Immobilization of enzymes on spheron: 1. Trypsin and glucoamylase by the titanium-chelation method

In a preliminary study, trypsin (EC 3.4.21.4) and glucoamylase (exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) were immobilized on Spheron by the titanium-chelation method. The activity of trypsin immobilized on Spheron P100 000 was higher against tosyl-l-arginine 4-nitroanilide than against casein. The variation in the specific activities of glucoamylase immobilized on Spherons of different porosities to wards substrates of different molecular weights was examined.     

Immobilization of dextranase on bentonite

Dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) from Penicillium aculeatum culture has been immobilized on a bentonite support. The matrix-bound enzyme could be stored as acetone-dried powder or as a suspension in acetate buffer, pH 5.6, for about three weeks at 4°C without any loss of activity. There was no change in the specific activity of the enzyme on immobilization and the enzyme yield was 0.1–0.6 mg/g bentonite matrix. In the presence of sucrose, thermal stability of the immobilized enzyme was high and the bound enzyme could be used for about six cycles.     

糖芯片制备技术研究进展

糖芯片是一种分析糖-蛋白质相互作用最直接有效的方法.对目前糖芯片的各种制备方法进行了综述,并分析了影响糖芯片质量的一些关键因素及其对底片材料和固定方法的整体要求,进而对该领域发展方向和广阔的应用前景进行了展望.     

Site-directed chemically-modified magnetic enzymes: fabrication,improvements, biotechnological applications and future prospects

Numerous enzymes of biotechnological importance have been immobilized on magnetic nanoparticles (MNP) via random multipoint attachment, resulting in a heterogeneous protein population with potential reduction in activity due to restriction of substrate access to the active site. Several chemistries are now available, where the modifier can be linked to a single specific amino acid in a protein molecule away from the active-site, thus enabling free access of the substrate. However, rarely these site-selective approaches have been applied to immobilize enzymes on nanoparticles. In this review, for the first time, we illustrate how to adapt site-directed chemical modification (SDCM) methods for immobilizing enzymes on iron-based MNP. These strategies are mainly chemical but may additionally require genetic and enzymatic methods. We critically examine each method and evaluate their scope for simple, quick, efficient, mild and economical immobilization of enzymes on MNP. The improvements in the catalytic properties of few available examples of immobilized enzymes are also discussed. We conclude the review with the applications and future prospects of site-selectively modified magnetic enzymes and potential benefits of this technology in improving enzymes, including cold-adapted homologues, modular enzymes, and CO₂-sequestering, as well as non-iron based nanomaterials.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis

Chemical protein biotinylation and streptavidin or anti‐biotin‐based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine‐tuning of critical parameters is important to reduce biotinylation in a protein binding site.     

EFFECT OF FERMENTATION PARAMETERS ON BIO-ALCOHOLS PRODUCTION FROM GLYCEROL USING IMMOBILIZED Clostridium pasteurianum: AN OPTIMIZATION STUDY

This article addresses the issue of effect of fermentation parameters for conversion of glycerol (in both pure and crude form) into three value-added products, namely, ethanol, butanol, and 1,3-propanediol (1,3-PDO), by immobilized Clostridium pasteurianum and thereby addresses the statistical optimization of this process. The analysis of effect of different process parameters such as agitation rate, fermentation temperature, medium pH, and initial glycerol concentration indicated that medium pH was the most critical factor for total alcohols production in case of pure glycerol as fermentation substrate. On the other hand, initial glycerol concentration was the most significant factor for fermentation with crude glycerol. An interesting observation was that the optimized set of fermentation parameters was found to be independent of the type of glycerol (either pure or crude) used. At optimum conditions of agitation rate (200 rpm), initial glycerol concentration (25 g/L), fermentation temperature (30°C), and medium pH (7.0), the total alcohols production was almost equal in anaerobic shake flasks and 2-L bioreactor. This essentially means that at optimum process parameters, the scale of operation does not affect the output of the process. The immobilized cells could be reused for multiple cycles for both pure and crude glycerol fermentation.     

Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption

In this article, surface coatings derived from homo-bifunctional tri(ethylene glycol) (EG₃) and hexa(ethylene glycol) (EG₆) molecules which have two terminal aldehyde groups are reported. These homo-bifunctional molecules can be used to functionalize amine-terminated surfaces through crosslinking one aldehyde group to surface amine groups, while leaving the other aldehyde group available for covalent immobilization of proteins. Best of all, after reducing remaining aldehyde groups on the surface with a reducing agent, sodium borohydride, the surface becomes oligo(ethylene glycol) (OEG)-terminated. The OEG-terminated surface can resist nonspecific protein adsorption, a feature that is often required for many biosensors and biomedical devices. Although some mixed self-assembled monolayers formed from two different organothiols also permit covalent protein immobilization and resist nonspecific protein adsorption, the procedure reported herein requires only one type of homo-bifunctional molecule and can be applied to both silicon and gold surfaces.     

Site-directing an intense multipoint covalent attachment (MCA) of mutants of the Geobacillus thermocatenulatus lipase 2 (BTL2): Genetic and chemical amination plus immobilization on a tailor-made support

Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst⁻¹ min⁻¹) as the reference in the production of PUFAs.     

高效删除标记基因的Bhlea2植物表达载体的构建

为培育去除选择标记基因的耐旱转基因植物,同时利用Cre/Lox和FLP/frt系统,构建一个能够高效删除标记基因的Bhlea2基因植物表达载体.拟南芥rd29A启动子是在低温、干旱、高盐胁迫下的快速应答启动子,玉米ubiquitin启动子可有效驱动外源基因的转录,拟南芥pAB5启动子是花粉及胚胎等发育早期特异表达的启动子,利用上述启动子构建了表达Bhlea2基因并能够删除标记基因的植物表达载体.该表达载体包括重组酶表达元件pAB5-FLP、Bhlea2抗旱基因表达元件rd29A-Bhlea2和bar标记基因表达元件ubiquitin-bar.