KR810002146B1 - Process for preparation of immobilized saccharifying enzyme - Google Patents

Abstract

A method is described for immobilization of amyloglucosdase and maltogenic α-amylase in particle form by co-crosslinking the enzyme in a mixt. of casein particles and egg albumin. Thus, 9g of relatively large casein particles(100-500μ) was mixed with 1.2g of dried egg albumin. Then, 6.5ml of an aq. amyloglucosidase soln. was added to the particle mixt. together with 1.2ml of a 50% aq. glutaraldehyde soln. The mixt. was kneaded in a mortar and left to stand fo 1 hr. The resulting aggregate was pulverized into a granular product and dried at room tepm. for 1 day. The product(11.4g) had an activity of 425 units/g.

Description

Preparation of insoluble glycosylase product

The present invention relates to a process for preparing insolubilized enzyme and insoluble enzyme product.

More specifically, the present invention is a physically stabilized form of insoluble glycosylase of microparticles usually selected from the group consisting of amyloglucosidase and maltogenic alpha-amylase of fungl origin. And methods for preparing such insoluble enzyme products.

Insolubilization (or inactivation) of water-soluble enzymes in cells and within cells, i.e., the immobilization of catalytically active (natural) enzymes in the type or typeable structure of insoluble solids or semi-solids As a means of making enzyme products reusable in batch-type methods or as a means of making them suitable for continuous operation in so-called enzymatic reactors, they have increased economic importance as well as engineering importance.

Recently, extensive efforts have been made in the development of immobilization techniques in the starch processing industry, particularly with regard to the production of products such as high dextrose and high fructose syrup. In recent years, the relative importance of high fructose syrups in food and related industries has increased because high fructose syrups are an advantageous substitute for sucrose and high dextrose syrup (less sweet). to be. Two additional syrups based on starch hydrolysis and both having a substantial maltose content are increasing in use, particularly in the sugar and hard canning industries.

The whole industrial method of converting starch into high fructose syrup via high dextrose syrup is based on three successive processes: starch dilution to dextrin under the action of alpha-amylase of acid and / or bacteria. The method of liquefaction consists of saccharification to high dextrose syrup followed by conversion of high dextrose syrup to high fructose syrup under contact with specific enzymes, namely amyloglucosidase and glucose isomethase, respectively. Recent industrial development has made it possible to use the continuous method for the first and third processes of the manufacturing series, but industrial saccharification is still primarily a batch method. In addition, the starting material of the high maltose-type syrup is liquefied starch prepared by any one of the above-mentioned methods. In this case, however, the subsequent saccharification process resulting in a syrup of high maltose content (and usually contains a relatively small amount of glucose) is carried out by maltose amylase, preferably by fungal alpha-amylase. As with glucose genic saceharification, the corresponding maltoseification method is usually carried out batchwise using soluble enzymes.

Although it is evident that it is convenient to have a whole series of preparations in one treatment, the development of industrial enzyme reactor techniques involving the use of insoluble amyloglucosidase in the saccharification process is only a preliminary step.

The prior art includes many references to insolubilization of amyloglucosidase, such as for example binding the enzyme to an insoluble inorganic or organic carrier. However, only a few of these methods seem to have been developed beyond the laboratory scale. Among the methods exceeding this step, it is necessary to specifically note the inactivation of amyloglucosidase by covalent bonding to porous particles of glass or porcelain material.

[Reference book about this is D.D. See a recent paper by Lee's colleagues in Die Starke, Vol. 27 (1975), page 387].

In a continuous process of feeding this particular enzyme product to a Pilotplant-scale column and supplying a commercially available dextrin solution, the drug generally required in a batch glycosylation process with soluble amyloglucosidase. A call to dextrose close to a 92% minimum conversion rate was achieved. However, the product analysis indicated that the reverse reaction, ie, repolymerization of maltose, isomaltose and glucose to higher oligomers under amyloglucosidase catalysis occurs more frequently in the column method than in the batch free enzyme method.

The increase in adverse reactions associated with this type of immobilized enzyme product appears to be an inherent drawback, at least in part, when using porous enzyme carrier materials.

Clearly, the porous structure contributes to a very substantial extent with respect to the overall surface area and binds to the enzyme itself, consequently contributing to the maximum enzymatic activity that can yield insoluble enzyme product.

However, high concentrations of amyloglucosidase adhering to the carrier pore surface inevitably result in locally high glucose concentrations as the rate of diffusion in the pores decreases, thereby facilitating enzymatic treatment with high Km-values. The preferable conditions are comprised. This is certainly the case with undesirable adverse reactions, especially when isomaltose and isomaltrios are formed.

Insoluble maltosized alpha-amylase products prepared by binding soluble enzymes to aminoethylcellulose by glutaraldehyde are disclosed in US Pat. No. 4,011,137 (Thomson's colleague). However, according to this document, the intended use of inactivated enzymes is limited to increasing the extent to which dextrinized starch is converted to glucose by similarly insoluble amyloglucosidase preparations, and the glycosylation process involves By a mixture of insolubilizing enzymes. There is no mention in this patent of the use of insoluble maltosized alpha-amylases in the preparation of high maltose syrups.

An additional drawback of using particulate inactivated glycosylating enzymes that bind enzymes to a substantially uniform porous or networked material throughout the particulate structure is the heterogeneity of the molecular size of the dextrinized starch that acts as a substrate. Can be predicted. All dextrins produced by starch acid or enzymatic hydrolysis contain a significant portion of glucose oligomers.

Typically, when the average chain length of the hydrolyzate is in the range of 6-10 glucose residues, the starch dilution process is stopped, which results in the formation of larger dextrin molecules into enzymatic sites embedded above a certain depth in the porous or reticulated particle structure. It can be imagined that simple stereoscopic obstacles prevent diffusion.

One object of the present invention is to provide an insolubilized product of the prior art, as described above, by providing an insolubilized product having similar processing characteristics to that of a soluble enzyme, regardless of whether the inactivated product is used in a batch or continuous process. It is in overcoming or at least alleviating the significant shortcomings presented with regard to.

In the case of immobilized amyloglucosidase, this requirement requires that the continuous saccharification process can be carried out under practically acceptable process conditions (eg in terms of composition and flow rate of the dextrin feed). Produce a glucose concentration of at least 92%, wherein the total concentrations of these sugars (maltose and isomaltose) and trisaccharides (mainly pannose and isomaltolithose) do not exceed 4% and 1%, respectively.

In the case of unmobilized maltose alpha-amylases, the corresponding conversion factor required is about 40-60% maltose, 25-35% maltotriose, and preferably up to 10% glucose.

Although commercially available soluble glycosylase is relatively inexpensive and very active, it is not necessary, but another preferred object of the present invention is to provide an economically advantageous insoluble product. What is needed in the pursuit of this purpose is the use of carriers and other auxiliary materials which are relatively inexpensive and readily commercially available, and furthermore achieve a substantial recovery of the enzymatic activity in the inactivation process. In addition, from a toxicological point of view, all materials used must be acceptable for food processing purposes.

In short, these aims are to crosslink glutar aldehyde with enzymatically inert, insoluble in water, to form an immobilized glycosylation product in particulate form substantially coated with a liquid permeable protein layer onto which glycosylation is immobilized. In accordance with the present invention provided.

Attempts to produce such insolubilization products, in which the protein layer consists of glutaraldehyde-crosslinked glycosylation enzymes, have not been successful on their own in that they result in incompletely immobilized products that rapidly leak enzymes regardless of the choice of core material. It was. Similar problems have arisen in inorganic (eg mineral or ceramic) strains of water-insoluble particulate carrier materials or certain types of proteinaceous core materials (such as granular soy protein), which may indicate that the number of crosslinking sites at these particle surfaces It was thought to be insufficient.

However, if granular casein is used as the core material and furthermore, the glutaraldehyde crosslinking of the enzymes in the coating layer is carried out in the presence of a water-soluble non-enzymatic binder protein such as egg albumen, the selection of such a material is clearly a sufficient number of proteins. It has been found that the above and other drawbacks are overcome because they constitute a desirable combination for forming enzyme stabilizing crosslinks therebetween, and the enzymatic properties of the non-synthetic enzyme coating products are greatly improved.

Therefore, in more detail, according to another embodiment of the present invention, in the non-glycosylated glycase product of the particulate form, the glycosylase is selected from the group consisting of amyloglucosidase and maltose alpha-amylase, The product comprises a carrier particle composed mainly of granular casein, wherein the glycosylase is substantially covered by a liquid permeable proteinaceous layer which is crosslinked jointly with the egg albumen by glutaraldehyde. Is provided.

The relative amounts of constituent glycosylase, casein and albumen can vary widely, especially depending on the unit activity of the soluble glycosylase starting material (as described below). However, in one preferred embodiment of the present invention, the ratio of glycosylase to casein is in the range of 1: 200-1: 3, preferably 1: 20-1: 5, and the amount of glycosylase is at least 50%. It is estimated based on a standard. Similarly, the preferred range of glycosylation enzyme to egg albumen is 1: 5-1: 0.05, more preferably 1: 2-1: 0.2.

According to another embodiment of the present invention, in the method for preparing an immobilized glycosylase product in particulate form, the glycosylase is selected from the group consisting of amyloglucosidase and maltose alpha-amylase, and the method is a glycosylation enzyme. A method is provided by substantially covering carrier particles composed primarily of granular casein having a proteinaceous layer that is crosslinked jointly with egg albumen by glutaraldehyde.

More specifically, the method involves vigorously stirring a dry mixture of granular casein and egg albumen powder with an aqueous mixture consisting of glycosylated enzyme and glutaraldehyde dissolved in PH 4-7, wherein the albumen is mixed with casein in a dry state. Instead, it consists of a wet step of appropriately dissolving together with these components and a step of completing the carrier coating and protein crosslinking step by holding the obtained wet mixture in a stationary state at normal ambient temperature.

Mixing can be carried out manually, for example by mixing and kneading completely in mortar, or mechanically, such as in a plow-level mixer (Gebr, Lodige Maschinenbau G. mbH Paderborn, West Grmaany) or an industrial mixer of similar formation. can do.

The ratio of glutaraldehyde (generally added to 50% (W / W) aqueous solution) to coating protein (enzyme + albumen) can vary considerably and is in the preferred range of 0.1-1. The water content of the aqueous solution is usually controlled in the range of 30-60% of the layer mixture.

The nature of the granular casein product selected as carrier material is important for achieving the object of the present invention. Thus, the casein granules must have sufficient physical stability to withstand substantial deformation when deposited under packed bed column conditions. In addition, the degree of flatness in water should be reasonably low, preferably not exceeding 200%. An example of a product that meets this need is acid precipitated granular casein. Preferred food grade products of this type with particle diameters of 100-500 microns can be purchased, for example, from the French company Scerma S.A. Scanning electron microscope showed that the surface of these particles had a very nonuniform and irregular layered structure similar to the appearance of pumice. It is conceivable that the microstructure of this type of surface will expose a number of glutaraldehyde defect sites.

It is essential to the practice of the present invention that a substantial portion of the albumen used as binder protein should be able to dissolve in water almost instantaneously. Therefore, preferred grades of albumen are generally available spray-dried products.

The mixing process involves a certain amount of coagulation of the coated particles, and when the crosslinking reaction is completed, a wet coarse lump product is produced. This product can be further disintegrated, for example, by granulation, to form a particulate product having a particle size within a preferred range. Granulation can be performed on vibratory granulators of the type provided by various companies (Diaf, Copenhagen or Manosty, Liverpool). The granular product passing through the granulator screen with holes of 1-2 mm can remove fine material by conventional means. The product thus obtained can be washed if necessary and dried to contain the desired amount of water.

Prior to use in the saccharification step, the dried enzyme product is usually controlled by pre-immersion in an aqueous solution. At this stage and during the initial stages of the next use of the product, specific leakage of enzymatic activity can be observed. In some cases incorporating additional crosslinking processes in connection with the dipping process serves to substantially reduce the loss of this enzyme activity. Thus, according to a preferred embodiment of the present invention, the pretreatment of the enzyme product is taken up in an aqueous solution containing 0.5-5% glutaraldehyde and an appropriate amount, followed by general washing with water to remove excess reagents and if necessary dried. To recover the product.

It was further observed that pretreatment of the enzyme product with salts of sulfurous acid can achieve a significant increase in the degree of conversion (expressed as the dextrose equivalent or as a percentage of the DE-value) of the saccharified product. The action of sulfite is expressed as the opening up of a particular network structure, which includes a proteinaceous enzymatic active layer but obviously (and surprisingly) does not affect the binding to the enzyme, thus facilitating access to higher oligosaccharides to the enzymatic activity site. Most preferably. Thus, in another preferred embodiment of the present invention, the enzyme product is treated with an aqueous solution of sulfite dilute, preferably with an aqueous solution containing 1-2.1% sodium sulfite at pH about 4-5. The sulfite treatment is preferably carried out at ambient temperature, usually terminated after 120 minutes by washing with water, and if desired, the dried product is recovered.

The particulate enzyme product prepared according to the method can be used in a batch saccharification process involving separation and reuse of the enzyme product or in a continuous process in an enzyme reactor.

The microbiological source of glycosylase is not part of the present invention. Preferred commercial amyloglucosidase products, however, can be obtained by culturing strains belonging to the genus Aspergillus or Rizopus, such as Aspergillus niger or Rizopusdelema. Likewise maltosized alpha-amylases are preferably obtained from strains of Aspergillus Oryzea.

[Measurement of Amyloglucosidase Activity]

Unit activity and inactivation of soluble amyloglucosidase, defined as the amount of enzyme or enzyme product that hydrolyzes an aqueous solution containing 30% (W / V) maltose at an initial rate of 1 micromole maltose per minute The unit activity of the enzyme is assayed under standard conditions of pH 4.5 and 55.0 ° C. The activity of the inactivated enzyme is assayed in batches on a sample held in suspension by a shaker.

[Measurement of Alpha-amylase Activity of Bacteria]

Soluble bacteria, defined as the amount of enzyme or enzyme product that hydrolyzes an aqueous solution (6.95 g / 1000 ml) containing soluble starch (Merck Amylum Soluble, DAB, Erg. B VI) at an initial rate of 5.26 mg of starch per hour. The unit activity of alpha-amylase and the unitase of inactivated enzyme are assayed under standard conditions of pH 5.6-5.7 and temperature of 37.0 ± 0.05 ° C. and in the presence of 4.3 mmol Ca ⁺⁺ ions. Iodine is used to generate blue to track the reaction. During starch decomposition, the dichroic color becomes weaker and gradually turns reddish brown. Changes in color are visually inspected by comparison with colored glass standards. The activity of the inactivated enzyme is assayed under conditions similar to those indicated for amylobacilli coosidase.

Soluble glycosylase is commercially available. Examples of such products are amyloglucosidase NOVO 150 and FUNGAMYL 1600 (Pungarme® is a registered trademark). The former is commercially available in aqueous solution and is usually converted to a spray-dried powder before use for the purposes of the present invention. If necessary, the drying step is usually performed by ultrafiltration combined with a washing step by a preparation for removing low molecular weight contaminants. Such concentration and washing processes well known to those skilled in the art can be used to easily obtain a solid amyloglucosidase product having 5000 units or more of activity per gram.

Fungamil 1600, which is commercially available in powder form, has 1600 units of activity per gram. This commercial product can serve as a satisfactory starting material in itself. However, if desired, it is possible to intervene, for example, cleaning and concentration processes of the kind described above.

The overall recovery of activity in the inactivated glycosylating enzyme product is a function of the detailed parameters of the inactivation process, but is usually not less than 40% and sometimes higher.

Substrats of the saccharification process performed with the immobilized enzyme product according to the present invention may be any oligosaccharide that is glycosylated in the presence of the corresponding soluble enzyme. Examples of preferred substrates include dextrins having a DE-value in the range of 5-40 obtained by liquefaction of starch with acids and / or enzymes; High maltose syrup (DE-value 35-60); And residual oligosaccharides present in the mother liquor from dextrose crystallization or in raffinates obtained from fructose-glucose fractionation. Dextrin solutions with dry solids in the range of 20-55% (W / V) are preferred.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1

Spray-dried egg eggs commercially available in a relatively coarse granular hydrochloric acid precipitated casein [9 g of Caseine alimentaire® (Scerma SA, France), consisting of 100-500 micron particles with about 60% of 300-500 micron particles present] Mix with bumen (1.2 g). This mixture contains amyloglucosidase powder [6.5 ml of 18.5% (w / v) aqueous solution containing a total of 12,500 amyloglucosidase units] and glutaraldehyde [1.2 ml of 50% (w / w) aqueous solution]. The mixed solution was added. The amyloglucosidase powder was an ultrafiltered spray-dried product made by AMYLOGLUCOSICASE NOVO (Novo Industries A / S, Denmark).

The mixture was mixed carefully in mortar and then left at room temperature for 1 hour. The obtained aggregate was pulverized to obtain a granular product, which was dried at room temperature for one day.

The dried granular product (11.4 g) had 425 units of activity per gram, thus recovering 38.8% after inactivation.

Example 2

Spray-dried albumen (120 g) and 20 liters of granular acid-filled casein (comprising 1500 g of type S60 (Scerma SA), consisting of 100-500 micron particles with about 70% 150-350 micron particles present) Mixing was carried out in a plow-type horizontal mixer (manufactured by West German company Gebr.Lodige Machinenbau Gmb H).

Ultrafiltration concentrate of amyloglucosidase prepared from amyloglucosidase novo (concentrate, 650 g of solution containing about 3200 units of soluble amyloglucosidase per gram of dry matter) The mixture was mixed thoroughly with glutaraldehyde (180 ml of 50% (w / w) aqueous solution) at a temperature of pH 4.9 and 18 ° C., and then the obtained solution was poured into a mixing apparatus. The mixing was continued vigorously for an additional 0.5-1 minutes and then the wet particulate product was removed from the mixer. The wet product was left to stand for about 45 minutes to complete the crosslinking process, resulting in the formation of agglomerated particles. Granulation of the product was carried out in a vibratory granulator with 1.5 mm diameter screen (supplied from Diaf A / S company in Copenhagen).

The granules were washed with de-ionized water (10 L) for 10 minutes, collected by filtration and dried over fluid bed. The microproducts were removed by sieving the dry product (1600 g, 450 amyloglucosidase units per g).

Example 3

A solution of spray-dried amyloglucosidase powder (105 g, activity: 11.400 units per gram, prepared as in Example 1) and spray-dried albumen (150 g) was prepared in tap water (400 ml) and cooled It was left overnight at 6-7 ° C. The pH of the solution was 5.4.

After adding glutaraldehyde (100 ml of 50% (w / w) aqueous solution), the resulting solution was poured into a vigorously stirred batch of granular hydrochloric acid casein (725 g, the same grade used in Example 1) for 3 hours. It transferred and mixed in the same industrial mixer as used in Example 2. The reaction mixture was stirred vigorously for an additional few minutes and left until about 1 hour until the crosslinking reaction was completed. The resulting aggregated mass was granulated in a vibratory granulator through a screen of 2 mm diameter.

The granules were washed with aqueous sodium acetate (pH 4.2) and then dried in vacuo at 35 ° C. The dried granular product (925 g) had 800 units of activity per gram and showed a recovery of 67.8% after inactivation.

Example 4

The non-immobilized amyloglucosidase product (20 g) prepared according to Example 3 was immersed in an aqueous solution of glutaraldehyde (500 ml of a 1% solution adjusted to pH 7) for 1 hour at ambient temperature followed by a dry product (20 g). Was recovered.

The activity of this product is 625 units per gram. Thus, 78% recovery from the second inactivation step and 53% overall recovery from soluble amyloglucosidase.

Example 5

Aqueous solution of maltose alpha-amylase [2 ml of 5% solution (w / v) of FUNGAMYL 1600) (Novo Industries A / S, Denmark) in deionized water with pH 6.2] (0.3 ml of 50% (w / v) solution) was then added quickly to a dry mixture of granular acid precipitated casein (4.0 g type M60) and commercially spray-dried egg albumen (0.5 g). The product was mixed in the mortar and left for 1 hour at room temperature. The mass was disintegrated in the mortar and then dried for 1 day at room temperature.

Example 6

The powdered amyloglucosidase product (5.7 g) prepared according to Example 1 was placed in a coated column with an internal diameter of 15 mm maintained at 55 ° C. 0.2% (w / v) sodium sulfite was added to a down-flow feed consisting of 30% (w / v) commercially available acid / enzyme-hydrolyzed dextrin (CPC Snow Flake Maltodextrin 01915 from DE20) having the following composition: Was adjusted to 4.5 and added to the column at a constant flow rate of 15 ml per hour.

The effluent was analyzed by high pressure liquid chromatography (HPLC) to obtain the following results.

Example 7

Into a coated column having an internal diameter of 25 mm, kept at 55 ° C., was filled with the inactivated amyloglucosidase product (20 g) prepared according to Example 2.

An up-flow substrate feed having the same concentration, composition and pH as used in Example 6 was added to the column at a constant flow rate of 50 ml per hour. The glucose content rate (% DP ₁ ) was analyzed by the hexokinase method, and the following results were obtained.

Example 8

Into a coated column having an internal diameter of 25 mm held at 55 ° C. was filled with the inactivated amyloglucosidase product (25 g) prepared in Example 2. Up-flow substrate feed of the same composition as used in Example 6 was added to the column. This feed (pH 4.5) had a dry solids content of 30% (w / v) and potassium sorbate (2 g / 1) was added as preservative. The flow rate was adjusted to maintain a nearly constant degree of conversion. As a result of analyzing the effluent by HPLC, conversion of glucose (DP1) to time was obtained as shown in the following table.

After the reaction was completed, there was no change in the hardness or other physical properties of the column material.

Example 9

Using the method of Example 7, but using the product of Example 4 instead of the inactivated amyloglucosidase product of Example 2, the following results were obtained.

Example 10

Inactivated maltosized alpha-amylase (4 g) prepared according to the method of Example 5 was charged into a coated column maintained at 45 ° C. and sodium sulfite (0.2%) as a 30% (w / v) dry solid and preservative. Down-flow feed of commercial textrin (CPS Snow Flake Maltodextrin 01913) containing%) was added. The pH of the feed was adjusted to 4.5 and the flow rate was adjusted to 15 ml per hour. The table below shows the composition of the column effluent: DP ₂ mainly indicates the maltose content.

Example 11

160(노보 인더스트리 에이/에스, 덴마크)를 사용하여 전분을 액화시켜 제조하고, 하기 조성을 갖는 덱스트린 건조물 함유율을 기준으로 31%(w/v)의 하향유동 공급물을 컬럼에 가하였다.A. A immobilized amyloglucosidase (12 g) prepared by the method of Example 2 was charged to a coated column having an internal diameter of 15 mm maintained at 55 ° C. Dermamil (THRMAMYL)

Prepared by liquefying starch using 160 (Novo Industries A / S, Denmark), a 31% (w / v) downflow feed was added to the column based on the content of dextrin dry matter having the following composition.

This feed contained sodium sulfite (0.2% w / v) as preservative and had a pH of 4.5. Flow was adjusted to maintain a constant conversion (by% DP1). Analysis of the effluent by HPLC gave the following results.

B. Dextrin substrates prepared according to the method described above in (A) and having the composition

Was treated for 2 hours with 0.01% (based on total solids) maltosized-alpha amylase (Fungamil 1600, Novo Industries A / S) at pH5.0 and 50 ° C. After treatment, the substrate had the following composition.

Two coated columns of 15 mm internal diameter maintained at 55 ° C. were charged with unimmobilized amyloglucosidase (10 g), prepared by Example 2, respectively.

Composed of 30% (w / v) of two forms of dextrin prepared as described above, 0.2% (w / v) of potassium sorbate was added thereto and a downward flow feed with a pH of 4.5 was added to the column. The same result was obtained.

Example 12

Portions (10 g) of unmobilized amyloglucosidase prepared according to the method of Example 2 were immersed in 100 ml of solution containing different concentrations of sodium sulfite. The pH of the solution was adjusted to 4.5 by adding acetic acid. After 2 hours immersion at ambient temperature, the enzyme sample was washed with deionized water and charged into a coated column maintained at 55 ° C. with an internal diameter of 15 mm. Dextrin (30% (w / v)) prepared according to the method of Example 11A, to which potassium sorbate (0.2% (w / v)) was added and the pH of the downflow feed was adjusted to 4.5 Was added.

The maximum DX-value of the effluent obtained by adjusting the flow rate of each column is as follows.

Example 13

A high maltose (D.E. about 42) effluent collected from the unimmobilized Fungal Mill column of Example 10 was used as feed to the unmobilized amyloglucosidase column of Example 6. The flow rate was adjusted (to 73 ml per hour) to obtain an effluent with a high rate of solidification (D. E. about 65) syrup of the following composition.

Example 14

The immobilized amyloglucosidase product (20 g) prepared according to Example 3 was placed in a coated column having an internal diameter of 25 mm held at 55 ° C.

An upflow substrate feed consisting of a mother liquor (or raffinate) obtained from Protos-glucose fractionation and having the following composition and adjusted to pH 4.5 was added to the column at a flow rate of 250 ml per hour at a concentration of 25% (w / v).

The composition of the effluent determined by HPLC was as follows.

Claims (1)

As detailed herein, in the preparation of insoluble glycosylase products in particulate form, the glycosylase is selected from the group consisting of amyloglucosidase and maltose alpha-amylase, the method being predominantly precipitated. The carrier particle composed of casein is substantially a protein layer in which the glycosylated enzyme in the protein layer is crosslinked with the egg albumen by glutaraldehyde with the egg albumen by glutaraldehyde. Preparation of insoluble glycosylase products characterized by coating with.