TW201107479A - Fabrication method of microbial cell treated material - Google Patents

Abstract

A microbial cell treated material containing an ionized nitrile-hydratase is fabricated by breaking a microbial cell which generates the nitrile-hydratase through homogenizer, and then treating the broken microbial cell by acid. Using the microbial cell treated material as a catalyst to fabricated amide, such as (methyl) acrylamide. According to the present invention, high quality amide, such as (methyl) acrylamide, can be fabricated without purification when fabricating the useful (methyl) acrylamide in manufacturing industry by catalyzed reaction of nitrile-hydratase. Besides, an efficient fabricating method of the beneficial catalyst is also provided.

Description

[Technical Field] The present invention relates to a method for producing a bacterial body treated product comprising a nitrile hydratase free from a bacterial cell, and using the bacterial cell A method for producing a (mercapto) acrylamide of a treated material, and a method for producing a polymer using the (meth) acrylamide. [Prior Art] A technique for producing a corresponding guanamine compound by hydrating a nitrile group of a nitrile compound to form a corresponding amine group, which has been previously known in the presence of an acid or a test, or in the presence of a copper catalyst A chemical method of heat treatment of a nitrile compound. In addition, in recent years, it has been found that a nitrile hydratase which is an enzyme having a nitrile hydration activity which is converted into a mercapto group by hydration of a nitrile group, has been used for a nitrile compound by using a microbial cell containing the enzyme or the like. A method of producing a corresponding guanamine compound was studied. Compared with the prior chemical methods, this manufacturing method is known to have advantages such as a high conversion rate and a high selectivity from a nitrile compound to a corresponding acid amine compound, and is considered to be a powerful industrial because it is an extremely easy process composition. The above system is as described in Patent Documents 1 to 5). According to the prior art documents, it is described that not only bacteria can be used, but also bribes can be processed by ultrasonic treatment, and the body is broken, the body is broken, and the enide enzyme and the part (punfied enzyme) are prepared after the body is broken. Or a bacterial treatment such as a purified enzyme, and the acrylonitrile is converted into (f-based) acrylamide. In the above-mentioned cutting technique, the company has disclosed that (7) acrylamide can be produced in the same manner as the bacterial cells, but high-quality (meth) propylene is produced in order to use the bacterial treatment. Lai, there is still room for improvement. In addition, when a reaction such as a protein or a bacterium is left in the case of the reaction of the bacteria, the bacterium, or the like, the following conditions may occur: For example, (meth) acrylamide is easy to foam, etc. It adversely affects the quality of the manufactured (indenyl) glycerol. A method of purifying acrylamide using a microbial catalyst which is reduced as such an impurity, and a method of purifying a microbial bacterium, acrylamide, by activated carbon (for example, polyhydrazine, ? , Patent Document 6, 7), or Dreams by Lao Lu, such as the reference to the patent document 8), etc. by the method of 屯^ (in the crystallization step, in the above-mentioned purification method, in the pure process of the process)发 种 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需 无需The acrylamide aqueous solution is used as a raw material and the amine is used as a raw material (for example, refer to Patent Document 10). Patent Document 2 Patent Document 3 Patent Document 4 Patent Document 5 1 Japanese Patent Laid-Open No. Hei 09-275978, Japanese Patent Laid-Open No. Hei 11-253168 Japanese Laid-Open Patent Publication No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. 02-009022 JP-A-2004-298155, JP-A-2002-199495, JP-A-2007-277563, JP-A-2006-277563, JP-A No. JP-A----- A problem is to provide a method for producing a cell treated product having a nitrile hydratase activity capable of producing high-quality (meth) acrylamide, and a method for producing (mercapto) acrylamide using the cell treated product, and A method for producing a (mercapto) acrylamide-based polymer using the (meth) acrylamide. The present inventors have solved the above problems (4) (methods for the production of a base-based thin amine and a polymer thereof). Anti-Weiyi study. It was found that if the body of the microorganism producing nitrile hydratase was broken by homogenization II (hGnu>genize〇, the ship pair was broken (four) body riding treatment, the insoluble product was inspected, etc., used The obtained (four) body treatment product produces (mercapto) propylene-amine, and the product contains the protein money to produce a high-quality polymer core-quality (meth) acrylamide; thus completing the hair That is, the present invention is as follows. (Ό-species method for the treatment of cells containing free nitrile hydratase] is based on: production of nitrile hydratase-containing acid to treat the broken product The crushed material is subjected to alkali treatment. The method for the use of acid at 201107479 (r) is a well-known method for treating a bacterial body containing a free nitrile hydratase, which comprises: coating (1) producing a microorganism producing nitrile hydratase a step of the bacterial body disrupted product, (2) a step of acid-treating the bacterial cell disrupted material, (3) a step of removing the insoluble matter formed during the acid treatment of the above step (7), and (4) a pair of passing The acid used in the alkali treatment of the cell disrupted material in the above step (3) is a weak acid. (II) The method for producing a bacterial body treated product according to the above (1), wherein the acid used in the above (I), that is, the step (7) (III) of the above (1), is as defined in the item (II). The method for producing a bacterial cell treated product, wherein the weak acid is an antimony selected from the group consisting of acrylic acid, acetic acid, and phosphoric acid.

.1+ ^ / Wang Yi:, 冉Use resin or the same type for post-treatment. The above filter aid is diatomaceous earth. +, #, H (1) The method for producing a bacterial cell treated product (VI) The fungus described in the item (V) is Celite. (VII) The compound according to the item (IV) The method for producing a body treated product, wherein the resin is an ion exchange tree. (VIII) The method for producing a carcass treated product according to the item (I'), and the like, wherein the resin is (1) to (VII) The method for producing a bacterial cell treated product according to any one of the above-mentioned 201107479, wherein the microorganism producing a nitrile hydratase is a form of a water-sucking S# gene which is twice expressed by the microorganism in the host of the Rensi (cl〇njng). a transformant (transf〇rmat); and one or more of the constituent amino acids of the nitrile hydratase are replaced, deleted, removed, or inserted by other amino acids by a recombinant DNA technique. A shape-transformer of a variant nitrile hydratase which is further improved in a guanamine-containing compound or a nitrile-resistant compound and a temperature-increasing property. (IX) The method according to any one of (1), (1) to (VIII) The production method, wherein the microorganism producing nitrile hydratase is a microorganism belonging to the genus Pseudonocardia. And a method for producing a (meth) acrylamide, which is obtained by using the method according to any one of the items (I) and (I) to (IX), (Methyl) acrylonitrile (mercapto) acrylamide. (XI) - (mercapto) acrylamide obtained by the production method according to item (X). (XII) - (X) an aqueous solution of (meth)acrylamide obtained by the production method described in which it is 40 wt% (weight%) to 80 wt% of acrylamide or 1 wt% to 4 wt% per 1 L of L The solution of % mercapto acrylamide and water contains 0.1 mg to 50 mg of protein. (XIII) A method for producing a (meth) acrylamide polymer, which is used in the item (XI) The (meth) acrylamide or the (meth) acrylamide aqueous solution described in the above (XII) is used to produce a (meth) acrylamide polymer. Advantageous Effects of Invention According to the present invention, purification is not required The above-mentioned and other objects, features and advantages of the present invention can be produced by producing a high-quality 201107479" polymer. The following is a detailed description of the preferred embodiment, and the following is a detailed description of the following: [beta] [Embodiment] The nitrile hydratase-producing microorganism which can be used in the present invention may be exemplified by the following genus. Nocardia, Corynebacterium, Badllus, Thermophilic genus, Pseudomonas, Micr〇c〇 Ccus ) is a genus of Rhodococcus, Acinet〇bacter, Xanthobacter, and Strept〇myces represented by rhodochrous species; Rhizobium ( Rhizobiimi), Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter, colorless A genus of Achromobacter, Agr〇bacterium or Pseudonocardia, Bacteridium, and Brevibacterium represented by thermophila. Further, a shape conversion body of a nitrile hydratase gene which is selected from the microorganisms in any host; and one or two amino acid constituting the nitrile hydratase by recombinant DNA technology may be mentioned. In the above, a shape-converting substance of a variant nitrile hydratase exhibiting resistance to guanamine compound, nitrile-resistant compound, and temperature resistance is further improved by substitution, deletion, removal or insertion of other amino acids. 201107479 In addition, as an example of the example described below, Escherichia coli is used as a representative example, but it is not limited to coliforms, and may include rods such as Bacmus subtilis. Other microbial strains such as bacteria, yeast or actinomycetes. An example of the above-mentioned bacteria is: MT_10822 (This strain was deposited in the city of Tsukuba, Ibaraki Prefecture on February 7, 1996 under the registration number pERM BP_5785, based on the Budapest Treaty relating to international recognition of microbiological deposits in patent procedures. 1T, 1st, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd

Among these microorganisms, in terms of a highly active and highly stable nitrile hydratase, it is preferably a microorganism belonging to the genus Pseud〇n〇cardia and highly expressed in any host. The shape transformant of the nitrile hydratase gene selected by the microorganism, and the morphological transformant of the variant nitrile hydratase. In addition, the above-mentioned shape transforming body further improves the stability of the nitrile hydroenzyme and the activity of each of the cells is higher, and preferably D additionally 'the Rhodococcus rhodochrous which can highly express the nitrile hydratase in the microorganism. (Rhodococcus rhodochrous) J-1. The shape-converting body 2 of the nitrile hydratase gene which is produced by the microorganism in any of the hosts is preferred. The cells of the above-described nitrile hydratase-producing microorganism can be prepared by a general method 77 known in the fields of molecular biology, bioengineering, and genetic engineering. For example, it can be prepared by culturing the microorganisms in a usual liquid medium of LB medium, Yangmei et al 201107479, at a suitable culture temperature (usually 20 ° C ~ 耽, in the case of thermophiles) In the present invention, the microbial cell produced by the above-described nitrile hydration-microorganism is broken up to produce a bacterial cell disrupted product (step (丨)). The form of the microbial cell of the broken microorganism includes the production of a hydrating enzyme. The microbial cells are not particularly limited, and examples thereof include a culture solution containing the cells, a collection and collection of the culture solution by centrifugation, and the collection of the cells by physiological saline. The obtained bacteria, etc. The apparatus for breaking the stone into the above-mentioned cells can be broken as long as it can break the bacteria, for example, an ultrasonic breaker, a French pressure pot, a vibration machine (with _. plus), a homogenizer, and beads. Grinding machine L), a crushing device such as a cool mill, and the like. In terms of being able to expand the scale (SCaI,) at a low cost, it is a state of two. In addition, the so-called homogenizer refers to the gap between the plungers (_) of the pump (the outlet of the pump, the h〇m〇genizing valve) by the screw or the hydraulic pressure == piston (PiSt〇n). There are shears, slams, holes (cavitati°n), IZUMIFOO]^. This homogenizer is not less than a thief when the carcass is broken, such as MIF00D MAC_ERY Co., Ltd., etc., and it is better to be below the pit. ^ is 〇C or more, when the cell is broken, the π value of π is 4 or more and 1 〇 or less, and more preferably, the positive value is m. 11 201107479 The use of the scallops to break the bacteria is only repeated. , then __ 彳, _ job Pa above 3 ==, better for the object a above 〇嶋 〇嶋 below. After the preparation of the bacterial cell disrupted material as described above, that is, ^^", the bacterial cell disrupted material is treated with acid (step ()). The acid treatment can be carried out by the acid treatment, and there is no particular limitation on the effect of the present invention. The sulfuric acid is used in the second aspect, but the enzyme activity is less reduced in the acid treatment. In terms of §, it is preferably a weak acid. - The above weak acids include propylene glycol, acetic acid, scaly acid, etc. These weak acids are secreted by acetic acid and Wei Lin's highly toxic substances. The positive value at the time of the treatment is preferably 4 or more and 6 or less, and when the pH is 4.5 or more and 5.5 or less, the pH of the whole bacterial cell is uniform, and it is preferable to make the pH of the whole cell uniform. Further, the acid is slowly added. Further, before the acid treatment, the bacterial body is subjected to heat treatment. When the heat treatment is processed, it is easier to make the heat treatment temperature of the crushed product not more than the price. / more than 50C and 60. 〇 below. The heat treatment time of the broken body of the bacteria is more than 10 minutes or less, more preferably 5 minutes or more and 6g minutes. The above-mentioned acid treatment, that is, the cells subjected to the above step (7) Broken 201107479: Acid treatment The precipitated insoluble matter is removed (step (1)). It is presumed that if it is contained in (meth) acrylamide, it will affect the (meth) propyl group, and it is easy to make (meth) propyl (four) vesicles. A substance derived from a cell or a culture solution having a foaming property. When the foaming property of (meth)-Si amine is large, for example, when the compound f is polymerized (methylnaamine or the like), in order to eliminate two: Substances that are adversely affected, etc., sometimes enter the gland ~ / after the reaction, and the reaction of the beak into the c-amine treatment. At this time, the gas is blown into the (methyl) According to this, the amine has high foaming property, which leads to subsequent operation changes; in the operation of the device: in the operation of the amine, the (indenyl) propylene π: yl) propylene = material method can be listed, for example; The removal operation of the treated material may be carried out after the acid is treated, and the butyl ketone is preferably treated for a period of Λ 1 , (4) hours or less, more preferably 3 hours or more and ^^ hours or more. The opening limit is preferably carried out at 72. If the person wants to remove the lingering material, there is no special time. The following time is better for 48 hours =: step: : After the above step (1), the insoluble matter caused by the acid enters the test, and the test used in the process of 13 m. 201107479 can be suppressed as long as the effect of the present invention is not impaired. There is no particular limitation, and in terms of an inexpensive test, sodium hydroxide is preferred. The pH at the time of the alkali treatment is preferably a pH of 6 or more and 8 or less. The alkali treatment is the above step (4). Thereafter, the transition may be further carried out by using a microfiltration membrane (microfiltration membrane) and/or an ultrafiltration membrane (ultrafiltration membrane). The pore diameter of the MF filtration membrane is preferably 〇μμ1 or more and 2 μιη or less. More preferably, it is 0.1 μιη or more and 〇45 μιη or less, and more preferably 0.1 μιη or more and 0.2 μπι or less. Further, the material of the microfiltration membrane (MF membrane) and the ultrafiltration membrane (UF membrane) membrane is preferably a material which is adsorbed by a nitrile hydratase, and specifically, preferably polyvinylidene fluoride or polyethersulfone. Polysulfone, polyacrylonitrile. Method) and so on. Further, the filtration method using the above microfiltration membrane (MF membrane) and ultrafiltration membrane (UF membrane) is not particularly limited, and examples thereof include a dead end filtration method and a sweep filtration method (cr〇). Ss_fl〇w flltmti〇n, where there is a more reliable removal of the above insoluble matter, and the use of this micro-film (MF membrane), ultrafiltration membrane (UF membrane)

The performance of the properties affects the tendency of impurities. In addition, by the above MF film, and/or UF

When the UF film is treated, it is not only filterable, but also treated with (meth)acrylic acid amine and/or UF film. When treated with a resin or filter aid in the treatment of a boring bed, a fluidized bed or a suspension bed, there is an improvement in 201107479, f and the more reliable removal of the insoluble matter and the (meth) acrylonitrile Impurities that affect the performance of the amine. It is preferable to use an ion exchange resin for the resin to be used for the treatment of the tree sap, and it is more preferable to exchange the (4) cation exchange. The buffer solution in the case where the above-mentioned ion-retaining resin is used may, for example, be a hydrochloric acid buffer acid buffer or the like. In addition, when the cell treated material containing the nitrile hydratase is separated from the impurities by the ion exchange resin, the nitrile hydration _8 body treatment substance can be separated under the conditions of the nitrile hydratase adsorption, and the surface substance can be adsorbed. The bacterial material containing the nitrile hydratase is separated from the impurities under the conditions. When the cell treated material containing nitrile hydratase is adsorbed and eluted, a salt can be used, and the salt is preferably sodium chloride or chlorinated. The concentration of the salt is a concentration at which the nitrile hydratase is dissolved, and is preferably 2000 mM or less, more preferably 2 mM or more and 1 mM or less. Examples of the auxiliary agents include: Shixiazao soil, activated carbon 'barite, and the like. The above filter aid is preferably Shishizao soil in terms of a small loss of nitrile hydratase activity. The diatomaceous earth used in the treatment with diatomaceous earth is preferably Cd such as (a mixture of sodium carbonate and calcined diatomaceous earth), preferably in CeUte.

HYFLO SUPERCEL and Celite 5〇5 manufactured by Celite. Further, in the present invention, the above treatment of the treatment filter using a resin can be combined. The guanamine compound can be produced from a nitrile compound by using a free hydrated lysate containing the material obtained in the above manner, for example, from (meth) 15 201107479 acrylonitrile (T-based) acrylamide. The production of the above (mercapto) acrylamide is usually carried out in an aqueous vehicle (so called this vehicle). Here, the term "aqueous vehicle" refers to water, which contains a buffer for phosphorus bismuth, and dissolves an inorganic salt such as a sulfate or a carbonate, a hydroxide of an alkali metal, or a guanamine compound at an appropriate concentration. Aqueous solution. In the present invention, the concentration of the (meth)acrylonitrile in the aqueous medium is not particularly limited as the concentration of the nitrile compound at the start of the reaction, but when the supply of the nitrile compound is too large, the reaction is required to be completed. A large amount of catalyst, a reactor having an excessively large volume, and an excessively large heat exchanger for removing heat have an economic burden in terms of equipment. Therefore, when the supply concentration of (meth)acrylonitrile is in the case of acrylonitrile, and when all of the acrylonitrile is the corresponding acrylamide, it is preferable to use propylene oxime relative to the total mass of the reaction aqueous solution in the reaction. The theoretical production liquid of the amine is added in a range of 40 wt% to 80 wt%, and more specifically, it is preferably added in an amount of 〇.4 by weight relative to the hydrazine part by weight. A portion of acrylonitrile in a range of ~1.5 parts by weight. Further, in the case of mercapto acrylonitrile, when all of the mercaptopropyl nitrile becomes the corresponding methacrylamide, it is preferably a mercapto propylene relative to the total mass of the reaction aqueous solution in the reactor. Methyl acrylonitrile is added in a manner in which the theoretical solution concentration of guanamine is in the range of 10 wt% to 40 Wt%. Preferably, the mercapto acrylonitrile is added in an amount of from 0.08 parts by weight to 0.5 parts by weight based on 1 part by weight of the water in the above mixture. Further, the reaction time in the above reaction may be affected by conditions such as the amount of catalyst used or temperature, and is usually in the range of 丨 hours to 12 〇 hours.

A continuous reaction can be carried out. In addition, any of the moving bed and the like. In addition, the combination. The reaction form is not special, it can be batch type or semi-batch type, and the reactor can be a suspended bed, a fixed bed, and various types of reactors can be used. "The amount of the medium used depends on the reaction conditions. Usually dry by this microbe

Further, the reaction temperature is particularly limited in the case of ice (d) which is aqueous (d), and is usually preferably 0. It is carried out in the range of 〇~5叱, and is further in the range of 10C to 4 (TC). Alternatively, the reaction may be carried out in a state in which the product is crystallized in a liquid crystal. In addition, the above hydration reaction The pH of the reaction liquid is not particularly limited as long as the nitrile hydratase activity can be maintained, and it is preferably in the range of pH 6 to 1 Torr. Further, by the above operation, it is possible to obtain a protein contained in an aqueous solution per liter of L. Preferably, it is 0.1 mg to 50 mg, more preferably 5 mg to 20 mg of an aqueous solution of (meth) acrylamide. Further, in the present invention, the protein concentration is used as a standard accompanying bovine serum albumin (albumin). It is obtained by Protein Assay (manufactured by Bi〇_Rad Co., Ltd.) Even if the protein of the above range is contained, the obtained foam of the 17 201107479 (meth)acrylamide aqueous solution is not observed, and the workability is excellent. Further, the (meth)acrylamide polymer obtained by using the (meth)acrylamide is of high quality. Further, in the above (meth)acrylamide aqueous solution, it is preferably acrylamide k'. For containing 40 wt% to 80 wt% of acethanide When it is a methacrylic amine, it is preferably a fluorenyl acrylamide containing from wt% to 4% by weight. The (meth) acrylamide polymer of the present invention can be produced by the following means: The methacrylamide obtained by the method is homopolymerized or copolymerized with at least one unsaturated monomer copolymerizable with (fluorenyl) acrylamide. Examples of the unsaturated monomer copolymerized with fluorenylamine can be exemplified by unsaturated acid such as acrylic acid, methacrylic acid, itaconic acid, maleic acid, and fumaric acid, and salts of such acid retardants; , styrene-refluoric acid, propylene-based amino-propionic acid and salts of the same; methacrylic acid-N,N-dimethylamino ethyl methacrylate, sulfhydryl An alkylaminoalkyl (meth) acrylate such as N,N-diethylaminoethyl acrylate or N-N-dimethylaminoethyl acrylate or the like Ammonium derivative; N,N-dimethylaminopropylmercaptopropenylamine, n,n-dimethylaminopropylpropenylamine, etc., hydrazine-dioxane Aminoalkyl (meth) acrylamide, or a quaternary ammonium derivative of the (meth) acrylamide; acetone acrylamide, hydrazine, hydrazine-dimethyl methacrylate, hydrazine, hydrazine Hydrophilic women such as methyl methacrylate, Ν-ethyl methacrylate, Ν-ethyl propyl amide, νν_ diethyl propyl uranium, Ν-propyl propyl amide Stuffed amine.

18 201107479 N-acrylic acid*, N-propyl_yl«, (4)_N-acryloyl morpholine; methyl acetoacetate by acetylacetate Propyl ester, hydroxypropyl acrylate; methoxy polyethylene glycol (meth) acrylate, N-ethylene-2, ketone; N, N-di-n-propyl acrylamide, N• n-butyl Propylene amine, N_n-hexyl acrylamide, N-n-hexyl decyl propylamine, & amine, N-n-octyl methacrylamide, N, trioctyl propylene fluorene, :: Pyridyl propylene giamine, & n-dodecyl-6-f-yl (fluorenyl) acrylamide derivative; heart N-n-based n, n-, glycidyl acrylamide (n, n_ ΓΓ:?), N With diglycidylmethylpropanolamine, N-(4'shrinked 1 oleyl butyl butyl) propylene, N-(4' glycidoxybutyl) methyl propylene, N_(5'四 甘油 氧基 钱 ) ( 四 四 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( Methyl acrylate, ethyl (meth) acrylate, (methyl) acetonate, ( Methyl)lauryl acrylate, 2-ethylhexyl (meth) acrylate, (meth) propylene, condensed glucosinolate, etc.; <acrylic guess, methyl propyl Nitrile, vinyl acetate, vinyl chloride, vinylidene chloride, _, butyl hydrazine and other dilute hydrocarbons such as styrene, α-mercapto, butadiene, isoprene, etc. These monomers may be used alone or in combination of two or more. 19 ^ t. 201107479 Polymerization method of these monomers Among them, there are liquid solution polymerization, emulsion polymerization, and the like for the aqueous solution. , added injury list === from the base polymerization initiator. Γ Γ 却 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Redox (re- can be used alone or in combination with the above-mentioned initiators, and the amount of the polymerization agent is usually in the range of ～5 wt% relative to the total amount of the monomers. 勹υ.υιη wt/ 〇~合斗显度#, when it is a single polymerization initiator, it is usually 〇ΐ 产 Ϊ Ϊ Ϊ 〜 听 听 ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ ～ In the case where the polymerization proceeds appropriately, the polymerization heat tends to rise, so that it may be cooled as occasion demands. The ambient gas during the polymerization is not particularly limited, and it is preferably from the viewpoint of rapid progress. For example, in the case of nitrogen (tetra), the concentration of the nitrogen is reduced, and the polymerization is carried out. (4) ~ 201107479. In addition, the pH of the aqueous solution during polymerization is not particularly limited, and it is necessary to carry out polymerization by adjusting the value. The positive value of the yang can be praised, such as sodium hydroxide, potassium hydroxide, ammonia and other alkali; phosphoric acid, sulfuric acid, citric acid Inorganic acid; organic acid such as formic acid or acetic acid, etc. The molecular weight of the polymer obtained by the present invention is not particularly limited, and the flux is in the range of 100,000 to 50 million, preferably 50,000 to 10,000. In the above-mentioned manner, the (fluorenyl) acrylamide-based polymer of the present invention has a water-soluble and high molecular weight polymer, and has excellent color tone, and is suitable as a coagulant and an additive for papermaking. Examples of petroleum time agents and the like Hereinafter, the present invention will be more specifically described based on examples, and the present invention is not limited to the following examples. Unless otherwise specified, % and ppm are based on mass standards. [Propylene amide, sulfhydryl group Analysis of acrylamide] High performance liquid chromatography (HPLC, high perf〇nnance liquidchr〇mat〇graphy) analysis in each example and comparative example and reference example is

Using Finepak SIL C18_5 (25 (4) 6 〇 Na) manufactured by JASCO Corporation, as a column column, a 1 mM % water-cooled liquid containing 4 0.001% by volume of acetonitrile was used as a developing solution. In addition, acrylamide and methyl propylamine were detected by absorbance at 220 nm. [Production Example 1] [Preparation of the bacterial cell] According to the method described in Example 1 of Japanese Patent Laid-Open No. 2.-91, the ρρτ• brain quality 21 201107479 DNA obtained in the Japanese Patent Application No. The Νο·3 bacterium selected in Table 3 of Example 1 of JP-A-09-275978 was obtained. The Escherichia coli having the pPT-DB1 plastid is the MT-10822 strain (Accession No. FERM BP-5785), which is deposited in the Patent Biological Depository Center as described above. Ibicillin was added in a LB medium containing 〇.1 g/L of FeS〇4 and 0.05 g/L of CoCl2 by steam sterilization in the south to achieve 0.1 mg/L. 200 ml of the medium for pre-itching was prepared. The No. 3-selected cells of the bacterium, which was incubated with the bacterium, were pre-cultured at a temperature of 33 °C. The turbidity measurement method was used to grasp the amount of growth of the cells at the time of pre-culture, and the pre-culture was terminated at a concentration of 3. G to 6. G at a concentration of 66 〇 nm. The pre-cultured culture solution is sterilized in 5 L of the medium used for the maintenance of the high-pressure steam-reduced table, at a temperature of -1 〇 = air to culture_ventilation - The broth was carried out for 48 hours to obtain a culture solution (cell suspension). In addition, the pH of the culture solution was monitored from the beginning of the test, and 15 ml of 500 g/L of glucose was added to the horse for 30 minutes when the positive value was 9 ° and the Prt value was 7.45 or more. The calendar [Table 1]

201107479 [Example 1] [Preparation of a bacterial material containing free nitrile hydratase from a bacterial cell] (The bacterial cell was disrupted by a homogenizer) A homogenizer H50' manufactured by SANWA ENGINEERING Co., Ltd. was used at a temperature of 15. Under the conditions of a crushing pressure of 50 MPa and a crushing time of 1 minute, the cells contained in the culture solution prepared in the above Production Example 1 were crushed, and the nitrile hydratase contained in the cells was dissolved in the solution. The broken solution in the medium. (Heat treatment) The above-mentioned crushed solution was transferred to a 10 L glass beaker, and the glass beaker was immersed in 5 Torr while the crushing bath was sufficiently stirred. The water bath is in the bath. After the liquid temperature of the crushing solution reaches 5 (TC), the impregnation is continued for 15 minutes, and then the beaker is taken out and cooled in the air. (Precipitation of impurities by acid) Slowly add 2 Μ acetic acid in the crushed solution after the above-mentioned heat treatment which is sufficiently disturbed The aqueous solution was allowed to stand until the pH of the solution reached 5 Torr, and the solution was mixed for 3 hours to precipitate impurities. (Removal of the crushed material by centrifugation, etc.) Using a centrifuge (manufactured by Hitachi Industrial Co., Ltd.: CR22G), f 15 < 〇, (10) (8) Inoculum, under the condition of 15 minutes, the above-mentioned crushed liquid subjected to acid treatment was centrifuged, and the impurity precipitated by the addition of acid was removed to obtain a centrifugal supernatant. (Neutralization of Centrifugal Supernatant) Slowly add 1 Μ of 23 in the above-mentioned centrifugation supernatant mixed with a knife. 2011/07479 X丄

NaOH until the pH of the solution reaches 7 ο Α , • until the centrifugation supernatant neutralizes 0. In addition, in the case of non-compliance with this neutralization υ ^ , w month / where, due to the precipitation of impurities can not enter MF filtration after a step in Kenting. (MF filtration of neutralizing solution) <7 450 (DFC capsule type), centrifuge supernatant to filter. Using the only one manufactured by Poul, the neutralization (concentration of the filtrate) was carried out under the conditions of 0,5 MPa using the ^^, manufactured by Asahi Kasei Co., Ltd.; Pennel was used for AHP-0013, at an average pressure〇 .1MPa is called Τ; ,.y, preparation speed is 1.5 mm/sec. Under white conditions, 5 times concentration is obtained to obtain bacterial treatment (measurement of protein concentration). The protein Han of the body treatment is used (4) Company _ conduct the measurement. The standard is to use the accompanying bovine blood > lunar albumin. The result is that the protein concentration in the bacterial treatment is % mg/ml. [Production of acrylamide] A 1 L glass flask equipped with a stirrer was prepared as a first reactor, and a TeflQn (registered trademark) tube 2 () m having an inner diameter of 5 mm was prepared as a second reactor. 4 g of water was previously charged in the first reactor. The cell treatment 〇 3 wt% prepared by the above method was suspended in a 0.3 mM aqueous solution of NaOH. While stirring this suspension with acrylonitrile, the suspension was continuously fed to the i-reactor at a rate of 49 g/h and 31 g/h, respectively. Next, the reaction liquid was continuously withdrawn from the first reactor at a rate of 80 g/h in a manner of 24 S·201107479 in which the liquid level of the second reactor was kept constant. The extracted reaction liquid was continuously fed to the second reactor at a rate of 80 g/h, and the reaction was further carried out in the second reactor. Both the α 苐 1 reactor and the 2 reactor were immersed in a temperature-producing water bath of 1 〇 ° c 2 2 〇〇 c so that the liquid temperature inside each reactor was 15. The temperature is controlled in a sly way. ^ Adjusting the addition amount of the steroid treatment to the 0.3 mM-NaOH aqueous solution so that the conversion rate of the first reactor outlet to acrylamide is 9% or more, and the first reaction is the outlet acrylonitrile concentration for detection. Below the limit (below ppm). The conversion to acrylamide was determined based on the analysis results of HpLC. To the obtained aqueous solution of acrylamide, 2 v [acrylic acid was added to adjust the pH to 7. Further, the obtained acrylamide aqueous solution was subjected to analysis of the contained protein. The protein concentration was measured by dialysis and removal of acrylamide contained in an aqueous solution of acrylamide using a semipermeable membrane (Spectra/Por molecular weight cutoff of 1000), and then measurement was carried out using a Protein Assay (Bi〇-Rad Co., Ltd.). The amount of protein present in 1 kg of the obtained aqueous solution of acrylamide was 10 mg, which was the same as the value calculated based on the protein concentration of the cell-treated material and the amount of the cell-treated material. [Foaming test of aqueous acrylamide solution] 300 g of an aqueous solution of acrylamide obtained by the above method was placed in a 500 ml measuring cylinder. Into the acrylamide aqueous solution, a wood-type glass ball filter 503G was introduced, and the bottom of the measuring cylinder was passed through the filter, and air was blown at 900 ml/min, and the height of foaming at the time of 5 minutes was measured. As a result, the foaming of the aqueous acrylamide solution was less than 1 Torr and almost no observed in 201107479, which was a very good result. [Example 2] A cell treated product and an aqueous solution of acrylamide were obtained by the same operation as in Example 1 except that the aqueous solution of 2 hydrazine was used instead of the aqueous solution of acetic acid. The amount of protein present in 1 kg of the obtained acrylamide solution was 1 〇 mg. In the same manner as in Example 1, a foaming test of an aqueous solution of acrylamide was carried out, and as a result, the foaming of the aqueous solution of the potassium sulphate was less than 1 〇 mm and was hardly observed, which was a very good result. [Example 3] A steroid treated product and an aqueous solution of propylene sulphate were obtained by performing the same procedure as in Example 1 except that the aqueous solution of 2 hydrazine phosphate was used instead of the aqueous solution of hydrazine acetate. The amount of protein present in 1 kg of the obtained acrylamide solution was 1 mg. In the same manner as in Example 1, the foaming test of the aqueous acrylamide solution was carried out. As a result, the foaming of the aqueous solution of the acrylamide was less than 1 〇 mm and was hardly observed, which was a very good result. [Example 4] The same operation as in Example 1 was carried out, except that a 2 Μ aqueous solution of sulfuric acid was used instead of the aqueous solution of acetic acid in Example 2, and the same procedure as in Example 1 was carried out to obtain a cell treated product and an aqueous solution of acrylamide. The amount of protein present in 1 kg of the obtained acrylamide solution was 8 mg. In the same manner as in Example 1, the foaming of the aqueous acrylamide solution was carried out, and as a result, the foaming of the aqueous solution of the acrylamide was less than 10 mm and almost no

26 S 201107479 Observed, very good results. Further, when impurities were precipitated by acetic acid, acrylic acid or phosphoric acid (weak acid) in Examples 1 to 3, no decrease in nitrile hydratase activity was observed. On the other hand, in the present Example 4 in which impurities were precipitated by sulfuric acid (strong acid), it was observed that the hydrolytic enzyme activity was decreased by about 25%. [Example 5] The same operation as in Example 进行 was carried out except that the heat treatment of Example 1 was not carried out, and a bacterial cell treated product and an aqueous solution of acrylamide were obtained. The amount of protein present in 1 kg of the obtained acrylamide solution was 1 〇 mg. In the same manner as in Example 1, a foaming test of an aqueous solution of acrylamide was carried out, and as a result, foaming of the aqueous solution of acrylamide was less than 1 mm and almost no observation was made as a very good result. [Example 6] ^ After the neutralization of the centrifugation supernatant in Example 1, the same operation as in Example 1 was carried out, except that the following treatment was carried out, resin treatment was carried out, and MF filtration of the neutralization liquid was performed. The obtained bacterial material and the aqueous solution of the acrylic acid amine were obtained. The protein present in the lkg of the resulting acrylamide solution was 5 mg. &, in the same manner as in Example 1, the foaming of the aqueous acrylamide solution was carried out, and the foaming of the aqueous solution of the aqueous solution of the phthalocyanine to the guanamine was less than 10 mm, which was hardly observed, and was a good result. The child is not used (green child is replaced by resin). It is used as a strong anion exchange resin.

0 sphere Q manufactured by Bio-Rad. Prepare a resin tower with a resin content of 5 〇〇 blood, with % 瓜% 27 5.

XX 201107479 Phytic acid buffer (pH 7.0) 2.5 L for adequate equilibration. Then, the same operation as in Example 1 was carried out, and 2.5 L of the obtained centrifuged supernatant was supplied to the above resin column to adsorb the nitrile hydratase. Then, after washing with 2.5 mM of 5 mM phosphate buffer (pH 7.0), NaCl was added in a 50 mM sulphate buffer (pH 7.0) to achieve 0·2 藉. The solution was allowed to elute the nitrile hydratase to obtain an ion exchange resin-treated centrifugal supernatant. [Example 7] After the neutralization of the centrifugation supernatant in Example 1, the Celite treatment described below was carried out, and MF filtration of the neutralization liquid was carried out, except that the same operation as in Example 1 was carried out to obtain a bacterial cell. The treated material and the aqueous solution of acrylamide. The amount of protein present in 1 kg of the obtained acrylamide solution was 9 mg. In the same manner as in Example 1, the foaming test of the aqueous acrylamide solution was carried out. As a result, the foaming of the aqueous acrylamide solution was less than 10 mm and almost no observation was made, which was a very good result. (Celite processing)

Celite is a HYFLO SUPERCEL manufactured by Celite. The same operation as in Example 1 was carried out, and HYFLO SUPERCEL was added in the obtained neutralized centrifugal supernatant to a ratio of 3 wt%, and stirred at 15 ° C for 3 hours, and then, using Toyo Filter Paper No. 2 The filtrate was filtered under suction to obtain a Celite-treated centrifugal supernatant. [Example 8] The Celite used was replaced with Celite 505', except for

28 201107479. Only the same wood was used in Example 7, and the bacterial material treatment and the acrylonitrile water = liquid were obtained. The amount of protein present in 1 kg of the resulting propylamine solution was 9 mg. The foaming test of the aqueous solution of acrylamide was carried out in the same manner as in the case of the shell example 1. As a result, the foaming of the aqueous solution of the aqueous solution of the azamine was less than 10 mm and was hardly observed, which was a very good result. [Comparative Example 1] The same procedure as in Example i was carried out without using the acid-precipitating impurities and the centrifugation supernatant in Example 1, and the same conditions as in Example i were carried out to obtain a bacterial material: a physiological substance, and an aqueous solution of propidium. The protein (minus) of the obtained (iv) treated product was 300 mg/m. Further, the amount of protein present in i of the obtained acesulfame solution was 6 〇 mg. & A foaming test of the aqueous acrylamide aqueous solution was carried out in the same manner as in Example 1. As a result, the foaming of the aqueous acrylamide aqueous solution was (10) or more, which was not preferable because it was not used as a product. [Reference Example 1] - The same operation as in Comparative Example 1 was carried out, and an aqueous solution of 2 Torr of acrylic acid was added to the produced acrylamide aqueous solution to prepare an aqueous solution having a pH adjusted to 5, and was added to the aqueous solution. 〇 4 wt% of activated carbon (powder activated carbon pM_sx manufactured by Sancang Chemical Co., Ltd.) at 25. Stir under the arm for 5 hours. Then, the activated carbon was removed by filtration using a filter paper, and a 1 Torr aqueous NaOH solution was added thereto, and the pH of the obtained aqueous solution was adjusted to 7 Å to prepare a neutralized filtrate. The protein concentration in this neutralized filtrate was determined. The protein concentration was 29 201107479. After dialysis and removal of the acrylamide contained in the filtrate by a semipermeable membrane (Spectra/Por molecular weight cutoff of 10 Torr), Amic〇n uiira_15 (Millpore's molecular weight cutoff 3 〇〇〇) was used. The concentration was 1 time, and the measurement was performed using a Protein Assay (manufactured by Bio-Rad Co., Ltd.). The amount of protein present in 1 kg of the obtained aqueous solution of acrylamide is less than mg. In the same manner as in Example 1, the foaming test of the aqueous solution of the acrylamide aqueous solution was carried out. As a result, the foaming of the aqueous acrylamide solution was 1 mm, which was a good result. [Reference Example 2] In the aqueous solution of acrylamide obtained by the same operation as in Reference Example 1, the protein concentration in 1 kg of the aqueous solution of acrylamide was 10 mg, and Comparatively, the cell treated material prepared by the same operation was used. The foaming test of the acrylamide aqueous solution was carried out in the same manner as in Example 1. As a result, in the case of the cell treated material which was not subjected to the acid treatment, the foaming of the aqueous acrylamide solution was 100 mm or more, and it was impossible to use the product. bad. [Reference Example 3] A 52 wt% aqueous solution of acrylamide was prepared using a 0.3 mM aqueous solution of NaOH and an acrylamide (purity: 99.9%) for electrophoresis manufactured by First Chemicals Co., Ltd. 'In this aqueous solution, 2 Å of acrylic acid was added. An aqueous solution was obtained to obtain an aqueous acrylamide solution having a pH adjusted to 7. In the same manner as in Example 1, a foaming test of an aqueous solution of acrylamide was carried out, and as a result, the foaming of the obtained aqueous solution of acrylamide was less than 10 mm.

30 201107479 Almost unobserved, very good results. [Example 9] In the aqueous solution of acrylamide obtained by the same operation as in Example 1, 'the removal of the protein using the activated carbon in the phase (f) of Reference Example 1 was carried out, except that the same procedure as in Example 丨 was carried out. The operation was carried out while obtaining an aqueous solution of acrylamide. The foaming test of the aqueous acrylamide solution was carried out in the same manner as in Example 1. As a result, the foaming of the aqueous acrylamide solution was 1 mm, which was a good result. [Example 10] When the MF filtration of Example 1, Example 6, Example 7, and Example 8 was carried out, the filterability was evaluated. Evaluation is done by: using Mmip〇re

Express SHC 0.5/0.2UM (membrane area 13.8 cm2) was compared for filtration at 0.5 MPa. When the amount of filtration in Example 1 was set to 1, 'Example 6 was 7, Example 7 was 15, and Example 8 was 50. [Reference Example 4] In the aqueous solution of acrylamide obtained by the same operation as in Reference Example 1, the ratio of the protein present in 1 kg of the aqueous solution of acrylamide was 10 mg, and the addition and comparison were carried out. The bacterial material preparation prepared in the same manner as in Example 1. The foaming test of the aqueous acrylamide solution was carried out in the same manner as in Example 1. As a result, the foaming of the aqueous acrylamide solution was 100 mm or more, which was unsuitable for use as a product. [Example 11] 31 201107479 - [Production of acrylamide-based polymer] Water was added to an aqueous solution of acrylamide obtained in the above examples and comparative examples to prepare a acrylamide aqueous solution having a concentration of 20% by weight. 500 g of this 2 wt% acrylamide aqueous solution was added to a 1 L polyethylene container while maintaining the temperature at 18 ° C - the dissolved oxygen in the solution was removed by nitrogen gas, and immediately added to the foamed styrene. Insulation block. Further, nitrogen gas was allowed to flow over the solution in the polyethylene container so that oxygen did not dissolve in the solution. Then '20〇xl〇_6 mpin (relative to the molar ratio of acrylamide) 4,4'-azobis(4-cyanovalerate), 2〇〇xl〇-6 mpin The mercaptoaminopropionitrile and the ammonium persulfate of 8〇xl〇_6 mpm were respectively dissolved in a small amount of water, and sequentially injected into a 1 L·polyethylene container. Nitrogen gas was previously supplied to the tanning agents, and a small amount of nitrogen gas was introduced into the polyethylene container at the time of injection and before and after the injection to prevent oxygen from entering. ^ If these reagents are injected, it is found that the internal temperature of the polyethylene thief rises after a few minutes of induction period, thus stopping the supply of nitrogen. In the insulation environment, the polyethylene container is kept in the above state for about 100 minutes, and the internal temperature of the polyethylene container reaches about 7 (TC. Therefore, the polyethylene is removed from the insulation by the block. The poly-reaction was carried out by immersing in water at 97 ° C for 2 hours, and then immersed in cold water for cooling, and the polymerization reaction was stopped. < The aqueous gel of the acrylamide polymer obtained in the above manner was self-polymerized. Take out the 'five pieces' and divide them into small pieces and grind them with a meat grinder. Dry the hydrogel of the ground chopped amine resin in a hot air of loot: 2 small

32 S 201107479 : Next, pulverization was carried out by a high-speed rotary knife pulverizer to obtain a dry powdery acrylamide polymer. The obtained dry powdery acrylamide polymer was sieved and the polymer of 42 mesh was obtained. This fractionated acetamide polymer was evaluated by the 4-test of propylene-based polyamines described later. The evaluation is shown in Table 2. 'Test method for acrylamide polymer> The polymerization rate at the time of production of the above polymer sample was evaluated by the time until the highest temperature was reached. Further, the water solubility and standard viscosity of the polymer sample obtained above were evaluated in the following manner. Water-soluble: Water-soluble is 600 mL of water in a 1 L beaker, stirred at 25 ° C in a prescribed shape, and a polymer sample of 0.66 g (polyacrylamide pure bifurcation · 6 g) is added. After stirring at 4 rpm for 2 hours, the obtained solution was filtered through a 150-mesh gold net, and water solubility was judged based on the amount of insoluble components and the filterability. That is, the completely dissolved solution is set to AA', and the solution which is nearly completely dissolved is set to bb, and the solution which has insoluble components but can be separated by filtration is set to CC, and the filtrate is passed through the slow, insoluble component. The filtered solution was set to DD. Standard viscosity: The filtrate obtained by the above water solubility test is an aqueous solution of a polymer having a concentration of 0.1 wt%, to which sodium chloride equivalent to 1 Torr is added, and a BL adapter is used at 25° by a BL type viscometer. C. The viscosity (standard viscosity) was measured at a rotor speed of 60 rpm. The standard viscosity obtained by this method is used as a value having a correlation with the molecular weight. An- 33 201107479 [Table 2] Ί------ Lean white matter concentration Cmg/kg . aam Water sputum) Foaming (foaming height) Polymerization speed (relative value) Polymer soluble polymer water soluble ash ' Viscosity (mPa · s) Example 1 Example 2 10 No observed 1 AA 5.5 ' 10 No observed 1 AA 5.5 ^' Example 3 10 No observed 1 AA 5.5 ~~~' Example 4 8 Unobserved 1 AA 5.5 Example 5 1〇 No observed 1 AA 5.5~ Summer Example 6 '5~~~ . No observed 1 AA 5.5 H column / ^9~~~~~ No 1 AA 5.5 'Bei was observed. J δ Example 9 Comparative Example 1 9 ----- No observed 1 AA 53 _ Snack color 1 10 mm 1 AA 5.5 Reference example 1 OU —---- 100 mm or more - - - Reference example 2 Reference example ^ Less than 0.1 10 mm 1 AA 5.5 ' __10 Less than 0.1 100 mm or more to the chain fil 1 A Λ ---. Not pass L belt jade J 1 AA 5.5 ' ------ Registration number PERM BP-5785 Limited hair has been The preferred embodiments are disclosed above, but they are not intended to be used in the scope of the present invention. The present invention is not limited to the scope of the present invention. [Main component symbol description] 34

Claims (1)

201107479f VII. Patent application scope: 1. A method for manufacturing a bacterial body treatment product, wherein the biological treatment product is hydrated from the nitrile __ treatment, and the preparation method of the bacterial treatment material comprises: (1) preparing a microorganism producing nitrile hydratase (4) a step of crushing the object; (2) a step of acid-treating the broken material of the cell; (3) a step of removing the acid obtained by the step (2); and a step (1) of (4) The step of performing the treatment of the broken body of the bacteria. 2. The method for producing a bacterial material treated product according to the above paragraph (4), wherein the acid used in the above step (2) is a weak acid. 3. The method for producing a bacterial cell treated product according to the second aspect of the invention, wherein the lion is at least one selected from the group consisting of acrylic acid, acetic acid, and miscellaneous. The method for producing a bacterial cell treated product is described in which at least one of the resin or the filter aid is post-treated after the above-mentioned step (4). The method for producing a bacterial cell treated material according to the fourth aspect of the invention, wherein the filter aid is diatomaceous earth. 6. The method for producing a bacterial cell treated product according to claim 5, wherein the diatomaceous earth is Celite. 7. The method for producing a bacterial body treated product according to the fourth aspect of the invention, wherein the resin is an ion exchange resin. 8. The method of producing a cell treated product according to claim 1, wherein the microorganism producing a nitrile hydratase is a form of a nitrile hydratase gene highly expressed by the microorganism in any host. a transformant; and one or more of the constituent amino acids of the nitrile hydratase by a recombinant DNA technique, which are substituted, deleted, deleted or inserted by other amino acids, thereby exhibiting melamine-resistant compound or nitrile-resistant compound A shape-transformer of a variant nitrile hydratase which is further improved in temperature and temperature resistance. 9. The production method according to claim 1, wherein the microorganism in which the nitrile hydratase is produced is a microorganism belonging to the genus Pseud〇n〇cardia. A method for producing a bismuth (mercapto) acrylamide, which is obtained by using the method according to any one of the first to ninth aspects of the invention, wherein Methyl)acrylonitrile (mercapto) acrylamide. A (meth) acrylamide obtained by the production method according to claim 10 of the patent application. 12. An aqueous solution of (meth) acrylamide, which is obtained by the production method of claim 10, and the (meth)acrylamide aqueous solution is 40 wt% to 80 per 1 L. A solution of wt% acrylamide or 10 wt% to 4 〇 wt ° / hydrazine decyl acrylamide, and water contains 〇 j mg 〜 5 〇 protein. A method for producing a (meth) acrylamide-based polymer, which comprises using (mercapto) acrylamide as described in claim 11 or as claimed in claim 12 A (mercapto) acrylamide-based polymer is produced by a (hydrazino) acrylamide aqueous solution. 36 201107479 IV. Designated representative map: (1) The representative representative of the case is: No (2) The symbol of the symbol of the representative figure is simple: No. 5. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: 201107479 ί__________ For the Chinese manual No. 99123033, there is no sizing correction page correction date " _ _ 4 positive ' _ . . . L_ Fu 丨 丨 合物 合物 合物 合物 合物 合物 合物 。 。 。 。 。 。 The above and other objects, features, and advantages of the present invention will become more fully understood from v β [Embodiment] The nitrile hydratase-producing microorganism which can be used in the present invention may be exemplified by microorganisms belonging to the subordinates, such as the genus Nocardia, and the genus Corynebacterium, Bacillus. ), genus, thermophilic bacillus, pseudomonas (genus pseudomonas), micrococcal (five) stagnation (10)) genus, rhodococcus (Rho〇ochrous) species represented by Rhodococcus (Rh〇d〇C〇CCUS) Genus, Adnet〇bacter, Xanthobacter, streptomyces, Rhizobium, Klebsiella, Enterobacter , genus Erwinia, genus Aeromonas, citr〇bacter, Achromobacter, Agrobacterium D genus or thermophila The species is represented by the genus Pseudonocardia, the genus Bacteridium, and the genus Brevibacterium. Further, a shape-converting substance which highly expresses a nitrile hydratase gene selected from the microorganisms in any host; and one or more amino acids constituting the nitrile hydratase by recombinant DNA technology may be mentioned. A shape-transformer of a variant nitrile hydratase which exhibits resistance to guanamine compounding or nitrile-resistance and temperature resistance is further improved by substitution, deletion, removal or insertion of other amino acids. 201107479 Date of revision: 99th, the 8th of the next month, the 99th, the Chinese manual, the no-line correction page. In addition, the arbitrary host (h〇st) here, as the example described later, can be cited as Escherichia coli. As a representative example, it is not particularly limited to coliform bacteria, and may include other microbial strains such as a genus of the genus Bacillus, such as a bacterium, a yeast, or an actinomycete. An example of the above-mentioned bacteria is: MT_10822 (This strain was deposited in Ibaraki Prefecture on February 7, 1996 under the registration number FERM Bp_5785, based on the Budapest Treaty relating to the international recognition of the micro-material registration of the patent procedure. Boss City, 1st, 1st, 1st, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd ). The microbial towel preferably has a high sorghum and high sulphur nitrile hydratase, and is preferably a Pseud〇n〇cardia genus, and is highly expressed in any host. The synthase base of microbial selection _ shape (four) health, silk is now variegated to hydrated _ shape. In addition, the above-mentioned morphological conversion is to improve nitrile hydratase, sex, and the activity of each cell is higher. Preferably, 'In addition, the Rhodococcus rhodochrcms can be expressed in the microorganisms, and the shape of the nitrile hydratase gene in the host, the ancient J. Also preferred is J ^ The above-mentioned microbial cell producing a nitrile hydratase-producing microorganism can be prepared, for example, by a method known in the fields of branching, biological process, and hydrazine, for example, by the following method: Medium, _ medium, etc. 201107479 is the 99123033 Chinese manual without a stencil sheet Revision date: 99 years, February 8th, the best range of 2 hours to 48 hours. f should be no specific form, can be batch: owed Half batch, also two ^Continuous reaction. In addition, the reactor can be any of a slant bed, a fixed bed, or a bed. In addition, multiple (four) reactors can be combined. Ο The amount of catalyst used depends on the reaction conditions. Usually, according to the microbial dry mass ratio, the amount of the catalyst used is relative to the mass of the reaction solution = 鹏~5_鹏, preferably 5G ppm~3_〇ppm. In addition, the hydration reaction is usually in the usual ink or It is carried out in the vicinity of normal pressure, and the solubility of the nitrile compound in the water-based towel may be carried out under pressure. Further, the reaction temperature is particularly limited if it is an ice-based method, and it is usually preferred. It is carried out in the range of S叱~pit, and is further in the range of 10 ° c to 4 (TC). Alternatively, the reaction may be carried out in a slurry state in which the product is crystallized in the reaction liquid. The pH of the reaction liquid in the hydration reaction is not particularly limited as long as the activity of the hydration enzyme is maintained, and it is preferably in the range of pH 6 to 1 Torr, more preferably in the range of 7 to 9 pH. By the above operation, it is possible to obtain an egg per 1 L of the aqueous solution. The white matter is preferably 0.1 mg to 50 mg, more preferably 5 mg to 2 mg (the aqueous solution of methotrexate. In addition, in the present invention, the protein concentration is used as a standard accompanying bovine serum albumin (albumin). , obtained by the company PlOteinAss.) d Even if the protein of the above range is included, no result is observed. 17 201107479 ^正臼期:99年二月8曰为99123033号The (meth) propylamine aqueous solution is foamed, and the operability is excellent.) The (meth) acrylamide-based polymer obtained by acrylamide is high-quality (A, B, Methyl) Acrylic amine in the aqueous solution of enamines in the amphibious sputum < soil for § has 4 〇 plus 0 / 〇 ~ 8 〇 plus % of cimelamine, for carbamide when Xiaojiao contains 1 () pain ~ 4_ Jane methyl propylene fondue ^. The (meth) acrylamide polymer of the present invention can be produced by homopolymerizing (meth) acrylamide obtained in the above manner. The (fluorenyl) acrylamide is copolymerized with two less unsaturated monomers copolymerizable with (meth) acrylamide. Examples of the unsaturated monomer copolymerizable with (meth) acrylamide: unsaturated unsaturated acid such as acrylic acid, methacrylic acid, itaconic acid, maleic acid, fumaric acid, and salts of the carboxylic acids; Sulfonic acid, styrene sulfonic acid, acrylamidopropane sulfonic acid and salts of the same; dimethyl acrylate, N, N-dimethylamino ethyl methacrylate, methyl Acrylic acid - hydrazine, hydrazine diethylaminoethyl ester, C An alkylaminoalkyl group of (meth)acrylic acid such as an enoic acid-hydrazine, anthracene-didecylaminoethyl ester or a quaternary ammonium derivative of the same; N,N-dimethylamine N,N-dialkylaminoalkyl(meth)acrylamide such as propyl methacrylamide or N,N-didecylaminopropyl acrylamide, or these (methyl) Tetra-ammonium derivatives of acrylamide; acetone acrylamide, N,N-dimercapto acrylamide, hydrazine, hydrazine-dimethyl methacrylamide, hydrazine-ethyl decyl acrylamide, N - Hydrophilic acrylamide such as ethyl acrylamide, hydrazine, hydrazine-diethyl acrylamide, hydrazine-propyl acrylamide; 18 201107479 Revision date: November 8, 1999 is No. 99123033 Chinese manual襄 (Line correction page In addition, the pH of the aqueous solution during polymerization is not particularly limited, and it is necessary to adjust the pH to carry out polymerization. The positive value can be used at this time: enumerated: sodium hydroxide, hydroxide, ammonia, etc. ; sulphuric acid, sulphuric acid, and other inorganic acids; organic acids such as citric acid, acetic acid, etc. The molecular weight of the polymer obtained by the present invention is not particularly limited, and is usually 100,000 to 50,000. Range '5〇 preferably in a range of ten thousand million to fiber. In the above-mentioned manner, the (meth)acrylamide-based polymer of the present invention has a water-soluble and high molecular weight polymer and has excellent color tone, and can be suitably used as a coagulant, an additive for papermaking, and oil recovery. Agents, etc. > Port Example Hereinafter, the present invention will be more specifically described based on examples, but the present invention is not limited by the examples. Unless otherwise specified, 〇/ is the quality standard. [Analysis of acrylamide and methacrylamide] The HPLC 'high performance liquid chromatography' analysis in each of the examples and comparative examples and reference examples is ◎Finepak SIL manufactured using the spectrometer C18_5 (25〇χ4 6 φ let) As a column, an aqueous solution of i〇mM containing 4v〇1% (by volume) of acetonitrile was used as the developing solution. In addition, acrylamide and mercapto acrylamide were detected by absorbance at 220 nm. [Production Example 1] [Preparation of the cells] According to the method described in the example of JP-A-2001-340091, the pPT-DB1 quality 21 201107479 obtained in Japanese Patent Laid-Open No. 09-275978 is No. 99123033 The Chinese version of the book has no scribe line correction date. The date of the correction of the date: 99 years, the date of the month, the body DNA is the template, and the n〇.3 bacterium is described in Table 3 of Example 1 of Japanese Patent Laid-Open Publication No. 9_275978. The Escherichia coli having the pPT_DBl plastid is the MT-10822 strain (registered number FERMBP-5785), which is deposited in the patent bio-consignation center as described above. Ampicillin was added in an autoclaved LB medium containing g1 g/L of FeS〇4 and 〇.〇5 g/L of CoCl2 to achieve 0.1 mg/L. 200 ml of the medium used for the pre-culture was prepared, and the No. 3-selected cells of the bacterium, Platinum ear, described in the medium, were precultured at a temperature of 33 °C. The turbidity measurement method was used to grasp the amount of growth of the cells at the time of preculture, and the preculture was terminated at the time when the absorbance at 66 Torr was in the range of 3.0 to 6.0. The culture solution obtained by the pre-culture is sterilized in a medium of 5 liters of the medium used for the sputum and the cultivating time indicated by the autoclave sterilized at a temperature of 33 t: -1, 1 〇 = air is cultured The mixture was stirred while being ventilated in the liquid, and cultured for 48 hours to obtain a culture solution (cell suspension). In addition, the pH of the culture solution was monitored from the beginning of the normal gas culture, and 15 ml of 5 g/L glucose was added for 30 minutes at a pH of 7.45. Tmz ----- 55~Τε)ΪΛ~~---- Polypept〇ne 7Ό —____ FeS04 · 7H20 ' nno"------ CoCl2 · 6H20 005*~-~-- - ~~~ Fine W2SO4 ' ' ~~'~~ L5 --- MgS04 · 7H20 Z0 --- KH^ ----- Σο~ —-- K-2HP〇4 ADEKA NOL ---- 22 201107479 No. 99123033 Chinese manual without scribe correction page Revision date: 99 years U month § day [Example 1] [Preparation of bacterial cells containing free nitrile hydratase from cells] (Using a homogenizer to break the cells) Using S The homogenizer 1150 manufactured by ANWA ENGINEERING Co., Ltd. is at a temperature of 15. 〇 〇 压力 压力 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 The nitrile hydratase contained in the solution is dissolved in a solution of the solution. ~ ^ (Heat treatment) The above-mentioned crushed solution was transferred to a 10 L glass beaker, and while the crushed solution was thoroughly stirred, the glass beaker was immersed in a water bath of 5 〇t:. After the liquid temperature of the crushed solution reached 50 〇c, the dipping was continued for another 15 minutes: Then the beaker was taken out and cooled in the air. In the knives (precipitating impurities by acid), a 2 Μ acetic acid aqueous solution is slowly added to the crushed solution after the above-mentioned heat treatment which is sufficiently stirred until the pH of the solution reaches 5 ,, and the solution is stirred for 3 hours to make impurities. Precipitate. (Removal of the crushed material by centrifugation, etc.) Using a centrifuge (manufactured by Hitachi Koki Co., Ltd.: cr22(j), f 5C 8000 rpm, for 15 minutes, the above-mentioned crushed liquid by acid is removed. Centrifugal supernatant was obtained by removing impurities such as precipitated by adding acid. (Neutralization of Centrifugal supernatant) Slow addition of strontium in the above-mentioned centrifugation supernatant which was sufficiently disturbed 23 201107479 Revision date: &quot On November 8th, the Chinese manual No. 99Π3033 has no scribe line correction page NaOH until the pH value of the solution reaches 7.0, and will be centrifuged on '.Main, wide and 0 other' without performing this neutralization. MF filtration in the latter step was not possible due to the precipitation of impurities. ' (MF filtration of neutralizing solution) The above-mentioned neutralization centrifugation was carried out under the conditions of 〇 5 MPa using suporlife450 (DFC capsule type) manufactured by Poul Co., Ltd. The supernatant was passed. ' (Concentration of filtrate) Using a Pencil-type hollow fiber membrane AHP-0013 manufactured by Asahi Kasei Co., Ltd., the cells were treated with an average pressure (U MPa, cycle speed 丄 5 mm/sec) to obtain a bacterial body treatment. (Measurement of protein concentration) The protein concentration of the body treatment was measured using Pr〇tein ASSay (manufactured by Bio-Rad Co., Ltd.) The standard was to use the accompanying bovine serum albumin, and the result was that the protein concentration in the cell treatment was 5 〇 mg/ml. . [Production of acrylamide] A 1 L glass flask equipped with a stirrer was prepared as a second reactor, and a tube of 20 mm of a Teflon (registered trademark) tube of 5 mm was prepared as a second reactor. 4 g of water was previously charged in the first reactor. The cell treatment 〇 3 wt% prepared by the above method was suspended in a 〇·3 mM-NaOH aqueous solution. While the suspension and the acrylonitrile were stirred off, the first reactor was continuously fed at a rate of 49 g/h and 31 g/h, respectively. Then 'to keep the level of the liquid level of the first reactor fixed 24 201107479 依弟; yyiz3033 Chinese patent scope is flawless [| line correction this revision period __99 year I] month 8 〇〇 seven, the scope of patent application A method for producing a bacterial cell treated product comprising a cell treated product of nitrile hydratase, and a method for producing the cell treated product, comprising: producing a microorganism capable of producing a nitrile hydratase (IV) a step of pulverizing the body; (2) a step of subjecting the cell body to acid treatment; (3) a step of removing the insoluble matter formed during the acid treatment of the above step (2); and (4) performing the above step (3) The step of performing the treatment of the broken body of the bacteria. 2. If you apply for a patent scope! The method for producing a bacterial body treatment according to the above aspect, wherein the acid used in the above step (2) is a weak acid. The method for producing a bacterial cell treated product according to the second aspect of the invention, wherein the weak acid is at least one selected from the group consisting of acrylic acid, acid 6 and linonic acid. 4. The manufacturer of the bacterial material treated as described in the patent application scope is called! After the alkali treatment in the above step (4), it is post-treated with at least one of a resin or a filter aid. 5: A method for producing a bacterial material treated product according to item 4 of the patent application, wherein the above-mentioned filter aid is diatomaceous earth. For example, the method for producing a bacterial material treated product according to claim 5, wherein the diatomaceous earth is Ceiite. The method for producing a bacterial material treated product according to the fourth aspect of the invention, wherein the resin is an ion exchange resin. 8. As described in the scope of application of the patent scope (4) Method of manufacturing processed materials 35 201107479 is the Chinese patent scope of No. 99123033 No revision ii line revision date: 99 years 丨] month S曰 method 'where nitrile is produced The hydratase-producing microorganism is a shape-transformer of a nitrile hydratase gene highly expressed by the microorganism in any host; and one or more of the ones of the amino acid constituting the nitrile hydratase by the recombinant DNA technique A shape-transformer of a variant nitrile hydratase which is substituted, deleted, deleted or inserted by other amino acids to exhibit further resistance to guanamine compound or nitrile-resistant compound and temperature resistance. 9. The method for producing a bacterial cell treated product according to claim 1, wherein the microorganism producing the nitrile hydratase is a microorganism belonging to the genus Pseudonocardia. A method for producing a bismuth (mercapto) acrylamide, which is obtained by a method for producing a bacterial cell treated product according to any one of claims 1 to 9 Manufactured from (meth)acrylonitrile (mercapto) acrylamide. A (meth) acrylamide obtained by the method for producing (meth) acrylamide as described in claim 1 of the patent application. ^ I2. An aqueous solution of (meth) acrylamide, which is obtained by the method for producing (meth) acrylamide as described in claim 10, and the aqueous solution of methacrylamide is in every i L 40 wt% to 80 wt% of acrylamide or 10 wt% to 40 wt% of decyl acrylamide, and water solution containing 〇·1 mg~50 mg of protein. A method for producing a methyl methacrylate-based polymer, i is a (meth)acrylamide as described in claim n, or a sulfhydryl group as described in claim 12 A propylene mixed aqueous solution and a (meth) acrylamide polymer.