TW200914378A - Use of hydrophobins as auxil auxiliaries in the crystallization of solids - Google Patents

Abstract

Use of hydrophobins as auxiliaries in the crystallization of solids, in particular the use for producing gypsum from an aqueous phase.

Description

200914378 IX. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to the use of hydrophobin as a supplement in the solid crystallization process, specifically for the purpose of producing gypsum from an aqueous phase. [Prior Art] The properties of fine solids are mainly determined by the crystal size and characteristics of the constituent solids. The size and characteristics primarily affect the rheological properties of the solid suspension, the ease with which the solid suspension is removed (e. g., filtered), the dry product treatment, and the nature of the solid itself. For example, the color strength or dispersibility of a colorant is mainly determined by the size and characteristics of a single crystal. In principle, it is known that the use of suitable inorganic or organic additives affects the size and characteristics of the crystals formed during precipitation or crystallization from the solution. See, for example, New York 2006, Weinheim, Wiley-VCH, Electronic, 2006, 7th edition, Ullmann, s Encycl〇pedia 〇f (d) her...

Chemistry"Crystallization and Precipitati〇n_4 4 Crystal

Habit Modification". The effect on the size and properties of gypsum crystals is an in-depth study (see example G.A. Bertoldi, Zement-Kalk-Gips, Nr. 12, 1978). It is seen that in the flue gas desulfurization, an aqueous suspension of a large amount of gypsum is produced for further use, and the gypsum needs to be separated from the aqueous phase. The gypsum is usually needle crystal. 1^4, 183, 908 and 〇 8 5, 246, 677 disclose a method of crystallizing gypsum in the form of dense crystals by promoting the removal of gypsum from the aqueous phase via the addition of polyphosphates, organophosphates or phosphonates. 131092.doc 200914378 Hydrophobin is a small protein that occurs in filamentous fungi (such as about 10 to m amino acids in the genus (4). These usually have 8 cysteines alone. The hydrophobin can be isolated from the source of the <is such that it can also be obtained by genetic engineering methods such as disclosed in WO 2006/082251 or WO 2006/131564. The prior art suggests the use of hydrophobins in a variety of applications. 96/41882 recommends the use of hydrophobins as emulsifiers, thickeners or surfactants, imparting hydrophilic properties to hydrophobic surfaces, improving the resistance of hydrophilic matrices, preparing oil-in-water emulsions or water-in-oil emulsions. Further recommendations include Pharmaceutical applications such as the preparation of ointments or creams and cosmetic applications such as the manufacture of skin care products or shampoos or conditioners. Μ 1 252 516 discloses the use of hydrophobics at temperatures of 30 to 8 Torr. The solution of the protein is coated with a variety of matrices. 'Step construction 匕 3 for example as a demulsifying agent (WO 2006/103251), evaporative retarder (W〇2006/128877) or stain inhibitor (w〇2嶋(10) L ) use of 7 hydrophobins The use of a crystallization adjuvant has not been disclosed so far. [Invention] The object of the present invention is to provide a novel adjuvant which affects crystallization. [Embodiment] It has been found that the purpose of the hydrolytic protein in the solid crystallization process is The use of the adjuvant is achieved. 进 The further embodiment of the invention is a method for separating the solid formed from the aqueous phase from the aqueous phase, wherein the aqueous phase has been added two 131092.doc 200914378. The total amount of the aqueous phase is 0.001. 。, ε, soil/〇 to at least 1% of at least one auxiliary agent soluble in the aqueous phase, at least one of the auxiliary λ-seeking assistants comprising hydrophobin. In the present invention - preferred embodiment In the method, the method of making a gypsum is particularly preferred as one of the steps of the flue gas desulfurization method. The details of the invention are as follows: The term as used herein, hydrophobin " has the following structural formula (1) Peptide

VCi-X1.50.C^X〇-5^.X1.1〇〇.C4.Xi i〇〇c^Xi ^ce x〇s C7_x ^ (丨) where X is 20 natural amino acids (Phe , Leu, Ser, Tyr, CyS,

Trp, pr. , Hls, Gin, Arg, I, Chest, w, Fine, _,

Any of Va Bu Ala, Asp, Glu, Gly). Each x may be the same or different. In each case, the index of the χ next to the χ is the number of amino acids, and the deuterated feta fe S is owed to alanine, serine, glycine, guanine or sulphamine. The k-restriction condition is that at least four of the residues represented by C are semi-deaminized S text, and the indices 11 and 〇1 are respectively a natural number between 〇 and 500, preferably between 15 and 300. The polypeptide of the formula (1) is further characterized in that the contact angle of the water droplets (after coating the surface of the glass) is at least 2 Å, preferably at least 25, greater than the contact angle formed by the water droplets of the same size and the surface of the uncoated glass. And better at least 30. Each of these measurements was carried out at ambient temperature. The amino acid representing C to C8 is preferably cysteine; however, these may be replaced by similar amounts of other amino acids, preferably alanine, serine, sulphate, methionine. Or glycine. However, at least four of the C1 to C8 positions, 131092.doc 200914378 are preferably at least 5, more preferably at least 6, and most preferably at least? One consists of a semi-phosphoric acid. The protein towel cysteine produced according to the present invention may exist in a reduced form or form a disulfide bond with each other. The formation of the c_c bond in the molecule is optimal, and in particular comprises at least one 'preferably two, more preferably three and most preferably four guaia bonds in the knife. When the above-mentioned similar volume of amino acid is used for 35 耽 (4), it is advantageous to form intramolecular disulfide bonds at positions involved in the pair exchange.

When the position of the X-form is cysteine, serine, alanine, glycine, methionine or threonine, the value at each C-position in the formula can be changed accordingly. A preferred embodiment of the present invention can use the hydrophobin of the general formula (11) X«-C1.X3-25-C2-X〇, 2-C3-X5.6〇-C-»-X2.3S.C5 .X2.15-Ce.X0.2-C?-X3.35.C8*Xm (Ιί) where X, c and the index immediately adjacent to X and C are as defined above, and the index ηΛιη represents the value between 〇 and 350. Preferably, it is between 15 and 3 Torr, and the proteins are further distinguished by the above contact angle change, and at least six of the residues represented by c comprise cysteine. It is particularly preferred that all C residues contain cysteine. It is especially preferred to use the hydrophobin of formula (III)

Xn-C1-X^9-C2-C3-Xii.3rC4-X2-2rC5-X5-rC6-C7-X&.i8-C8-Xm <") where X, C and the index immediately adjacent to X are as defined above The index n&m represents a value between 〇 and 2 ,, the proteins are further distinguished by the above contact angle change and at least six of the residues represented by C are cysteine. It is particularly preferred that all residues represented by c are cysteine. 131092.doc 200914378 J-based Xn and Xm are peptide sequences that are naturally linked to hydrophobins. However, the medium or both may be self-sequenced with the hydrophobin. Also included is a ^β^Jn η or Xm residue, wherein the peptide sequence naturally occurs in = white, and the (etc.) residue is extended naturally by the hydrophobic egg peptide. In the case of a peptide sequence which is not naturally linked to a hydrophobin, the A acid is at least 2G amino acids, preferably at least 35 amine groups. For example, this calculation is 20 to 500, preferably 30 to 400, and most preferably 35 to 1 amino acid ^ ^ ^ # -λ-, which is not naturally linked to hydrophobin. Also refers to the following fusion objects. This shows that the protein consists of at least one hydrophobin moiety with a non-gentleman ^ , , , , , . The composition of the fusion object is composed. The fusion hydrophobin formed by the fusion of W and the hydrophobin portion of the Japanese gems, & _ human knives, is disclosed in, for example, 〇wo 2〇〇6/082253, WO 2006/131564 〇 2: The "minus can be selected from a variety of proteins. There may be only one fusion pair", the hydrophobin moiety is linked or there are a plurality of fusion objects that can be linked to the hydrophobin moiety, such as the amino terminus of the 火"1 fire protein quinone (xn And carboxyl lignin (into the junction. However, one side of this, for example, two fusions are linked to a position (mm) in the protein according to the invention. A particularly suitable fusion object is a protein that is present in the microorganism. , especially 2 Escherichia coli or Bacillus subtilis. Examples of such fusion objects are Γ (6/082251 in seqidn〇: i6), (4) 细细细51, +...丨 to emulsify reduced protein. Extremely suitable is also... Or a derivative thereof, which comprises only a portion of the sequence, such as I31092.doc 200914378, such as 70% to 99%, preferably 5% to 5%, and more preferably 1% to 4%, or an individual thereof Amino acids or nucleotides have been modified relative to the above sequences, such The fractions are each calculated as the amount of amino acid. In another preferred embodiment, the fusion hydrophobin has not only the fusion object described above but also an affinity functional site in the form of an affinity tag/tail, such as a base in xn or xm a group or a terminal component such as the group. The affinity tag or tail (previously known) comprises an anchor group which interacts with some of the complementary groups and facilitates processing and purification of the protein. Examples of such affinity functional sites A natural number between the 3 (His)k, (Arg)k, (Asp)k, (Phe)d (Cys)k groups, where k- is UH). (1) It is preferable that k is between 4 and 6 in the group. The xn and/or x„ group may consist solely of the affinity functional site or the X^Xm residue naturally or unnaturally linked to the hydrophobin is extended by the terminally located affinity functional site. According to the invention as a hydrophobin The protein used in the derivative or derivative thereof may further modify the polypeptide sequence, for example, by saccharification reaction, acetylation or chemical crosslinking, for example, crosslinking with glutarylene. Hydrophobin or a material thereof used according to the present invention The characteristic is that the surface properties change when the surface is coated with the proteins. The change in surface properties can be determined experimentally, for example, before the surface is coated with the protein and (4) the contact angle of the water droplets and the difference between the two measurements is determined. The person skilled in the art knows in principle how to measure the contact angle. This, J is related to the to/in, the water droplets and the glass plate used as the substrate. The precise experimental conditions suitable for, for example, the method of measuring the contact angle are illustrated in the experimental part. Under the conditions described herein, the properties of the fusion protein used according to the invention 131092.doc 200914378 are in each case the contact angle is equal to the size The contact angle of the water droplets to the uncoated glass surface is increased by at least 20. 'preferably at least 25. And optimally at least 30. The optimal hydrophobin in the embodiment of the invention is dewA, r〇dA, hyPA 'hypB Hydrophobins of the SC3, basfl, basf2 type. Hydrophobins comprising such sequences are disclosed, for example, in WO 2006/82251. Unless otherwise stated, the sequences mentioned below are disclosed in w〇2〇〇6/82251 Sequence correlation. A summary of the SEq_id number is given in w〇2〇〇6/82251 ('page 20. Particularly suitable for the polypeptide sequence in parentheses and the nucleotide sequence encoded thereby in accordance with the invention (especially a fusion protein yaad_Xa_dewA_his (SEQ ID n〇2()) according to the sequence of SEQ ID N〇: 19, 21, 23,

Xa-n>dA-his (SEQ ID NO: 22) or yaad_Xa_basfKhis (SEQ ID NO. 24). It is particularly preferred to use yaa (j_xa_dewA_his (SEQ ID NO. 20). Likewise, a particularly preferred embodiment is the exchange, insertion or removal of at least the polypeptide sequence set forth in SEQ ID N: 2〇, 22 or 24 A protein of up to 1 ,, preferably 5, more preferably 5% of the total amino acid and still having a biological property of at least 50% of the starting protein is the best example. Means that the contact angle changes by at least 2 〇. Particularly suitable derivatives of the specific examples of the invention are % 以 heart dewA-his (SEQ ID NO: 20). yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa_basfl_his (SEQ ID N〇: 24) a derivative derived from a fusion object. It may be advantageous to use a truncated yaad residue instead of a complete "fusion" 131092 with 294 amino acids. Doc -12- 200914378 ID NO: 1 6). However, the truncated residue should have at least 2, preferably at least 3, 5 amino acids. For example, a residue having a truncation of from 2 to 293, preferably from 25 to 250, more preferably from 35 to 150 amino acids (e.g., 35 to 1 amino acid) can be used. One of the examples of this protein is yaad40-Xa-dewA-his (SEQ ID ΝΟ·.26 in PCT/EP 2006/064720) having a yaad residue truncated to 4 胺 amino groups. The splitting point between the hydrophobin and one or more fusion targets can be used to separate the fusion target and release the underivatized pure hydrophobin (eg, BrCN splitting)

Methionine, Factor Xa cleavage, intestinal peptidase cleavage, thrombin cleavage, Ding ev splitting, etc.). The hydrophobin used as a crystallizing aid according to the present invention can be chemically prepared by known peptide synthesis methods, for example, via Merrifield solid phase synthesis. Naturally occurring hydrophobins can be isolated from natural sources via suitable methods. Reference may be made, for example, to w6sten et al., Eur. J Cell Bi〇 63, 122 129 (1994) or WO 96/41882. The gene guard method for producing hydrophobins from unblended subjects from Talar〇myces therm〇philus is described in the training. The human sigma protein & is prepared by a genetic engineering method, and the nucleoside sequence encoding the fused 5 protein and the nucleotide sequence encoding the hydrophobin moiety, and the gossip sequence are combined to make the combined nucleotide sequence Gene expression, I produce the desired egg... This manufacturing method is disclosed, for example, by side = ΓΓΓ: 〇 2 ° 253. These fusion targets are highly advantageous for the production of hydrophobins from unfused objects. , 4 large 131092.doc •13· 200914378 The fusion hydrophobin produced by the genetic engineering method from the host organism can be processed in a manner known in principle, and purified by chromatography. A preferred embodiment of ice is a simple treatment and purification method using w〇2_(10)22w. Therefore, the fine cells of the fermented liquid are not separated from the fermentation broth, and the cell debris is separated from the inclusion body by rupture. The latter is best separated. Finally, the inclusion bodies are broken in a manner known per se, for example, the release of the fusion hydrophobin by acid, alkali and/or detergent. Inclusion bodies with fused hydrophobins used according to Lin Ming can generally be "completely" solved, even with 0.1 μl of NaOH. The present invention is specifically practiced, and the resulting solution can be used without further purification. However, the fusion hydrophobin can also be isolated as solids from solution. Preferably, it is separated by spray drying as described on page 12 of Motion 2_(4)(5). The product obtained by the thinning process and the purification method usually contains about 8 ounces by weight. A to 重量 Weight % protein and cell debris residue. The fusion hydrophobin content typically ranges from 30% to 8% by weight of the total protein content' depending on the fusion construct and the fermentation conditions. The isolated product comprising the fused hydrophobic egg is stored as a solid and dissolved in a specific desired medium for use. The fusion hydrophobin is "pure" after cleavage and removal of the fusion object, and the hydrophobin is subjected to cleavage after isolation and dissolution of the inclusion body. According to the invention, the hydrophobin is used as an adjuvant in solid crystallization. In the presence of the hydrophobin, crystallization is affected. A preferred embodiment of the invention comprises crystallizing a solid from a liquid phase comprising one or more solvents, dissolved solids and/or the original 131092.doc for use in the manufacture. -14- 200914378 Water protein and other components (such as its substances) as needed. The choice of solvent or solvent mixture is not in principle... Solids and hydrophobins to be crystallized are sufficiently soluble in them. A suitable choice can be made depending on the solid to be crystallized. = Preferably comprises the aqueous phase. The term 'aqueous' is used to include at least 50% by weight of water, based on the total content of all solvents. The water contains ί:°% by weight' more preferably at least 9% by weight. Possible cosolvent packages 3 are water miscible solvents such as alcohols (e.g., methanol, ethanol alcohol). The solvent contains only water particularly well. Or those skilled in the art can select the phase of the liquid according to the characteristics of the solid to be crystallized and according to the desired properties of the solid. According to the present invention, a hydrophobin (preferably a fusion hydrophobin) is preferably used at a ton of 4, particularly preferably a range of (1) preferably between 5 and 13, more preferably between 6 and 12, most preferably between.人士 Those skilled in the art can select the amount of hydrophobin according to the characteristics of the crystalline solid to be used and the properties required for the solid. Usually, the amount is less than i% by weight based on the total content of all components of the aqueous phase. The hydrophobin is preferably used in an amount of from 1% by weight to 1% by weight, more preferably from 重量1% by weight to 〇2 by weight. In principle, all means of crystallization from the liquid phase are possible. A possibility is, for example, a crystallization method, and the effect of the UI section is to initiate solid crystallization from a saturated solution of solids from eight T and by evaporating the solvent, cooling or mixing another solvent which does not dissolve the solid. Another possibility is the reactive killing method in which only the soluble components react with each other to form a solid in the aqueous phase. A particularly preferred embodiment of the invention is the use of the above-described fusion hydrophobin. Example 13W92.doc -15- 200914378 For example, yaaci-Xa_dewA-his (SEQ ID ΝΟ··20), and especially a protein with a singularly truncated yaad residue, such as yaad40-Xa-dewA-hlS, can be used. It is preferred to use the product obtained by the above simplified purification method. The hydrophobin is suitable as an auxiliary for the crystallization of inorganic and organic solids from the liquid phase, especially as an adjuvant for crystalline gypsum (Cas〇4.2H2〇). It is known that the corpus callosum is not a needle-like crystal, but instead obtains more dense crystals which are more easily separated from the aqueous phase and have a significantly smaller length/thickness ratio. Hydrophobin is further used to crystallize carbonic acid.

In a preferred embodiment of the invention, the hydrophobin can be used in a process for making a solid by solidification formed by crystallization from an aqueous phase and removal from the aqueous phase. This method is particularly suitable for the method of removing gypsum. More than one describes the crystallization step and preferred conditions. The solid, preferably gypsum, can be removed by methods known to those skilled in the relevant art, for example, by a combination of various methods of filtering or separating liquids and solids. After separation, the wet solid is dried and further processed. The method of the invention may (iv) include a flue gas desulfurization process. In the (four) gas desulfurization process, the 'stage-stage includes the reaction of the gaseous kisses present in the flue gas with the CaC()3 aqueous suspension to form caS〇3, in a secret device known to those skilled in the art. Oxidation by 〇2 forms CaS〇4, which is precipitated as Cas〇42. Therefore, the stone f is continuously formed in the aqueous phase via the reaction. To control B. T, add hydrophobin to the treated water at the above concentrations. The flue gas desulfurization method is a related skill; 4 people know. Such as S02 and

The CaCCh reaction can be carried out in a countercurrent manner without the separation of the concentrated gypsum suspension in the lysate separator, which is formed by the concentrated gypsum suspension, and then filtered and rinsed through 131092.doc -16· 200914378. And dry to recover solids. Further details refer to New York 2006, Weinheim, Wiley-VCH, Electronic Release, 2006 7th Edition,

"Calcium Sulfate-3, 2 Flue Gas Desulphurization (FDG) Gypsum", and references cited therein in Ullmann's Encyclopedia of Industrial Chemistry. The formed gypsum is subsequently fired (forming an anhydrate) and used in a known manner for example in construction In the materials industry, the following examples illustrate the invention: Providing hydrophobins. The invention utilizes a fusion hydrophobin with a complete fusion target yaad (yaad_Xa_dewA_hi, hereinafter referred to as hydrophobin a) and a fusion object with a cutoff of 40 amino acids. a fusion hydrophobin of yaa (j4〇-Xa-dewA-his (hydrophobin B). These were prepared according to the procedure described in WO 2006/082253. The product was treated via the simple purification method of Example 2 of WO 2〇06/82253, And spray dried according to the same reference example 1. In each case, the total protein content of the obtained dry product is about 70% to 95% by weight, and the hydrophobin content is about 40% of the total protein content. 90% by weight. These products were used for experiments. Performance test: Determination of the characteristic matrix of the fusion hydrophobin by the change of the contact angle of water droplets on the glass Glass (Window Glass, Siiddeutsche Glas, Mannheim) In these experiments, a spray-dried product containing fused hydrophobin was dissolved in sodium acetate containing 50 mm pH 4 and 0 gt; 1 wt% polyoxyethylene (2 〇) sorbose 131092.doc 200914378 Alcoholic anhydride monolaurate (Tween®20) in water. Product concentration: 100 pg/mL aqueous solution. Procedure: - Glass slide culture overnight (8 〇 art), then wash the coating with distilled water, - Then incubated for 10 minutes / 8 〇. 〇 / 1% sodium dodecyl sulfonate (SDS) in distilled water, - rinsed with distilled water

The sample was dried in air and the contact angle (in degrees) of 5 μl water droplets was measured at room temperature. The contact angle was measured via Dataphysics Contact Angle System OCA 15+, software SCA 20.2.0 (U-2002). Make measurements according to the instruction manual. The untreated glass has a contact angle of 15. To 30. ±5. . The fusion hydrophobin yaad. Xa-dewA-his6 coating increased the contact angle by 3 〇. Above; fusion of hydrophobin yaad40-Xa-dewA-his coating also increased the contact angle by 3〇. the above. Gypsum Crystallization Experiment General Procedures Prepare a saturated solution of CaS〇4.2H2 in fully deionized water (concentration approx. 2 g"). Each solution is mixed with hydrophobin A and hydrophobin b, respectively, so that the concentration of the spray dried product of the gypsum solution is about 1.1 8 / 卜 respectively by the two and NaOH to adjust the solution ρί^ by the folding filter (about 1 〇 Filter the aqueous solution/suspension. Then, transfer about 25 ml of each solution to the culture and evaporate the water at room temperature for about 24 hours. Add the solution as a control. Protein 131092.doc - 18- 200914378 Figure] $6 eight-mass form using a Hlympus BH-2 microscope to compare the microscopic images of the crystals obtained (40 times magnification - in the case of a particle size of about 0.01_0.lmm)

Figures 1 to 6 show that the gypsum crystal form can be affected by hydrophobin. The gypsum precipitated in the absence of an adjuvant and under the secondary 8 contains a needle-like body (10) 0 having a length/thickness ratio of about 1 。. Upon addition of hydrophobin A (Fig. 2) or hydrophobin B (Fig. 3), the gypsum precipitated at pH 8 did not have needles; conversely, a dense prism having a length/thickness ratio of about 2 to 3 was obtained. The length of the needle was significantly truncated compared to the experiment without hydrophobin. These compact particles have better filtration properties. The length of the needle is truncated by hydrophobin at all pH values (圊4 to 6). This effect is most pronounced in the alkaline range (Figures 1 to 3 and 6), where almost only prisms are obtained and no needles are present. [Simple description of the map]

Figure 1 pH 8, no hydrophobin added Figure 2 pH 8, add hydrophobin A Figure 3 pH 8, add hydrophobin B Figure 4 pH 4, add hydrophobin B Figure 5 pH 6, add hydrophobin B Figure 6 pH 1 〇 , adding hydrophobin B 131092.doc -19·

Claims (1)

200914378 X. Patent application scope: r—Use of hydrophobin as an auxiliary agent in the solid crystallization process. 2. The use of the requester, wherein the crystallization process comprises 曰 曰 从 0 from the liquid phase. 3. 3. 5. Ο 6· 7. 8. The use of claim 2 wherein the amount of the hydrophobin is The liquid phase totals 3: from 0.001% by weight to 1% by weight. The use of claim 2 or 3 wherein the liquid phase comprises an aqueous phase. The use of the item 4, wherein the pH of the aqueous phase is between 7 and 11. The use of claim 4, wherein the solid comprises gypsum. The use of claim 4, wherein the solid comprises calcium carbonate. The use of any one of claims 1 to 3, wherein the hydrophobin comprises a fusion hydrophobin. A method for producing a solid by crystallizing a solid from an aqueous phase and separating the formed solid from the aqueous phase, wherein at least one of 0.001% to 1% by weight of the total amount of the aqueous phase has been added to the aqueous phase. The adjuvant which is soluble in the aqueous phase 'at least one of the adjuvants comprises a hydrophobin. The method of claim 9, wherein the pH of the aqueous phase is between 7 and 11. 11. The method of claim 9 or 1 wherein the solid comprises gypsum. 12. The method of claim 9 or 1 wherein the solid comprises calcium carbonate. 13. The method of claim 11, which comprises a flue gas desulfurization procedure. 14. The method of claim 9 or wherein the hydrophobin comprises a fusion hydrophobin. 131092.doc 200914378 VII. Designated representative map: (1) The representative representative of the case is: (6). (2) A brief description of the symbol of the representative figure: (No description of the symbol of the component) 8. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: (none) 131092.doc