Enzyme degradation during drying

During drying of food materials a multitude of chemical reactions and/or physical changes may occur. In this article attention is focused on one of these, namely, inactivation of enzymes during drying. The prediction of enzyme retention during drying is of interest to the pharmaceutical industry for the production of dry enzyme preparations and to the food processing industry in drying operations of food materials containing enzymes. In this article calculated enzyme retentions are presented for different drying histories and shapes of drying particles. In the numerical calculations it is assumed that enzyme degradation kinetics are first-order reactions, of which reaction constants are known as a function of temperature and water concentration in the drying material. From the calculations, conclusions can be drawn about conditions favorable for high enzyme retentions, or for high enzyme degradations.     

Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.     

Estimation of kinetic parameters for substrate and inhibitor in a reaction with an enzyme sample containing different types of inhibitor

The method of kinetic analysis is developed to obtain the maximum velocity (V_m), the Michaelis constant (K_m) and the parameters characterizing the inhibitors in an impure enzyme reaction, contaminated with one of four types of inhibitor (competitive, noncompetitive, uncompetitive and mixed-type). Although the reaction rate decreases with the increasing concentration of the enzyme sample containing an inhibitor, the double-reciprocal plot of the rate against the sample concentration becomes linear. The slopes of these linear plots at several different concentrations of substrate provide K_m and the specific enzyme activity, which is proportional to V_m, in the sample. These linear straight lines intersect in a point, of which the coordinates give the unique parameters for the inhibitor. To prove the validity of this kinetic method, the model experiments were carried out with acetylcholinesterase and its inhibitors, phenyltrimethylammonium and trimethylammonium. The present method was applied to the measurement of the specific activity of galactosylceramide galactosidase in the mouse cerebral homogenate. In addition, a kinetic method is indicated for the inhibition of an enzymatic reaction by a contaminant which binds the substrate to reduce the fraction available to the enzyme.     

On the activity loss of hydrolases in organic solvents: I. Rapid loss of activity of a variety of enzymes and formulations in a range of organic solvents

Dehydrated enzyme powders have been used extensively as suspensions in organic solvents to catalyze synthetic reactions. Prolonged enzyme activity is necessary to make such applications commercially successful. However, it has recently become evident that the stability and thus activity of many enzymes is compromised in organic solvents. Herein we explore the stability of various hydrolases (i.e., lipases from Mucor meihei and Candida rugosa, -chymotrypsin, subtilisin Carlsberg, and pig-liver esterase) and various formulations (lyophilized powder, cross-linked enzyme crystals, poly(ethylene glycol)-enzyme conjugates) in different organic solvents. The results show a roughly exponential activity decrease for all enzymes and formulations studied after exposure to organic solvents. Inactivation was observed independent of the enzyme, formulation details, and the solvent. In addition, no relationship was found between the magnitude of inactivation and the value of initial activity. Thus, quite active formulations lost their activity as quickly as less active formulations. The estimated half-times (t_1/2) for all enzymes and preparations ranged from 1.8 h for subtilisin C. co-lyophilized with methyl-β-cyclodextrin to 61.6 h for the most stable poly(ethylene glycol)--chymotrypsin preparation. The data here presented indicates that the inactivation is likely not related to changes in enzyme structure and dynamics.     

Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

Novel Method for Enumeration of Viable Lactobacillus plantarum WCFS1 Cells after Single-Droplet Drying

Survival of probiotic bacteria during drying is not trivial. Survival percentages are very specific for each probiotic strain and can be improved by careful selection of drying conditions and proper drying carrier formulation. An experimental approach is presented, comprising a single-droplet drying method and a subsequent novel screening methodology, to assess the microbial viability within single particles. The drying method involves the drying of a single droplet deposited on a flat, hydrophobic surface under well-defined drying conditions and carrier formulations. Semidried or dried particles were subjected to rehydration, fluorescence staining, and live/dead enumeration using fluorescence microscopy. The novel screening methodology provided accurate survival percentages in line with conventional plating enumeration and was evaluated in single-droplet drying experiments with Lactobacillus plantarum WCFS1 as a model probiotic strain. Parameters such as bulk air temperatures and the carrier matrices (glucose, trehalose, and maltodextrin DE 6) were varied. Following the experimental approach, the influence on the viability as a function of the drying history could be monitored. Finally, the applicability of the novel viability assessment was demonstrated for samples obtained from drying experiments at a larger scale.     

Evaluating conditions for producing spray-dried formulations of Anagrapha falcifera nucleopolyhedroviruses (AfMNPV)

We investigated how altering parameters during the production of spray-dried lignin formulations affected the insecticidal activity of a baculovirus (AfMNPV), which was originally isolated from celery looper, Anagrapha falcifera (Kirby). Exposure to high temperature, varied pH of the dryer feedstock, substitution of formulation ingredients, and different commercial production lots of virus were evaluated for their effect on the insecticidal activity of AfMNPV. Insecticidal activities of treatments were determined by droplet-feeding assays using neonate Trichoplusia ni (Hübner) for dosage response or single-dosage comparisons. Unformulated virus exposed to 68^oC for 2 h showed no loss of insecticidal activity, whereas exposure to 90^oC for 30 min caused >20% loss of activity. Thus, residence at higher temperatures in drying systems could adversely affect virus activity. Spray-dried formulations made with Indulin AT lignin (pH 8.5, 9.5, and 10.5) lost insecticidal activity with increasing alkalinity of the dryer feedstock. In contrast to Indulin AT, the same formulations made with PC-1307 lignin did not lose insecticidal activity with increased alkalinity. Adding corn flour to spray-dried Indulin AT-based formulations improved insecticidal activity of the virus. These experiments demonstrated the importance of carefully selecting feed-stock ingredients and processing conditions when spray drying AfMNPV.     

Inactivation mechanisms of lactic acid starter cultures preserved by drying processes

The preservation of lactic acid starter cultures by drying are of increased interest. A further improvement of cell viability is, however, still needed, and the insight into inactivation mechanisms of the cells is a prerequisite. In this present work, we review the inactivation mechanisms of lactic acid starter cultures during drying which are not yet completely understood. Inactivation is not only induced by dehydration inactivation but also by thermal- and cryo-injuries depending on the drying processes employed. The cell membrane has been reported as a major site of damage during drying or rehydration where transitions of membrane phases occur. Some drying processes, such as freeze drying or spray drying, involve subzero or very high temperatures. These physical conditions pose additional stresses to cells during the drying processes. Injuries of cells subjected to freezing temperatures may be due to the high electrolyte concentration (solution effect) or intracellular ice formation, depending on the cooling rate. High temperatures affect most essential cellular components. It is difficult to identify a critical component, although ribosomal functionality is speculated as the primary reason. The activation during storage is mainly due to membrane lipid oxidation, while the storage conditions such as temperature moisture content of the dried starter cultures are important factors.     

两种杀虫真菌制剂对茶小绿叶蝉的田间防效评价

于2002年盛夏在浙江遂昌一高山茶园对茶小绿叶蝉(Empoasca spp．)进行了真菌杀虫剂的田间药效试验。所用菌剂为球孢白僵菌(Beauveria bassiana)和玫烟色拟青霉(Paecilomyces fumosoroseus)的纯孢子悬乳剂及其与3％吡虫啉10％可湿剂的混配剂．各菌剂稀释500倍喷雾2次,间隔12d．结果表明。两种真菌的混配剂明显优于纯菌剂,其中球孢白僵菌混配剂的最高防效达83．4％,而玫烟色拟青霉的最高防效为71．3％．根据25d期间历次调查结果计算平均防效,球孢白僵菌混配剂达66．8％,玫烟色拟青霉混配剂为62．1％,含微量吡虫啉的矿物油为50．3％,球孢白僵菌和玫烟色拟青霉的纯菌剂分别为49．5％和19．0％,结合试验期间气候及田间种群结构特征,讨论了各处理问差异的来源及提高真菌杀虫剂控制茶小绿叶蝉效果的可能途径。     

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled¹.     

Biomimetic Dissolution: A Tool to Predict Amorphous Solid Dispersion Performance

The presented study describes the development of a membrane permeation non-sink dissolution method that can provide analysis of complete drug speciation and emulate the in vivo performance of poorly water-soluble Biopharmaceutical Classification System class II compounds. The designed membrane permeation methodology permits evaluation of free/dissolved/unbound drug from amorphous solid dispersion formulations with the use of a two-cell apparatus, biorelevant dissolution media, and a biomimetic polymer membrane. It offers insight into oral drug dissolution, permeation, and absorption. Amorphous solid dispersions of felodipine were prepared by hot melt extrusion and spray drying techniques and evaluated for in vitro performance. Prior to ranking performance of extruded and spray-dried felodipine solid dispersions, optimization of the dissolution methodology was performed for parameters such as agitation rate, membrane type, and membrane pore size. The particle size and zeta potential were analyzed during dissolution experiments to understand drug/polymer speciation and supersaturation sustainment of felodipine solid dispersions. Bland-Altman analysis was performed to measure the agreement or equivalence between dissolution profiles acquired using polymer membranes and porcine intestines and to establish the biomimetic nature of the treated polymer membranes. The utility of the membrane permeation dissolution methodology is seen during the evaluation of felodipine solid dispersions produced by spray drying and hot melt extrusion. The membrane permeation dissolution methodology can suggest formulation performance and be employed as a screening tool for selection of candidates to move forward to pharmacokinetic studies. Furthermore, the presented model is a cost-effective technique.     

Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn²⁺ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

Drying and formulation of blastospores of Paecilomyces fumosoroseus (Hyphomycetes) produced in two different liquid media

Formulation matrices can play an important role in improving the storage survival and biocontrol efficacy of microorganisms used for the control of pest insects. In this study, liquid culture-produced blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with different inert and organic materials prior to air-drying. Paecilomyces fumosoroseus blastospores were produced in two different liquid media, a basal salts medium supplemented with Casamino acids and glucose (LM1) and a medium containing peptone of collagen and glucose (LM2). Blastospores produced in the two test media were formulated with various supports. The formulation supports were cornstarch, rice flour, talc powders, Mexican lime, calcined kaolin clay, and diatomaceous earth. Several of the supports were tested at different concentrations. The initial and long-term (after storage at 4 and 28 °C) survival of the formulated, air-dried blastospores were evaluated. Initial blastospore viabilities were affected by the formulation material and by the blastospore production medium. Medium composition, drying support and storage temperature had an impact on the long-term survival of the blastospores. Under the conditions of the study, LM1 produced higher concentrations of blastospores that not only survived drying better than blastospores produced in LM2 but also maintained viability longer during storage in the formulation supports tested. The nature of the drying supports was shown to have a significant impact on the storage stability of all blastospores, particularly those produced in LM1. Under the production, drying and storage conditions used in the study, calcined kaolin clay formulations stored at 4 °C had the best storage stability. In all formulations tested, spore survival over time was reduced for blastospore formulations stored at 28 °C rather than 4 °C.     

Rapid optimization of protein freeze‐drying formulations using ultra scale‐down and factorial design of experiment in microplates

Retaining biopharmaceutical proteins in a stable form is critical to their safety and efficacy, and is a major factor for optimizing the final product. Freeze‐dried formulations offer one route for improved stability. Currently the optimization of formulations for freeze‐drying is an empirical process that requires many time‐consuming experiments and also uses large quantities of product material. Here we describe a generic framework for the rapid identification and optimization of formulation excipients to prevent loss of protein activity during a lyophilization process. Using factorial design of experiment (DOE) methods combined with lyophilization in microplates a range of optimum formulations were rapidly identified that stabilized lactose dehydrogenase (derived from Lactobacillus leichmanii) during freeze‐drying. The procedure outlined herein involves two rounds of factorially designed experiments—an initial screen to identify key excipients and potential interactions followed by a central composite face designed optimization experiment. Polyethylene glycol (PEG) and lactose were shown to have significant effects on maintaining protein stability at the screening stage and optimization resulted in an accurate model that was used to plot a window of operation. The variation of freezing temperatures and rates of sublimation that occur across a microplate during freeze‐drying have been characterized also. The optimum formulation was then freeze‐dried in stoppered vials to verify that the microscale data was relevant to the effects observed at larger pilot scales. This work provides a generic approach to biopharmaceutical formulation screening where possible excipients can be screened for single and interactive effects thereby increasing throughput while reducing costs in terms of time and materials. Biotechnol. Bioeng. 2009; 104: 957–964. © 2009 Wiley Periodicals, Inc.     

Effect of different additives on the persistence and insecticidal activity of native strains of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The persistence and insecticidal activity of native strains of Bacillus thuringiensis was evaluated in formulations containing different additives such as arrow-root powder, carboxy methyl cellulose (CMC), gum acacia, non-food grade (NFG) starch, and soluble starch. Persistence of B. thuringiensis varied with different additives used in the formulations. Among the different additives used, NFG starch provided maximum protection to B. thuringiensis and native strain 42 showed maximum persistence (83%) which was higher than that obtained in commercial formulation. In commercial formulation, the persistence of B. thuringiensis was 47% only after 3 d of spray. The feeding trials conducted on second instar larvae of H. armigera using leaves sprayed with NFG starch formulation revealed 70% larval mortality while commercial formulation showed 50% mortality during the same period.     

Theophylline Cocrystals Prepared by Spray Drying: Physicochemical Properties and Aerosolization Performance

The purpose of this work was to characterize theophylline (THF) cocrystals prepared by spray drying in terms of the physicochemical properties and inhalation performance when aerosolized from a dry powder inhaler. Cocrystals of theophylline with urea (THF-URE), saccharin (THF-SAC) and nicotinamide (THF-NIC) were prepared by spray drying. Milled THF and THF-SAC cocrystals were also used for comparison. The physical purity, particle size, particle morphology and surface energy of the materials were determined. The in vitro aerosol performance of the spray-dried cocrystals, drug-alone and a drug-carrier aerosol, was assessed. The spray-dried particles had different size distributions, morphologies and surface energies. The milled samples had higher surface energy than those prepared by spray drying. Good agreement was observed between multi-stage liquid impinger and next-generation impactor in terms of assessing spray-dried THF particles. The fine particle fractions of both formulations were similar for THF, but drug-alone formulations outperformed drug-carrier formulations for the THF cocrystals. The aerosolization performance of different THF cocrystals was within the following rank order as obtained from both drug-alone and drug-carrier formulations: THF-NIC > THF-URE > THF-SAC. It was proposed that micromeritic properties dominate over particle surface energy in terms of determining the aerosol performance of THF cocrystals. Spray drying could be a potential technique for preparing cocrystals with modified physical properties.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-012-9883-3) contains supplementary material, which is available to authorized users.Key words: aerodynamic diameter, cocrystal, spray drying, surface energy, theophylline     

Thermoreversible gel formulation containing sodium lauryl sulfate as a potential contraceptive device

The contraceptive properties of a gel formulation containing sodium lauryl sulfate were investigated in both in vitro and in vivo models. Results showed that sodium lauryl sulfate inhibited, in a concentration-dependent manner, the activity of sheep testicular hyaluronidase. Sodium lauryl sulfate also completely inhibited human sperm motility as evaluated by the 30-sec Sander-Cramer test. The acid-buffering capacity of gel formulations containing sodium lauryl sulfate increased with the molarity of the citrate buffers used for their preparations. Furthermore, experiments in which semen was mixed with undiluted gel formulations in different proportions confirmed their physiologically relevant buffering capacity. Intravaginal application of the gel formulation containing sodium lauryl sulfate to rabbits before their artificial insemination with freshly ejaculated semen completely prevented egg fertilization. The gel formulation containing sodium lauryl sulfate was fully compatible with nonlubricated latex condoms. Taken together, these results suggest that the gel formulation containing sodium lauryl sulfate could represent a potential candidate for use as a topical vaginal spermicidal formulation to provide fertility control in women.     

Purification and properties of citrate lyase from Streptococcus diacetilactis.

Citrate lyase from Streptococcus diacetilactis has been purified to yield a protein that was homogeneous as judged by sedimentation velocity and sedimentation equilibrium experiments. The enzyme's sedimentation coefficient is 16.8 S and its molecular weight is around 585,000. It contains three nonidentical subunits of about 53,000, 34,000, and 10,000 daltons. The enzyme in its active form contains an acetyl group which turns over during the citrate cleavage reaction. Removal of the acetyl group inactivates the enzyme. The deacetyl enzyme can be partially reactivated by acetylation with acetic anhydride. The enzyme undergoes slow "reaction-inactivation." The rate of inactivation is first order and the rate constant of inactivation is much lower than that for a similar inactivation process of the citrate lyase from Klebsiella aerogenes. Like the latter enzyme it contains stoichiometric amounts of phosphopantothenate. The enzyme is inactivated at pH greater than 8.1 and the presence of citrate provides protection against this inactivation. Sedimentation studies of the enzyme at pH 8.7 indicate that the enzyme is dissociated, which may account for the inactivation. The enzyme is immunologically different from citrate lyases of K. aerogenes and Escherichia coli.     

Kinetics of inactivation of Ulva pertusa Kjellm alkaline phosphatase by ethylenediaminetetraacetic acid disodium

Ulva pertusa Kjellm alkaline phosphatase (EC 3.3.3.1) is a metalloenzyme, the active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory described by Tsou of the substrate reaction during irreversible inhibition of enzyme activity has been employed to study the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction at different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA indicated a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-EDTA complex is a relative rapid reaction, following by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.