Polymer requirements to achieve a particular floc character can vary greatly, depending on polymer dosing conditions and fermentation batch. The effect of polymer dosing conditions on the polymer requirement to obtain optimal floc character was evaluated. Polymer dosing method and calcium concentration were both found to have a significant effect (P < 0.0001) with continuous polymer addition and high calcium concentration requiring less polymer than did batch polymer addition and low calcium concentration, respectively. Polymer dosing concentration did not significantly affect polymer requirement for optimal flocculation. 相似文献
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杨缜 《生物化学与生物物理进展》1995,22(4):340-345
经氰尿酰氯和对硝基苯碳酸酯活化过的甲氧基聚乙二醇分别用来对枯草杆菌蛋白酶进行化学修饰．修饰后的酶在水溶液和有机溶剂中均保持活性．酶在水溶液里的kcat增加,Km不变.酶对温度和pH的稳定性都显著升高,但最佳反应温度不变． 相似文献
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Q103R subtilisin E was isolated following random mutagenesis and screening for improved activity in the presence of dimethylformamide (DMF). Our goal is to identify the mechanism(s) by which amino acid substitutions can enhance enzyme activity in polar organic solvents. A quantitative framework for comparing substrate binding and catalytic activities of mutant and wild-type enzymes in the presence and absence of DMF is outlined. Kinetic experiments performed at high salt concentration (1M KCl) reveal that the mechanism behind the Q103R variant's enhanced activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is both electrostatic and nonelectrostatic in origin. Favorable electrostatic interactions between the negatively charged succinyl group of the substrate and the positive charge on Arg 103 are responsible for tighter substrate binding. This conclusion is supported by kinetic experiments performed on the related substrate Ala-Ala-Pro-Phe-p-nitroanilide and the hydrolysis kinetics of the Q103E, Q103K, and Q103S variants constructed by site-directed mutagenesis. These results highlight the importance of the choice of the substrate used to screen for improvements in catalytic activity. 相似文献
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Akbar U Aschenbrenner CD Harper MR Johnson HR Dordick JS Clark DS 《Biotechnology and bioengineering》2007,96(6):1030-1039
A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed \"direct solubilization,\" this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation. 相似文献
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Carter B Squillace P Gilcrease PC Menkhaus TJ 《Biotechnology and bioengineering》2011,108(9):2053-2060
This study investigated the detoxification of a dilute acid pretreated Ponderosa pine slurry using the polyelectrolyte polyethyleneimine (PEI). The addition of polyelectrolyte to remove enzymatic and/or fermentation inhibitory compounds, that is, acetic acid, furfural, and 5-hydroxymethylfurfural (HMF), was performed either before or after enzymatic hydrolysis to determine the optimal process sequence. Negligible acetic acid, glucose, and xylose were removed regardless of where in the process the polymer addition was made. Maximum furfural and HMF separation was achieved with the addition of PEI to a clarified pre-enzymatic hydrolysis liquor, which showed that 88.3% of furfural and 66.4% of HMF could be removed. On the other hand, only 23.1% and 13.4% of furfural and HMF, respectively, were removed from a post-enzymatic hydrolysis sample; thus, the effects of enzymes, glucose, and wood solids on inhibitor removal were also investigated. The presence of solid particles >0.2 μm and unknown soluble components <10 kDa reduced inhibitory compound removal, but the presence of elevated glucose levels and enzymes (cellulases) did not affect the separation. The fermentability of detoxified versus undetoxified hydrolysate was also investigated. An ethanol yield of 92.6% of theoretical was achieved with Saccharomyces cerevisiae fermenting the detoxified hydrolyzate, while no significant ethanol was produced in the undetoxified hydrolyzate. These results indicate that PEI may provide a practical alternative for furan removal and detoxification of lignocellolosic hydrolysates, and that application before enzymatic hydrolysis minimizes separation interferences. 相似文献
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AIMS: The objective of this study is to actively express a novel fibrinolytic enzyme, subtilisin DFE (douchi fibrinolytic enzyme), in Escherichia coli. METHODS AND RESULTS: The DNA fragments encoding pro-subtilisin DFE was amplified and cloned into the vector pET32a to obtain N-terminal Trx fusion expression plasmid. The recombinant subtilisin DFE was successfully expressed and processed in the soluble fraction of E. coli BL21(DE3) in a similar fashion as the endogenous one of Bacillus amyloliquefaciens DC-4, resulting in an active enzyme. Moreover, active enzyme can also be refolded from inclusion body. CONCLUSIONS: Active subtilisin DFE can be expressed and processed in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidences that subtilisin DFE can be actively expressed in E. coli and the pro-peptide is essential for guiding the proper folding into the active conformation. As such, large quantities of recombinant subtilisin DFE can be produced for pharmacological and clinical research. 相似文献
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Subtilisin Carlsberg was covalently attached to five macroporous acrylic supports of varying aquaphilicity (a measure of hydrophilicity). Kinetic parameters of the transesterification of S and R enantiomers of secphenethyl alcohol with vinyl butyrate, catalyzed by various immobilized subtilisins, were determined in anhydrous dioxane and acetonitrile. Enzyme enantioselectivity in acetonitrile, but not in dioxane, correlated with the aquaphilicity of the support; a mechanistic rationale for this phenomenon was proposed. Although the catalytic activity of immobilized subtilisin in anhydrous solvents strongly depended on enzyme pretreatment, the enantioselectivity was essential conserved. (c) 1994 John Wiley & Sons, Inc. 相似文献
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高温高浓发酵技术作为一项新兴的啤酒生产技术,它为啤酒生产带来诸多利益的同时,也存在着发酵结束后酵母絮凝性下降、高级醇生成量过高等系列问题。为提高高温高浓发酵条件下酿酒酵母的絮凝性同时降低高级醇的合成能力,首先构建了以酿酒酵母BAT2基因为整合位点过表达FLO5基因的菌株,重组菌株S6-BF的絮凝性达到67.67%,比出发菌株S6提高了29%,而高级醇生成量仅降低5.9%;进一步构建以BAT2基因为整合位点再次过表达FLO5基因的菌株,与出发菌株S6相比,重组菌株S6-BF2的絮凝性提高了63%,达到85.44%,高级醇生成量下降至159.58 mg/L,降低了9.0%;通过弱化线粒体支链氨基酸转氨酶(BAT1)的表达,高级醇的生成量得到进一步的降低,达到142.13 mg/L,比原始菌株S6降低了18.4%,同时重组菌株S6-BF2B1的絮凝性没有受到影响;风味物质的测定结果表明啤酒中醇酯比例较为合理。研究结果对工业啤酒酵母发酵后的沉降分离和提高啤酒风味品质有着重要的意义。 相似文献
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Formation of noncovalent complexes between tyrosinase from mushrooms and a cationic polyelectrolyte, polybrene (PB, poly (1,5-dimethyl-1,5-diazaundecamethyelene) bromide), was shown to activate and stabilize tyrosinase in water-ethanol mixtures. In the reaction of catechol oxidation in aqueous solutions, catalytic activity (k(cat)) of tyrosinase-PB complex ([PB]/[tyrosinase] molar ratio 100:1, per mole of polymer) in a wide range of pH was higher than that of free tyrosinase. In aqueous solutions and in water-ethanol mixtures at moderate concentrations of ethanol (10-40% v/v), the value of k(cat) of tyrosinase-PB complex exceeded the activity of free enzyme, from 1.2-2-fold, accompanied by the essential (up to 10-fold) increase in the value of the specificity constant (k(cat)/K(m)). The results are of practical importance for the construction of biocatalysts working successfully in polar organic media. 相似文献
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血栓溶解酶产生菌及其培养条件的研究 总被引：11，自引：1，他引：11
通过对根霉１２号发酵液的分析及发酵条件的研究,发现其发酵液中含有能溶解血栓的物质,但不能分解血细胞。它在麸皮胰蛋白胨培养基（ｐＨ５．１）上３０℃振荡培养４８～６０ｈ,ｐＨ值达到７．４左右时产生血栓溶解酶的活力最高。 相似文献
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Over the last 40 years, the fermentation industry has provided facility planners, plant operators and environmental engineers with a wide range of residuals management challenges and resource/energy recovery opportunities. In response, the industry has helped pioneer the use of a number of innovative resource and energy recovery technologies. Production of animal feed supplements, composts, fertilizers, soil amendments, commercial baking additives and microbial protein materials have all been detailed in the literature. In many such cases, recovery of by-products significantly reduces the need for treatment and disposal facilities. Stable, reliable anaerobic biological treatment processes have also been developed to recovery significant amounts of energy in the form of methane gas. Alternatively, dewatered or condensed organic fermentation industry residuals have been used as fuels for incineration-based energy recovery systems. The sale or use of recovered by-products and/or energy can be used to offset required processing costs and provide a technically and environmentally viable alternative to traditional treatment and disposal strategies. This review examines resource recovery options currently used or proposed for fermentation industry residuals and the conditions necessary for their successful application. 相似文献
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During continuous alcoholic fermentation, Saccharomyces cerevisiae 38A floc concentration was monitored using an on-line impedance probe. Since the sensor response is linear and does not depend significantly on yeast particle size, an automatic technique of determining the yeast growth rate has been developed and validated against the conventional mass balance method. 相似文献
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Lucília Domingues António A. Vicente Nelson Lima José A. Teixeira 《Biotechnology and Bioprocess Engineering》2000,5(4):288-305
A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects—the basics of yeast flocculation, the development of “new” flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer's yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculentSaccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems. 相似文献
16.
Lousa D Baptista AM Soares CM 《Protein science : a publication of the Protein Society》2011,20(2):379-386
The phenomenon known as \"ligand imprinting\" or \"ligand-induced enzyme memory\" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation. 相似文献
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控制发酵参数对提高葡萄糖异构酶活力影响明显,尤其是通气比和ｐＨ影响更大。通过观察其菌丝形态变化能预测其胞内酶比例,有效确定放罐时间,提高产得率。 相似文献
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