Cost-efficient entrapment of ß-glucosidase in nanoscale latex and silicone polymeric thin films for use as stable biocatalysts.

ß-Glucosidase is an ubiquitous enzyme which has enormous biotechnological applications. Its deficiency in natural enzyme preparations is often overcome by exogenous supplementation, which further increases the enzyme utilization cost. Enzyme immobilization offers a potential solution through enzyme recycling and easy recovery. In the present work Aspergillus niger ß-glucosidase is immobilized within nanoscale polymeric materials (polyurethane, latex and silicone), through entrapment, and subsequently coated onto a fibrous support. Highest apparent activity (90 U g(-1) polymer) was observed with latex, while highest entrapment efficiency (93%) was observed for the silicone matrix. Immobilization resulted in the thermo-stabilization of the ß-glucosidase with an increase in optimum temperature and activation energy for cellobiose hydrolysis. Supplementation to cellulases also resulted in an increased cellulose hydrolysis, while retaining more than 70% functional stability. Hence, the current study describes novel preparations of immobilized ß-glucosidase as highly stable and active catalysts for industrial food- and bio-processing applications.

Enzyme-enabled responsive surfaces for anti-contamination materials.

Many real-life stains have origins from biological matters including proteins, lipids, and carbohydrates that act as gluing agents binding along with other particulates or microbes to exposed surfaces of automobiles, furniture, and fabrics. Mimicking naturally occurring self-defensive processes, we demonstrate in this work that a solid surface carrying partially exposed enzyme granules protected the surface in situ from contamination by biological stains and fingerprints. Attributed to the activities of enzymes which can be made compatible with a wide range of materials, such anti-contamination and self-cleaning functionalities are highly selective and efficient toward sticky chemicals. This observation promises a new mechanism in developing smart materials with desired anti-microbial, self-reporting, self-cleaning, or self-healing functions.

Poly(ethylene glycol) conjugated enzyme with enhanced hydrophobic compatibility for self-cleaning coatings.

Enzyme-based smart materials constitute a rapidly growing group of functional materials. Often the natively evolved enzymes are not compatible with hydrophobic synthetic materials, thus significantly limiting the performance of enzymes. This work investigates the use of a polyethylene glycol (PEG)-conjugated detergent enzyme for self-cleaning coatings. As a result, PEG conjugated α-amylase demonstrated a much more homogeneous distribution in polyurethane coatings than the parent native enzyme as detected by both fluorescent microscopy and scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (SEM-EDX). Additionally, the conjugated enzyme showed enhanced retention in the coating and much improved thermal stability with a halflife of 20 days detected at 80 °C and over 350 days under room temperature. Such coating-incorporated enzyme afforded interesting self-cleaning functionality against starch-based stains as examined through a slipping drop test.

Organic-soluble enzyme nano-complexes formed by ion-pairing with surfactants.

The solubilization of enzymes in organic solvents for non-aqueous biocatalysis has attracted considerable attention since the homogeneous distribution accounts for a drastically improved reaction efficiency compared to enzymes dispersed as aggregates in an organic phase. This chapter highlights ion-pairing as a valuable and facile method to make enzymes soluble in organic solvents. Ion-pairing denotes the formation of a nano-complex, in which a single enzyme molecule in the core is surrounded by counter-charged surfactant molecules. The special architecture of this nano-complex exposes the surfactant hydrophobic group toward the bulk solvent and renders the complex sufficiently soluble in organic media. This chapter also describes the underlying principle of ion-pairing as well as simple preparation and characterization techniques to yield highly active enzyme-surfactant nano-complexes. The general applicability of this technique is demonstrated on the base of the hydrolytic enzyme α-chymotrypsin (α-CT) and the redox enzyme glucose oxidase (GO( x )).

Entrapment of enzymes in nanoporous sol-gels.

The prerequisite for many successful enzyme-based biotechnologies is the preparation of highly stable and active biocatalysts, which can be achieved effectively by immobilization. This chapter introduces the immobilization of enzymes by entrapment in nanoporous silica particles made in a sol-gel process. These easily tailorable materials have been proven very beneficial for a broad variety of applications of biocatalysts. Besides the spatial confinement in silica sol-gels, another advantage is given by the easy possibility of fine-tuning the physicochemical properties of the matrix itself to provide the ideal environment for the reaction and the biocatalyst. Preparation details are demonstrated using the process of immobilizing a lipase in a sol-gel matrix, which is chemically modified by using methyl-, ethyl-, propyl-, and i-butyltrimethoxysilane. The transesterification of canola oil with methanol is used as a model reaction.

Nanoporous silica glass for the immobilization of interactive enzyme systems.

Recent pursuit on utilization of nanoscale materials has manifested a variety of configurations of highly efficient enzymic biocatalyst systems for biotechnological applications. Nanoscale structures are particularly powerful in effecting multienzyme biocatalysis. Inherent properties of nanomaterials--primarily, the high surface area to volume ratio and atomic scale 3D configurations--enable higher enzyme loadings, microenvironment control surrounding enzyme molecules, regulation on mass transfer, and protein structural stabilization of the biocatalyst as compared to traditional immobilization systems. This chapter introduces one versatile nanoscale immobilization method via details demonstrated using the case of nanoporous silica glass (30 nm diameter) for the concomitant incorporation of lactate dehydrogenase (LDH), glucose dehydrogenase (GDH), and the cofactor (NADH).

Encapsulation of synthetically valuable biocatalysts into polyelectrolyte multilayer systems.

Layer-by-Layer (LbL) technology recently turned out to be a versatile tool for the encapsulation of bioactive entities. In this study, the factual potential of this technology to encapsulate synthetically valuable biocatalysts, that is enzymes and whole cells expressing a specific catalytic activity, was investigated. The biocatalysts were embedded into a polyelectrolyte multilayer system involving poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS). The enzymes were adsorbed to CaCO3 or DEAE-cellulose previous to encapsulation. A slight increase (32%) of the catalytic performance was observed for lipase B from Candida antarctica when four layers of polyelectrolytes were applied. On the whole, however, the residual activity of the investigated enzymes after encapsulation was rather low. Similar results were obtained with whole-cell biocatalysts. It was found that the activity decrease can be attributed to mass transfer restrictions as well as direct interactions between polyelectrolytes and catalytically active molecules. Both effects need to be understood in more detail before LbL technology can be advanced to technically efficient biocatalysis.

Colorimetric assay for sensitive poly(styrene sulfonate) quantification in a template directed polyelectrolyte-assembling process.

Template directed Layer-by-layer (LbL) technology recently moved into the center of scientific attention, particularly as a versatile tool for bioencapsulation purposes. Its major advantages can be found in the striking simplicity of tuning wall properties and the complete control over layer thickness and permeability. Yet, for the most commonly applied pair of polyelectrolytes, poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS), the mandatory control of the successful deposition on plane and colloidal surfaces is currently only attainable by means of sophisticated and expensive equipment. Here we describe an alternative quantification method based on a simple colorimetric assay using the Bradford reagent, a cost-effective commercially available dye, and standard laboratory technical devices. The binding of the dye to PSS causes a distinct shift of the absorption maximum from 465 to 680 nm, providing a method for spectral quantification of submicrogram amounts of dissolved PSS during LbL coating with significant accuracy and excellent reproducibility. The method was successfully employed to quantify accurate polyelectrolyte loadings on several particles that have a general importance as LbL templates. Thus, this method can be recommended as standard laboratory technique for control of LbL encapsulation and will considerably broaden the applicability of this promising technology in biotechnology.

Volsurf computational method applied to the prediction of stability of thermostable enzymes.

A computational model for the quantitative prediction of protein thermostability has been developed by means of the Volsurf method. A data set of 22 enzymes of reported thermostability in water systems, for the most part coming from thermophilic and hyperthermophilic organisms, has been built up. Molecular descriptors of the protein surface have been calculated and their role in the stabilization of the macromolecule has been analyzed by a multivariate statistical approach. The resulting regression model has shown a good predictivity and it has been able to quantitatively identify some structural requirements correlated with protein stability. The method can be the basis for a new computational support tool in rational protein design, which is complementary to the existing methods based on the sequence analysis.