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There is a constant need to identify novel inhibitors to combat β-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM −1 ) and binding affinity ( K D = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV–vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a K i of 9.22 μM and a K i ′ of 33.12 μM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of β-lactamases and development of novel inhibitors of β-lactamases. [ABSTRACT FROM AUTHOR]

Highlights: [•] Hybrid cellulase aggregate consists of cross-linked cellulase layers physically adsorbed on silica gel. [•] Higher glucose production than cross-linked cellulase aggregates without silica core. [•] Reusable for cellulose and biomass hydrolysis. [Copyright &y& Elsevier]

Abstract: We report a biosensor for detecting organophosphates (OPs) by using liquid crystal (LC). The mechanism of the biosensor is based on detection of minute pH changes during the enzymatic hydrolysis of OPs. To hydrolyze OPs, paraoxonase 1 (PON1) is immobilized on a copper grid which also hosts pH-sensitive LC, 4-cyano-4′-pentylbiphenyl (5CB) doped with 0.3% of 4′-pentyl-biphenyl-4-carboxylic acid (PBA). Proximity of enzyme and LC ensures that H+ can be detected by the pH-sensitive LC before it is neutralized by buffer. Thus, the presence of OPs can be reported as color change in LC when they undergo enzymatic hydrolysis. For paraoxon detection, we can reach a limit of detection (LOD) of 1μM by using the naked eye. This type of LC-based biosensor also shows high specificity and does not respond to imidacloprid and ampicillin. [Copyright &y& Elsevier]

Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200 nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300 nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48 h and release reducing sugar concentrations up to 2.48 ± 0.08 g/l and 4.99 ± 0.09 g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of cellulose is often conducted in batch processes in which hydrolytic products tend to inhibit enzyme activity. In this study, we report a method for continuous hydrolysis of carboxymethyl cellulose (CMC) by using cross-linked cellulase aggregate (XCA) trapped inside a membrane. XCA particles prepared by using a millifluidic reactor have a uniform size distribution around 350 nm. Because of their large size, XCA particles in solutions can be filtered through a polyethersulfone membrane to collect 87.1 ± 0.9% of XCA particles. The membrane with impregnated XCA can be used as a catalyst for hydrolysis of CMC in a continuous mode. When the CMC concentration is 1.0 g/l and the flow rate is 2 μl/min, 53.9% of CMC is hydrolyzed to reducing sugars. The membrane with XCA is very stable under continuously flowing solutions. After 72 h of reaction, 97.5% of XCA remains inside the membrane. [ABSTRACT FROM AUTHOR]

Abstract: We report a liquid crystal (LC)-based sensor for real-time and label-free identification of phospholipase-like toxins. Beta-bungarotoxin exhibits Ca2+-dependent phospholipase A2 activity whereas alpha-bungarotoxin and myotoxin II do not exhibit any phospholipase activity. The sensor can selectively identify beta-bungarotoxin, when it hydrolyzes a phospholipid monolayer self-assembled at aqueous–LC interface, through orientational responses of LCs. As a result, optical signals that reflect the spatial and temporal distribution of phospholipids during the hydrolysis can therefore be generated in a real-time manner. The sensor is very sensitive and requires less than 5pg of beta-bungarotoxin for the detection. When phospholipase A2 inhibitors are introduced together with beta-bungarotoxin, no orientational response of LCs can be observed. In addition, the regeneration of the sensor can be done without affecting the sensing performance. This work demonstrates a simple and cost-effective LC-based sensor for identifying phospholipase-like toxins and for screening compound libraries to find potential toxin inhibitors. [Copyright &y& Elsevier]