Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.     

碳纳米管固定化纤维素酶的最佳工艺研究

纤维素酶在环保、医药、食品等领域都具有广泛的应用前景,但由于纤维素酶的生产成本较高,生物活性较低,使得纤维素酶的应用受到了限制。为了寻找一种固定化纤维素酶的方法,使酶可以重复多次使用,首次以多壁碳纳米管为载体固定化纤维素酶,研究功能化的多壁碳纳米管固定化纤维素酶的固定化条件,采用正交试验对酶固定化中的主要条件进行优化,并通过傅里叶变换红外光谱仪对多壁碳纳米管（multiwalled carbon nanotube,MWCNTs）、纤维素酶及固定化纤维素酶的结构进行表征。结果表明,固定化纤维素酶的最佳工艺条件为：酶浓度5 mg·mL-1,温度40 ℃,pH 5.0,固定化时间3 h;通过傅里叶变换红外光谱证实纤维素酶成功固定到多壁碳纳米管上。     

植物-固定化菌剂联合修复多环芳烃污染土壤

以火凤凰根际土壤中发现的3种优势菌[分枝杆菌(Ⅰ)、产黄纤维单胞菌(Ⅱ)、少动鞘氨醇单胞菌(Ⅲ)]构建的多菌剂体系为供试菌剂,针对大港油田原油污染土壤,将固定化供试菌剂接种于修复植物火凤凰根际,探讨供试菌剂强化火凤凰修复多环芳烃(PAHs)污染土壤的效果。结果表明: 处理ⅠⅢ(有效活菌数为10⁹ cfu·mL^-1)和ⅠⅡⅢ(有效活菌数为10⁷ cfu·mL^-1)对PAHs的降解有促进作用,PAHs降解率分别为32.2%和41.4%,均显著高于相应对照处理。此外,处理ⅠⅡⅢ对火凤凰的地下生物量有明显促进作用,比对照处理增加了31.2%。表明由3种优势菌构建的多菌剂ⅠⅡⅢ可以作为火凤凰修复PAHs污染土壤的强化手段,为微生物强化植物修复技术提供了新的修复思路及方法。     

Asymmetric ammonolysis of (R/S)-mandelic acid by immobilized lipases via direct amidation of mandelic acid in biphasic media

Abstract

We have investigated the direct enantioselective amidation of mandelic acid with ammonia, catalyzed by a variety of commercial lipases including those from Candida rugosa, Mucor miehei, Pseudomonas sp., Rhizomucor miehei, and Thermomyces lanuginosus covalently immobilized onto Florisil^® support via glutaraldehyde and polysuccinimide spacer arms. All the immobilized lipase preparations tested preferentially amidated the R isomer of mandelic acid. The highest amide yields were obtained for immobilized Pseudomonas sp. lipase preparations under the optimized reaction conditions. After 24 h of amidation, the reaction had proceeded with an excellent yield (50%) and enantiopurity (> 99%). The immobilized Pseudomonas sp. lipase preparations catalyzed the amidation reaction with the same yield and enantioselectivity. The enzyme immobilized via a glutaraldehyde spacer arm showed better reusability than that immobilized via a polysuccinimide spacer arm.

In view of these results, it is revealed that the direct amidation of mandelic acid catalyzed by the immobilized Pseudomonas sp. lipases is a facile and effective methodology for obtaining (S)-mandelic acid and (R)-mandelamide.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

Biodegradation of Phenol Using Immobilized Nocardia hydrocarbonoxydans in a Pulsed Plate Bioreactor: Effect of Packed Stages,Cell Carrier Loading,and Cell Acclimatization on Startup and Steady-State Behavior

The effect of the number of stages and cell carrier loading on the steady-state and startup performance of a continuous pulsed plate bioreactor with glass beads as the cell carrier material for biodegradation of phenol in wastewater using immobilized Nocardia hydrocarbonoxydans has been studied. It was found that the performance of the pulsed plate bioreactor during startup and at steady state can be improved by an increase in cell carrier loading, number of stages, total plate stack height, and with a decrease in plate spacing. The startup time for the continuous bioreactor can be decreased by increasing the number of preacclimatization steps for the cells. The attainment of steady effluent phenol concentration can be considered as an indication of steady state of the continuous bioreactor, as when phenol concentration attained a steady value, biofilm thickness, and the attached biomass dry weight also attained a constant value.     

A novel Aurora-A-mediated phosphorylation of p53 inhibits its interaction with MDM2

Purpose: Crosstalk between Aurora-A kinase and p53 has been proposed. While the genetic amplification of Aurora-A has been observed in many human cancers, how p53 is regulated by Aurora-A remains ambiguous. In this study, Aurora-A-mediated phosphorylation of p53 was analyzed by mass spectrometry in order to identify a new phosphorylation site. Subsequently, the functional consequences of such phosphorylation were examined. Experimental design: In vitro phosphorylation of p53 by Aurora-A was performed and the phosphorylated protein was then digested with trypsin and enriched for phosphopeptides by immobilized metal affinity chromatography. Subsequently, a combination of β-elimination and Michael addition was applied to the phosphopeptides in order to facilitate the identification of phosphorylation sites by MS. The functional consequences of the novel phosphorylation of p53 on the protein–protein interactions, protein stability and transactivation activity were then examined using co-immunoprecipitation, Western blotting and reporter assays. Results: Ser-106 of p53 was identified as a novel site phosphorylated by Aurora-A. A serine-to-alanine mutation at this site was found to attenuate Aurora-A-mediated phosphorylation in vitro. In addition, phosphate-sensitive Phos-tag SDS-PAGE was used to confirm that the Ser-106 of p53 is in vivo phosphorylated by Aurora-A. Finally, co-immunoprecipitation studies suggested that Ser-106 phosphorylation of p53 decreases its interaction with MDM2 and prolongs the half-life of p53. Conclusions: The inhibition of the interaction between p53 and MDM2 by a novel Aurora-A-mediated p53 phosphorylation was identified in this study and this provides important information for further investigations into the interaction between p53 and Aurora-A in terms of cancer biology.     

Proteome analysis of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae

The plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight, which is one of the most serious diseases of rice. Xoo has been studied for over one century, and much has been learned about it, but proteomic investigation has been neglected. In this study, proteome reference maps of Xoo were constructed by two-dimensional gel electrophoresis, and 628 spots in the gels representing 469 different protein species were identified with MALDI-TOF/TOF MS. The identified spots were assigned to 15 functional categories according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the annotations from the National Center for Biotechnology Information (NCBI) database. The data set has been deposited in the World-2DPAGE database (Database ID: 0044). In addition, comparative proteomic analysis revealed that proteins related to the TonB-dependent transportation system and energy metabolism are involved in the phenazine-1-carboxylic acid resistance in Xoo. In conclusion, we have established a proteome database for Xoo and have used this database in a comparative proteomic analysis that identified proteins potentially contributing to phenazine-1-carboxylic acid resistance in Xoo.