Joey N. Talbert

Enzymes on material surfaces.

Enzyme interactions with material surfaces are of interest for industrial food and pharmaceutical transformations, biosensors, artificial cells, cell free reactions, drug and nutrition delivery technologies, and imaging. When in contact with a material surface, an enzyme may lose or appear to lose activity due to the nature of the enzyme, the nature of the material, and/or the nature of the interface between the enzyme, material, and substrate environment. The purpose of this review is to survey recent advances that have been made towards the preservation, optimization, and enhancement of enzyme activity on material surfaces within the context of well-known concepts that describe the loss of activity after immobilization. This review breaks down the immobilized enzyme system to look at the individual components of the system-namely the enzyme, the material, and the interface. For each piece, possible causes for the loss of enzyme activity are described as well as strategies that have been applied to limit the affect. At the conclusion we identify areas of future research needed to overcome limitations in the current state-of-the art for immobilized enzyme systems.

Immobilization and Stabilization of Lipase (CaLB) through Hierarchical Interfacial Assembly

Nanostructure-enabled hierarchical assembly holds promise for efficient biocatalyst immobilization for improved stability in bioprocessing. In this work we demonstrate the use of a hierarchical assembly immobilization strategy to enhance the physicochemical properties and stability of lipase B from Candida antarctica (CaLB). CaLB was complexed with iron oxide nanoparticles followed by interfacial assembly at the surface of an oil-in-water emulsion. Subsequent ring opening polymerization of the oil provided cross-linked microparticles that displayed an increase in catalytic efficiency when compared to the native enzyme and Novozym 435. The hierarchical immobilized enzyme assembly showed no leakage from the support in 50% acetonitrile and could be magnetically recovered across five cycles. Immobilized lipase exhibited enhanced thermal and pH stability, providing 72% activity retention after 24 h at 50 °C (pH 7.0) and 62% activity retention after 24 h at pH 3.0 (30 °C); conditions resulting in complete deactivation of the native lipase.

Layer by Layer Assembly of a Biocatalytic Packaging Film: Lactase covalently Bound to Low‐Density Polyethylene

Active packaging is utilized to overcome limitations of traditional processing to enhance the health, safety, economics, and shelf life of foods. Active packaging employs active components to interact with food constituents to give a desired effect. Herein we describe the development of an active package in which lactase is covalently attached to low-density polyethylene (LDPE) for in-package production of lactose-free dairy products. The specific goal of this work is to increase the total protein content loading onto LDPE using layer by layer (LbL) deposition, alternating polyethylenimine, glutaraldehyde (GL), and lactase, to enhance the overall activity of covalently attached lactase. The films were successfully oxidized via ultraviolet light, functionalized with polyethylenimine and glutaraldehyde, and layered with immobilized purified lactase. The total protein content increased with each additional layer of conjugated lactase, the 5-layer sample reaching up to 1.3 μg/cm2 . However, the increase in total protein did not lend to an increase in overall lactase activity. Calculated apparent Km indicated the affinity of immobilized lactase to substrate remains unchanged when compared to free lactase. Calculated apparent turnover numbers (kcat ) showed with each layer of attached lactase, a decrease in substrate turnover was experienced when compared to free lactase; with a decrease from 128.43 to 4.76 s(-1) for a 5-layer conjugation. Our results indicate that while LbL attachment of lactase to LDPE successfully increases total protein mass of the bulk material, the adverse impact in enzyme efficiency may limit the application of LbL immobilization chemistry for bioactive packaging use.

Chemical modification of lactase for immobilization on carboxylic acid-functionalized microspheres

Abstract Surface interactions between an enzyme and support influence the retention of activity after immobilization. Chemical modification of enzymes prior to immobilization may be used to alter these interactions and enhance activity retention. Lactase (A. oryzae) was covalently conjugated to P(S/V-COOH) microspheres, with surface carboxylic acid densities of 9 μeq/g and 137 μeq/g, using carbodiimide chemistry. Under optimum pH and temperature conditions, activity retention was greater when the enzyme was conjugated to microspheres containing a lower density of surface carboxylic acid groups (32% activity retention) than when the enzyme was conjugated to microspheres having a greater density of surface carboxylic acid groups (11% activity retention). Chemical modification of lactase carboxylic acid groups with glucosamine prior to immobilization was evaluated as a means to increase activity retention. Under optimal conditions, modification resulted in a 17% decrease in soluble enzyme activity compared to the native enzyme. However, immobilization of the modified enzyme yielded 85% and 64% activity retention after conjugation to microspheres with a lower and higher density of surface carboxylic acid groups, respectively. The results suggest that increases in surface carboxylic acid density on the carrier promote the loss of lactase activity after immobilization, and chemical modification of the enzyme with glucosamine provides a means to retain catalytic activity after attachment to these supports.

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specific enumeration of E. coli in contaminated drinking water is critical to mitigate significant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane filtration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifically engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specific to cellulose. These constructs were then inserted into the E. coli-specific phage T7 which were used to infect E. coli trapped on a cellulose filter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose filter in proximity to the lysed cells. Following substrate addition, the location and quantification of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.

Strep-tag II fusion technology for the modification and immobilization of lipase B from Candida antarctica (CALB)

Fusion tags – amino acid sequences that are genetically coded to be expressed as attached moieties to a protein – have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB). The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli. Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based support through affinity binding, and the immobilized and free Strep-tag II CALB were compared to a commercial CALB. Following modification, the enzyme could be selectively purified from culture media with no observable non-specific binding. The catalytic efficiency of the purified fusion-tagged enzyme was significantly greater than that of the commercial CALB in its free form. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose support yielded a catalytically active enzyme; however, the kcat of the immobilized enzyme was significantly reduced compared to the free tagged enzyme. This work indicates that a C-terminus Strep-tag II fusion tag may be employed to improve the catalytic efficiency of free CALB, but may not be suitable for immobilized applications that employ binding of the enzyme to a Strep-Tactin-modified support.

Engineering bacteriophage for a pragmatic low-resource setting bacterial diagnostic platform.

ABSTRACT Bacteriophages represent multifaceted building blocks that can be incorporated as substitutes for, or in unison with other detection methods, to create powerful new diagnostics for the detection of bacteria. The ease of phage manipulation, production, and detection speed clearly highlights that there remains unrealized opportunities to leverage these phage-based components in diagnostics amenable to resource-limited settings. The passage of regulations like the Food Safety Modernization act, and the ever increasing extent of global trade and travel, will create further demand for these types of diagnostics. While phage-based diagnostics have begun to entering the market place, further research is needed to ensure the potential benefits of phage-based technologies for public health are fully realized. We are just beginning to explore the possibilities that phage-based detection can offer us in the future. The combination of engineered phages as well as engineered enzymes could result in ultrasensitive detection systems for low-resource settings. Because the reporter enzyme is synthesized in vivo, we need to consider the options outside of normal enzyme reporters. In this case, common enzyme issues such as purification and long-term stability are less important. Phage-based diagnostics were conceptualized from out-of-the box thinking and the evolution of these systems should be as well.

Effect of cleaning and sanitization agents on the surface characteristics of new and extended-wear produce picking bins.

BACKGROUND Although surface characteristics of food contact materials are known to alter the efficacy of cleaning procedures there is a lack of data establishing how cleaning/sanitization practices affect the surface characteristics of materials used for produce handling on-farm. The overall objective of this work was to characterize the effects of cleaning and sanitization procedures on the surface properties of new and extended-wear polyethylene bins used for produce harvest and handling. RESULTS Compared to detergent cleaned samples, chlorine and quaternary ammonium sanitization resulted in a decrease in advancing contact angle from 100° to 88° and 59°, respectively, after 2 min exposure. However, changes in surface chemistry were not observed. Increasing sanitization time to 144 min (representative of 4320 sanitization cycles) resulted in an increase in contact angle to 73° for quaternary ammonium sanitization and a decrease in contact angle to 75° for chlorine sanitization. Abrasion increased contact angle hysteresis due to enhanced surface roughness. The hysteresis effect of abraded material was reduced with quaternary ammonium treatment. CONCLUSIONS This work indicates that sanitizing agents employed in on-farm cleaning can alter the surface characteristics of polyethylene picking bins and should be considered in developing cleaning and sanitization procedures.