Amihay Freeman

In vitro enzyme evolution: the screening challenge of isolating the one in a million

The generation of large mutant libraries for in vitro enzyme evolution presents the challenge of effectively screening libraries of 10(4)-10(7) mutants on the basis of simultaneously assaying their biocatalytic activity. In this review, we highlight the main steps involved in this process, describe the alternative approaches to address this challenge, survey the state-of-the-art technology and assess achievements already made. It is anticipated that, as a result of the expected accomplishment of further improvements in high-throughput screening that will allow routine screening of whole libraries, the number of useful new and improved enzymes derived through in vitro enzyme evolution will expand rapidly in the near future.

Effect of processing parameters on the feasibility and operational stability of immobilized viable microbial cells

The use of aerated immobilized viable microorganisms despite their potential has so far been limited on the large scale except in waste treatment. This review assesses the information available on the effect of processing parameters on the behavior and operational stability of aerated immobilized viable cells being used for synthesis. It highlights the problems of adequate oxygen supply and a need to develop new reactors via a rational approach to process design and operation.

Electroless processes for micro- and nanoelectronics

Abstract Electroless deposition of metals has many applications in micro- and nanotechnologies. Currently, electroless Cu, Co, Ni, Ag and their alloys are used as materials for interconnects and packaging applications for ultralarge-scale integration (ULSI) as well as for microelectro-mechanical systems (MEMS). Electroless methods offer high-quality ultrathin films that are compatible with high-resolution patterns such as sub-100 nm interconnects, contacts and via contacts for ULSI and in high aspect ratio structures for MEMS. In this paper, we present an overview of electroless methods for microtechnologies applications. We also present Cu, Ag, and Co alloys that are designed to improve performance and reliability of the pure metals. Next, we present new concepts of electroless deposition methods that will be useful for nanotechnologies. We explore the possibility to use electroless methods that can be combined with self-assembly of organic compounds such as proteins to form metallic interconnect network. Silver deposition on organic substrates is described as a novel approach to produce high-quality Ag coating on features with 1–10 nm critical dimensions.

Self-Assembly of a Tetrahedral Lectin into Predesigned Diamondlike Protein Crystals.

Binding sites analogous to those of sp(3) carbon are presented by concanavalin A. This lectin has now been cross-linked with a bismannopyranoside which contains the C(2) spacer required to form the computer-modeled diamondlike three-dimensional protein lattice shown in the picture.

Whole-cell biochips for bio-sensing: integration of live cells and inanimate surfaces

Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.

Enzyme stabilization by bilayer “encagement”

Abstract A two-step simple procedure for enzyme stabilization—in solution—was developed. The enzyme is first coated, in a complementary way, by a monolayer of low-molecular-weight polymeric glutaraldehyde. Following removal of unbound polyaldehyde, this layer is crosslinked by a second layer made of polyacrylamide derivatives, bearing primary amine or acylhydrazide groups. Thus a bilayered, chemically crosslinked, synthetic “cage” is formed, surrounding the enzyme and increasing its stability to denaturation by rigidification of its structure. The “encagement” process results in a significant enhancement of the thermal stability as well as survival in the presence of water-miscible organic solvents. The “encaged,” stabilized enzyme could be readily immobilized by crosslinking into polyacrylamide-hydrazide gel, resulting in an additional stabilization effect imposed by this gel. The procedure worked out was demonstrated by the stabilization of two enzymes of analytical importance [glucose oxidase (E.C.1.1.3.4) and penicillinase (E.C.3.5.2.6)] as well as dramatic stabilization of carboxylesterase (from pig liver, E.C.3.1.1.1) one of the most useful enzymes in bio-organic synthesis.

Protein determination of cells immobilized in cross-linked synthetic gels

SummaryAn assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradfords procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems.

Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide.

Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.

Continuous Δ1-hydrocortisone dehydrogenation with in situ product recovery

Abstract A continuous aerated process for Δ 1 -hydrocortisone dehydrogenation by polyacrylamide-hydrazide (PAAH) bead-entrapped A. simplex cells was developed. The process allows for stable conversion of 1.6 g l −1 hydrocortisone (×5 the solubility in water), made possible by the incorporation of selected cosolvent [5% (v/v) triethyleneglycol]. A large difference in substrate and product solubilities in the cosolvent-buffer medium allowed for in situ product recovery in an aerated, fluidized-bed, immobilized-cell reactor by the controlled addition of fine product-adsorbing powder (microcrystalline cellulose). The product was recovered at the reactor outlet as a fine complex with the adsorbent. Stable continuous operation of at least 4 weeks was recorded for a prototype reactor configuration, followed by the exhibition of similar operational stability in a modified version of a commercially available 2.5-l airlift reactor. Our results demonstrate that in addition to an obvious desirable cosolvent effect on substrate solubility enhancement, it may also enable easy in situ product recovery by creating a large gap in the solubilities of the substrate and the product in the cosolvent-containing reaction medium.

Gel Entrapment of Whole Cells and Enzymes in Crosslinked, Prepolymerized Polyacrylamide Hydrazide

Immobilization of whole cells offers several advantages as a tool in biotechnology: easy separation of biocatalyst from the reaction mixture, reuse of biocatalyst, flexibility in reactor choice, high concentration of biocatalyst per unit volume of reactor, and improved storage, thermal, and operational stability. Throughout the last decade, these advantages led to the applications of immobilized cells as biocatalysts on a production scale’.’ as well as an analytical tool.’ Immobilization of whole cells may be achieved via several approachesadsorption, crosslinking, chemical binding, and gel entrapment. However, gel entrapment techniques seem to be the most popular approach employed for whole cell immobilization: Gel entrapment is usually based on suspending the cells to be immobilized in a solution containing water-soluble polymers or monomers, followed by the induction of gelation of the whole system via physical or chemical means. These include cooling, pH or salinity changes, initiation of polymerization, or the addition of an appropriate crosslinking agent.4 A process designed for the efficient entrapment of whole cells should allow for: