The coagulation of milk with immobilized enzymes: A critical review

The milk coagulation process is described including current understanding of the mechanism and kinetics involved with emphasis on aspects which impact on coagulation with immobilized enzymes. From the studies of coagulation with immobilized enzymes reported to date there is little evidence to suggest that immobilized enzymes can be used to coagulate milk.     

Einwirkung des immobilisierten Präparates „Sirenin”︁ aus Bacillus mesentericus auf Milch als Substrat

Comparative investigations for the possibilities of milk coagulation by the action of soluble and immobilized (on DEAE-cellulose by adsorption) bacterial enzyme preparation “syrenin” in periods from 30–120 minutes at 5°C are carried out. The milk clotting activities of both preparations are equalized after 40 minutes. The laboratory experiments for producing a bulgarian white brine cheese by the immobilized “syrenin” show a tendency to decrease the losses of substance into the whey and to increase the curdle yields in comparison with these made by soluble preparation.     

Continuous coagulation of milk using immobilized enzymes in a fluidized-bed reactor

Milk-clotting enzymes such as pepsin, chymosin, chymotrypsin, and M. miehei proteases were immobilized on porous, alkylamine glass and incorporated into a fluidized-bed continuous coagulation scheme. Only pepsin and calf rennet retained sufficient activity towards skim milk to warrant further studies. Comparison of kinetic data with fixed-bed reactors revealed the overall superior performance of fluidized beds; higher clotting activities were possible while avoiding plugging problems and high pressure drops common to fixed-bed reactors. Film diffusion and catalyst back-mixing appear to be significant factors in the overall kinetics. All enzymes lost activity on exposure to skim milk. The inactivation rates were lower at high substrate pH and insignificantly affected by reactor temperature. Nitrogen and sialic acid accumulation on the porous glass paralleled the loss in activity in the initial stages. Attempts to regenerate the immobilized enzymes were partially successful.     

High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity,Stability, Milk Coagulation Profile and Gel Development

This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Preparation and properties of soluble-insoluble immobilized proteases

In order to carry out an effective enzyme reaction, the preparation of soluble-insoluble immobilized enzyme was investigated. Proteases were selected as model enzymes, and their immobilization was carried out by using an enteric coating polymer as a carrier. Among the polymers tested, methacrylic acid-methylacrylate-methylmethacrylate copolymer (MPM-06) gave the most active soluble-insoluble immobilized papain. This immobilized papain showed insoluble from below pH 4.8 and soluble form above pH 5.8; it was also soluble in water-miscible organic solvent. It was reusable and more stable with heat and water-miscible organic solvents than native proteases. Furthermore, various proteases could be immobilized by using MPM-06 with high activity. Chymotrypsin immobilized by this method catalyzed the effective peptide synthesis in a heterogeneous reaction system containing water-miscible organic solvent.     

Milk Clotting Enzyme from Microorganisms

Out of some 800 strains of microorganisms, a potent fungus for milk clotting enzyme was isolated from soil during the course of screening tests and was identified as one of strains of Mucor pusillus Lindt. Satisfactory results were obtained in cheese making experiments with this enzyme which could be produced effectively by solid culture on wheat bran at 30°C for about 70 hrs.

The balance between milk clotting activity and proteolytic activity of this enzyme resembled very much to that of rennet.

Microbial rennet from Mucor pusillus F-27 was obtained with high productivity by solid culture followed by water extraction. The enzyme could be precipitated by salting out with ammonium sulfate and also by mixing with various water-miscible organic solvents such as ethanol, methanol or acetone.

This enzyme is one of acid proteases having its optimal pH for milk casein digestion around 3.5. The ratio of milk clotting activity to proteolytic activity of this enzyme resembled that of calf rennet than those of other proteases of fungal origin. This was more heat stable and more resistant against pH changes than animal rennet. Apparent activity of milk clotting was more affected by Ca ion concentration in milk than that of calf rennet.

The liberation of 12% TCA soluble nitrogen from casein fraction was a little less specific than that of calf rennet. The optimal temperature for milk clotting lay around 56°C.

Electrophoretic patterns of α-peak of casein treated with this enzyme showed the weak proteolysis which resembled that with rennet.     

Zymogens and cofactors of blood coagulation

Blood coagulation is a system in which a series of zymogens of serine proteases are sequentially activated. In this regard, there is little fundamental difference between coagulation and the activation of the homologous pancreatic zymogens. There are, however, several aspects unique to coagulation which are discussed in detail. These are (1) the requirement for a high-molecular-weight protein or lipoprotein cofactor for optimal reaction rates, (2) the requirement for membranes or a membrane-like surface which further distinguishes this system; (3) a metal ion requirement for most reactions (in contrast to the pancreatic serine proteases) relating to the content of the newly described amino acid gamma-carboxyglutamic acid in the four vitamin K-dependent proteins, regarding which recent data relating to the metal binding sites on prothrombin are discussed in detail; and (4) the uniqueness of the initiating reactions in comparison to those which activate the pancreatic zymogens, insofar as no enzyme corresponding to enterokinase has been identified. The implications of this phenomenon are analyzed with particular attention to the potential role of the endogenous activity of certain zymogens in initiating coagulation. The article deals finally with the specific problems attendant on analyzing a system in which many serine proteases lacking absolute specificity are generated and regulated.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV(WT), and Streptag II-TEV(S219V), respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV(WT) and Streptag II-TEV(S219V) were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step.     

Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. full hydrolysis of lactose in milk

The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.     

Ultrafiltration of raw sewage using an immobilized enzyme membrane

Ultrafiltration of raw sewage was performed using multiple enzymes immobilized on non-cellulosic, ultrafiltration membranes. An increase of 12% in the permeate flux rate at quasi-steady state was observed due to the action of the immobilized enzymes. Enzymes were immobilized by physical sorption to minimize the loss of ultrafiltration capability of the membrane, due to the immobilization process. A mathematical model based on diffusive transport and enzymatic action is presented. A standard Marquardt algorithm and a fourth-order Runge-Kutta integration routine were used for estimation of the non-linear parameters in the model. A comparison of data presented here with the data reported earlier on the ultrafiltration of NFDM (non-fat dry milk), showed that the enzyme-membrane has a longer half-life in the case of NFDM than for raw sewage. Furthermore, the first-order enzyme decay rate is much faster in the multiple enzyme system used in raw sewage filtration than in the single enzyme system used in the ultrafiltration of NFDM.     

Sol-gel immobilization of serine proteases for application in organic solvents

The serine proteases alpha-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of alpha-chymotrypsin and the resulting specific enzyme activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol in cyclohexane is 43 times higher than that of a nonimmobilized lyophilized alpha-chymotrypsin. The activities of trypsin and subtilisin Carlsberg are enhanced with 437 and 31 times, respectively. The effect of immobilization on the enzyme activity is highest in hydrophobic solvents.     

beta-Galactosidase immobilization for milk lactose hydrolysis: a simple experimental and modelling study of batch and continuous reactors

The experiment described in this paper introduces students to the practical use of an enzyme (beta-galactosidase, or lactase) acting on a natural substrate. The enzyme is immobilized onto a cheap support, and the immobilized derivative is used in a packed-bed reactor for continuous milk lactose hydrolysis. The results are compared to those obtained for discontinuous batch reactors with soluble enzyme. A mathematical model of the two types of reactors is run, and its results are compared with the experimental data obtained.     

Sulphydryl oxidase: Properties and applications

Sulphydryl oxidase has been isolated and purified from bovine milk and partially characterized. The enzyme is obtained from the whey fraction produced by chymosin (rennin) treatment of skim milk. Several procedures have been developed for its isolation, all of which use to advantage its concentration-dependent aggregate size. This enzyme exhibits a much broader substrate specificity than that shown by other ‘aerobic oxidases’ presently characterized which catalyse the formation of disulphides from thiols. Using molecular oxygen as an electron acceptor in a twoelectron reduction to hydrogen peroxide, the enzyme catalyses oxidation of cysteine and its analogues, some volatile thiols, peptides, and also thiols in proteins.Vis-a-vis protein-disulphide oxidoreductase and protein-disulphide isomerase, this enzyme does not catalyse thiol-disulphide interchange and thus functions only in de novo formation of disulphides. Enzyme-bound iron and most likely a free sulphydryl group are essential for catalytic activity; however, the carbohydrate moiety of this glycoprotein is probably not required. The broad substrate specificity suggests several potential industrial uses for the enzyme, including flavour modification in the food industry or synthesis of disulphides in pharmaceuticals. Consequently, immobilized forms were developed and characterized. Reactors containing sulphydryl oxidase covalently immobilized on porous glass and silica have been examined for their efficacy in eliminating the cooked flavour in ultra-high temperature sterilized milk. Both the degree of oxidation and cooked flavour were correlated with a normalized residence time in the reactor.     

Specificity of a Nuclease from Penicillium citrinum

The enzyme with high milk clotting activity produced by Irpex lacteus was partially purified by a CM-cellulose chromatography. Throughout the over-all process, the enzyme was purified approximately 9-fold from a crude powder with about 22.8% recovery of the original activity. The MCA/PU ratio of this fraction was 2.51 and the specific milk clotting activity was 188.7.

The purified enzyme is a sort of acid protease with optimum pH of 2.5 for casein digestion and 4.0 for hemoglobin digestion. The Lineweaver-Burk plot, when casein was used as a substrate, showed that the Km value of the enzyme was about 0.07% and the V_max value was 0.4. The molecular weight of the enzyme is about 34,000, the isoelectric point is pH 5.2 and a ultraviolet absorption maximum is at 277 mμ. The enzyme has not yet been crystalized but seems to be a sort of glycoprotein, because the Molish reaction was positive at the present purification stage.

Some enzymological properties of the enzyme was studied and compared with those of a calf rennet and Mucor rennet. In some respects such as pH optima, pH stability, thermostability and temperature optima, the enzyme is Mucor rennet alike. On the other hand, as to the increase in activity along with decrease in pH of milk and the increase in activity along with the addition of Ca ion, the enzyme is not very different from the calf rennet. However, proteolysis of milk casein by the enzyme was fairly higher than by the calf rennet.

As to the production of enzymes, I. lacteus can produce at least three types of proteases into liquid media. When, for example, R medium was used, only one type of protease, that is the fraction A, could mainly be produced and it was this enzyme that assumed to be a rennet like enzyme.     

Enhanced stability of immobilized keratinolytic protease on electrospun nanofibers

Abstract

Feathers from poultry industries are considered a major pollutant and its degradation is a challenging problem due to its recalcitrant nature. The high cost of energy and loss of essential amino acids by conventional methods have paved a way for an environmentally benign approach using microbial keratinolytic proteases. The widespread application of keratinolytic proteases is limited due to autolysis and denaturation of the enzyme upon storage. Immobilization overcomes these disadvantages by adsorbing the enzyme onto a solid support. Recently, electrospun nanofibers have been used due to their increased surface area and porous structure. The biocompatible and hydrophilic polyvinyl alcohol (PVA) has been blended with biodegradable chitosan for immobilization in electrospinning. The present study focuses on feather degradation by immobilized keratinolytic proteases on electrospun nanofibers. The keratinolytic protease production was enhanced by using a media containing hydrolyzed feather under optimized conditions. The immobilized keratinolytic protease on electrospun PVA chitosan (PVA-Ch) nanofibers (100–150?nm diameter) degraded the chicken feathers with 88% efficiency at the end of 72?hr.     

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

Immobilization of rennet from Mucor miehei via its sugar chain. Its use in milk coagulation

A successful strategy for the immobilization of rennet from Mucor miehei has been developed. The strategy is based on the immobilization of the enzyme, via their sugar chains at high ionic strength on aminated supports having primary amino groups with a very low pK value. The rennet was covalently immobilized via sugar chains (previously oxidized with periodate), which act as natural spacer arms and allow a very high percentage of rennet activity to be kept against small (H-Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe.TFA (98%)) and macromolecular substrates (k-casein) (78%). The use of tailor-made aminated support was critical to obtain good stability values, because using fully aminated supports achieved much lower thermostability values than using 50% aminated supports. The optimized derivative was utilized to hydrolyze casein in milk. To prevent the coagulation of the milk in the presence of the derivative, the reaction was performed at 4 degrees C (where hydrolyzed casein did not precipitate). Then the hydrolyzed milk was filtered and latter on heated to 30 degrees C, achieving a similar aggregate to the one achieved with soluble rennet.     

Kinetics of milk coagulation: III. Mathematical modeling of the kinetics of curd formation following enzymatic hydrolysis of kappa-casein--parameter estimation

A step function model of milk micelle agglomeration is proposed to explain the observed kinetics of milk clotting following rennet addition. The model ties together the primary and secondary phases of coagulation. The basis of the model is that no micelle flocculation takes place until ca. 75% of the kappa-casein in the milk is hydrolyzed, at which time flocculation occurs rapidly and the rate limiting step for the clotting process shifts to the kappa-casein hydrolysis reaction. Using such a model, it is possible to explain the clotting kinetics for both rapidly denaturing enzymes and stable enzyme systems. The average rate of the flocculation reaction can be obtained from clotting time-versus-reciprocal-enzyme-concentration data by extrapolating the data to infinite enzyme concentration. The critical conversion required for imminent flocculation can be found by extrapolating the enzyme concentration to zero. This approach indicates that the critical conversion necessary for gelation is temperature dependent changing from a limiting value of essentially 100% hydrolysis at temperatures below 15 degrees C to only 60% conversion at temperatures above 30 degrees C.