Separation of dilute aqueous butanol and acetone solutions by pervaporation through liquid membranes

A simultaneous extraction-stripping process is proposed for separating volatile products from fermentation broths, it is based on pervaporation through a liquid membrane supported with a hydrophobic porous membrane. The liquid membrane prepared with oleyl alcohol was selected as the most suitable for separating volatile products resulting from acetone-butanol fermentation. The separation performance and stability of the oleyl alcohol liquid membrane were investigated by using dilute aqueous butanol and acetone solutions. The oleyl alcohol liquid membrane was found to be superior by far in both selectivity and permeability of butanol to the better known silicone rubber membrane in its high selectivity for alcohols. Using the oleyl alcohol liquid membrane, the dilute aqueous butanol solutions of around 4 g/L obtained in acetone-butanol fermentation could be concentrated up to 100 times. The stability of this liquid membrane was also quite good as long as the surface tension of the feed solution was less than the critical surface tension of the support membrane. No change in the separation performance was found after the continuous usage in a long period of 100 h.     

Production of toluene cis-glycol by Pseudomonas putida in glucose feb-batch culture

Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.     

Immobilization of cellulolytic and hemicellulolytic enzymes on inorganic supports

Commercial cellulase preparations from Trichoderma viride and Aspergillus niger were immobilized on porous silica glass and ceramics such as alumina and titania with titanium tetrachloride (TiCl(4)) and on their silanized derivatives with glutaraldehyde (GLUT). The amounts of the immobilized enzymes were in the range 10-50 mg/g carrier (dry) depending on the kind of carrier and immobilization method. Their activities toward carboxymethyl cellulose (CMC), xylan, aryl-beta-glucoside, and aryl-beta-xyloside were 3-53% of those of the native enzymes. The optimum pH of the enzymes shifted to the acidic side in most cases, whereas the optimum temperatures were nearly the same as those of native ones. The activity of immobilized enzyme preparations towards CMC did not change significantly during continuous operation over a periods of 60 days. Finally, xylan was hydrolyzed with the immobilized enzymes, and the sugars formed were investigated.     

Distribution of heparinase covalently immobilized to agarose: Experimental and theoretical studies

An immobilized enzyme reactor has been developed to remove heparin, the anticoagulant that is required in all extracorporeal devices for patients undergoing open-heart surgery or kidney dialysis. The device uses the enzyme heparinase (EC 4.2.2.7), which is covalently linked to agarose with cyanogen bromide. A critical parameter in the development of a model for the degradation of heparin catalyzed by immobilized heparinase is the radial concentration profile of the enzyme within the agarose matrix. Experimental determinations of bound enzyme con centrations have been conducted previously for several enzyme systems using radioactive or fluorescent labels. For the development of the heparinase reactor it is necessary to use catalytically but not electrophoretically pure enzyme, and thus it is not possible to use the labeling techniques. To obtain information about the bound enzyme distribution, an experimental study of the intrinsic binding kinetics of heparinase to cyanogen bromide-activated agarose was conducted. The binding reaction was studied as a function of both the concentration of heparinase and the gel-reactive group. At conditions of functional group excess, the binding kinetics were pseudo first order in heparinase concentration with a rate constant equal to 0.12 C(c[triple chemical bond]n) (h(-1)), where C(c[triple chemical bond]n) is the gel-reactive group concentration. The reactive group concentration remained constant within the 2-4-h experiments. Competitive binding between heparinase and the protein contaminants was unimportant. A model was formulated for the immobilization procedure based on the diffusion of heparinase within the porous network and the binding kinetics as determined above. The model predicted the immobilization of heparinase to be kinetically controlled and the enzyme to distribute uniformly within the agarose matrix. These experimental techniques could be applied to predict the immobilized enzyme distribution for different enzyme systems that are not electrophoretically pure.     

Immobilized heparinase: In vitro reactor model

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 +/- 0.048 (N = 10). The immobilized enzyme has a K(m) of 0.15 +/- 0.03 mg/mL and an activation energy of 10.3 +/- 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 x 10(6) daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

Chloroplast genome evolution in the genus Avena

The genus Avena contains five different chloroplast genomes, I-V. A physical map of chloroplast (ct) DNA of Avena sativa (type I chloroplast genome) was constructed using three restriction endonucleases, PstI, SalI and SmaI. This genome is ca. 135.5 kbp in size, and contains two inverted repeats of ca. 22.5 kbp each, separated by a large (ca. 79.0 kbp) and small (ca. 12.5 kbp) single copy region. The rbcL gene which codes for the large subunit of ribulose 1,5-bisphosphate carboxylase, was located in the map. Restriction fragment patterns of all five chloroplast genomes were compared, and among them five fragment size and five restriction site mutations were disclosed. Four site mutations were found in two or more chloroplast genomes, the other site and five fragment size mutations were specific to one or another of the chloroplast genomes. A dendrogram showing phylogenetic relationships among the five chloroplast genomes, based on the distribution of the common and specific mutations among them, indicates that chloroplast genome divergence characterized by three restriction site mutations occurred first between two diploid groups, each carrying A and C genome (nuclear), respectively, followed by further speciation in each group.     

L-赖氨酸分批发酵连续补糖的研究

用L-赖氨酸产生株纯齿棒状杆菌PI-3-2(Hsc^-,AEC⁺)在8L自控发酵小罐上,用恒稀释率指数递增方式连续补加葡萄糖液进行L-赖氨酸分批发酵的研究。结果表明,一次投糖分批发酵时,较高糖浓度使比产酸速率Qp值下降,不能有效地提高产酸水平。采用连续补糖方式可以改变菌体竞争底物的能力或改善代谢途径,增大耗底物分数a2或真正产酸率yp,;从而增加表观产酸率Yp值,提高葡萄糖转化率。此方式的发酵属Caden动力学分类第I型,在发酵的中后期控制H等条件,可增加比产酸速率Qp值,提高发酵水平。PI-3-2菌株的产酸水平可由47．Mg／ml提高到64．2mg／ml(总糖浓度18．18％时),避离可达73．3mg／m1(总糖浓度22．73mg／ml时)。