Inactivation Of Β-galactosidase Research Articles

Enzyme inactivation kinetics: Coupled effects of temperature and moisture content

Enzymes are often dried for stability reasons and to facilitate handling. However, they are often susceptible to inactivation during drying. It is generally known that temperature and moisture content influence the enzyme inactivation kinetics. However, the coupled effect of both variables on enzyme inactivation over a broad temperature–moisture content range is still not well understood. Therefore, the inactivation of β-galactosidase in maltodextrin matrix is investigated using a newly developed method. An improved kinetic modelling approach is introduced, to predict the inactivation over a large range of temperature–moisture values. The model assumes a two-step inactivation mechanism involving reversible unfolding and irreversible inactivation. The model is able to describe the inactivation kinetics of β-galactosidase accurately, showing the temperature-dependent kinetic transition from reversible unfolding to irreversible inactivation limited. Application of this approach can provide immediate understanding of the effect of processing on enzyme inactivation and indicates the processes’ critical points, which offers the possibility for optimisation.

Genetic Suppressors and Recovery of Repressed Biochemical Memory

Many of the Reflections articles in this journal have focused on particular biological problems that the authors have devoted their careers to or on particular techniques that have facilitated their biological studies. Although I began my career as a biochemist, one of the most common threads to my research has been the exploitation of a genetic approach, the analysis of suppressor mutations. Particularly in the last 20 years, the success of this approach has led me into biological problems that required a remembering or relearning of my early training in biochemistry. This article will describe the voyage from chemistry and biochemistry to genetics and then to a recovery of remnants of my biochemical memory. As a graduate student working under Lowell Hager at Harvard in the late 1950s, my thesis research was chemical and biochemical, as I performed the chemical synthesis and studied the biosynthesis of the chlorinated mold product caldariomycin (2,2-dichloro-1,3-cyclopentanediol) (1–3). With his postdoctoral fellow Paul Shaw, Lowell had discovered an enzyme, chloroperoxidase, that was capable of utilizing chloride ion to chlorinate organic compounds (4). Part of my work was to determine how this enzyme might be involved in the biosynthesis of caldariomycin. Apparently, the work became somewhat well known. This I learned when, toward the end of my Ph.D. work, I attended a FASEB meeting in Atlantic City to give a short presentation. As I walked into the conference center on the first day, a scientist passing by me stopped to look at my badge, hesitated a moment, and then commented “Beckwith... hmmm, oh yes, I remember... chlorination!” Despite this really quite limited chemical/biochemical notoriety, I became more and more fascinated by bacterial genetics in graduate school. I proceeded to do postdoctoral work in several bacterial genetics laboratories.

Stabilization of β-galactosidase by amphiphilic additives during freeze-drying

The effects of amphiphilic additives on inactivation of β-galactosidase during freeze-drying were studied in comparison with those of sugars and amino acids. The activity loss was greater when the enzyme was freeze-dried from solutions of lower enzyme concentration without additives. Well-known cryoprotectants such as sugars and amino acids provided concentration-dependent preservation of enzyme activity. Hydroxypropyl-β-cyclodextrin (HP-β-CD), 2,6-di- O-methyl-β-cyclodextrin (DM-β-CD), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sucrose fatty acid monoester stabilized the enzyme at much lower concentrations than-sugars and amino acids. Polyethylene glycol 400 (PEG 400), polyoxyethylene 9 lauryl ether (polydocanol) and sodium dodecyl sulfate (SDS), however, were ineffective or rather induced inactivation. High-performance size-exclusion chromatography indicated the formation of soluble aggregates during freeze-drying without additives, which was inhibited by the sugar ester. SDS-PAGE indicated that the aggregates were formed by noncovalent bonding

The inactivation of β-galactosidase ( E. coli) by the carbodiimide reaction

1. 1. When β-galactosidase reacted with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC), activity was lost. The inhibitor, isopropyl-β- d-galactopyranoside (IPTG), decreased inactivation. 2. 2. Of 3 nucleophiles tested, incorporation was only decreased in the protected (IPTG added) enzyme when sulfanilic acid was the nucleophile but HPLC profiles of tryptic peptides were identical in protected and unprotected enzyme (except for magnitude). There were also no differences (except for magnitude) of HPLC profiles after 10 and 90 min of reaction and between active (soluble) and inactive (precipitated) enzyme. 3. 3. The data indicate that inactivation is not caused by reaction with a specific active site group. Inactivation probably occurs when a combination of groups are reacted.

The inactivation of β-galactosidase by N-bromoacetyl-β-D-glucosylamine

The inactivation of β-galactosidase by N-bromoacetyl-β-D-glucosylamine