• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A process for the production of xylose by enzymatic hydrolysis of xylan wherein an aqueous solution containing xylan is treated with a carrier having bonded thereto xylanase enzyme and a carrier having bonded thereto beta -xylosidase and, optionally, uronic acid-splitting enzyme.
    公开号:SU1011050A3
    申请号:SU772532400
    申请日:1977-09-28
    公开日:1983-04-07
    发明作者:Пулс Юрген；Зиннер Михаэль；Дитрихс Ханс-Херман
    申请人:Проектирунг Хемише Ферфаренстехник Гмбх (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to an improved method for the enzymatic production of XYLOSE, an important product of the food industry. A known method for producing xylose by enzymatic hydrolysis of aqueous xylan solution with xylanase and | -Xylosidase at room temperature for 6-8 hours. As a result of enzymatic hydrolysis, a hydrolyzate is obtained containing xylose and xylobiose in a ratio of 1: 1. A disadvantage of the known method is the low yield of the target product. The purpose of the invention is to increase the yield of the target product. The delivered chain is obtained by the method of producing xylose, which consists in the enzymatic hydrolysis of aqueous xylan solutions for k hours by treatment with xylanase and p) -sylozidase and, if necessary, with an enzyme that removes uronic acid, which are separately immobilized on an inorganic carrier, selected from the group: porous glass, silica gel or kieselguhr. As a result of enzymatic hydrolysis, only xylose without xylobiose impurities is obtained. The yield is 98.5%. Hydrolysis of xylan with an enzyme bound to a carrier is different from hydrolysis with free enzymes in that, due to the higher stability of the bound enzymes, higher temperatures can be chosen, resulting in a faster reaction. Temperatures in the range of 30-60 ° C, preferably in the range of,. give, as a rule, optimal results. The xylose yield in accordance with the proposed method is noticeably higher than then, when simultaneous binding of xylanase, α-xylozidase and the enzyme that causes the removal of uronic acid is carried out with the carrier and the enzyme hydrolysis of xylans is attempted due to the fact the xylan solution is treated with a carrier containing all these three enzymes. In the description and in the examples, when it comes to percentages, weight percentages are implied. Example 1: Isolation process (boiling). 400 g of red beech wood in the form of chopped chips are air-dried, treated on a defibrator with water vapor at 185–190 ° C for 6–7 min and under a corresponding pressure of 12 aYM for approximately tO s. The crude fiber material thus obtained is washed out of the defibrator, resulting in a l pulp that has been loaded onto the sieve. The yield of fibrous material is 83 based on the original wood (abs-i dry). The fibrous material washed and pressed on the press is then suspended at room temperature in 5 liters of 1% NaOH solution and 30 minutes later it is separated from the alkaline extract by Filtration and pressing. After washing with water, dilute acid and again with water, the output of the volatile material is 66% on the original wood (abs. Dry). Other types of wood are also treated appropriately, including in the form of large sawdust, as well as in the form of chopped straw. In tab. Figure 1 shows the average yields of the fibrous material (abs. Dry). Example 2. Hydrocarbon composition after aqueous and alkaline treatment. Part of the total amount of extract obtained in example 1, the products of aqueous and alkaline processes is subjected to complete hydrolysis (see Table 2). PRI me R 3. Separation and production of xylanase and / -xylosidase concentrates based on an enzyme preparation. 220 g of the enzyme preparation Celusibe (xylanase, xylosidase, enzyme splitting off "-O-methyl glucuronic acid) is dissolved in 4.8 liters of 0.02 M buffer solution of ammonium acetate buffer at pH 5. The insoluble residue is partially removed by passing through a porous glass filter. Then the enzyme solution is filtered with clarification through a Teflon filter. Next, an enzyme solution is ultrafiltered on a TCF-10 ultrafiltration device from the company Nfci Amincon (Lexington, Mass.) .I The following Amincon-Ultr filters are used (given in order of their use). The region of excretion mol. at. X M 100 A100000 X M 300300000 P M 3030000 D M 5500 The purified solution of the crude enzyme is initially filtered through a filter with a recovery area of mol. at. 100,000. After that, x lanaza is predominantly in the ultrafiltrate; j-Xylosidase (an unknown enzyme that causes cleavage of -0-methyl-glucuronic acid from acidic xylo-oligomers) is predominantly in the residue. This ultrafiltration residue is filtered through an ultrafilter separating the mol. at. 300000. At the end of the p-xylosidase operation, together with the active fermentation component that causes the removal of the uronic acid, it is determined only in the light solution of the ultrafiltrate, and the thick dark brown residue does not contain the active component-xylosidase and the uronic acid. The filtrate obtained at the first ultrafiltration is then further processed. Ultrafiltration on PM 30: Xyla Ease after this stage is again in the ultrafiltrate. Substances that do not possess xylanase activity are abundant. Ultrafiltration on D M 5: xylanase is in the residue; at this stage it is concentrated. At the same time, the largest amount of carbohydrates (in the starting material is 39%) is | B ultrafiltrate. In tab. Figure 3 shows the xylanase, P-xylosidase ji activity values of the enzyme that causes uronic acid cleavage. Presented values are denoted by arbitrary units. One conditional unit corresponds to the amount of enzyme that the sugar content of the MOMjpacTBOpe decomposition increases at 1 m1 mol xylose for xylanase and P-xylosydase and 1 μmol tO-methyl l glucuronic acid for the acid cleaving enzyme (decomposed solution -}% xylan beech wood for xylanase, 2; mmol of p-nitropene xylopyranoside for}) - xylozi 0 dazy, 0.02 µg / µl -0-methylglucuronosyl-xylotriose for the enzyme cleaving the acid). To measure the activity of pi-xylosidase, a dilute solution of p-nitrophenyl xyloside is reacted in a volume of 1.5 ml with 2 ml of 0.1 M borate pH buffer. The analysis of the released p-nitrophenol is carried out directly in the spectrum of 20 nm by its attenuation. The number of p-NITROPHENOL read on the calibration curve and recalculated for xylose. T-O-Methylglucuronosyl-xylotriosis serves as the substrate that cleaves the uronic acid. Example. The deposition of the enzyme on the carrier. As the carrier of the enzyme choose porous glass. Xylonolytic enzymes bind to the carrier via aldehyde glutaric acid. Overnight, 1 g of porous glass is heated under reflux with a 105% solution of gamma-amino-propyl-triethoxylan in toluene. Due to: this carrier receives a group; amine. Thereafter, intensive washing is carried out successively with toluene and acetone. Then, the carrier is vigorously stirred with 20 cl of a 5% solution of aldehyde glutaric acid in 0.02 M phosphate buffer at pH 6.5. Initially, for 15 minutes, stirring is performed under vacuum, and then further incubation is carried out at normal pressure. Next, suction is carried out, and the carrier material is thoroughly washed with 200 ml of buffer solution. On the basis of such an activated carrier material, two fergent preparations are obtained, bound to the carrier: a) 1 g of such activated carrier is mixed overnight with 5 ml of xylonase enzyme solution with an activity of 657 sr. unit, of the obtained Q according to Example 3. Next, a п wash through a porous glass filter with a solution of 1 M NaCl in a -0.02 M phosphate buffer with pH A, after which the washing was carried out with a 0.02 M solution phosphate with a pH of 5 to those 5 then, until the wash water does not contain the enzyme. The preparation 1 thus obtained contains on t g 6 sr. units -bound effective xylonase. b) The operation is carried out as indicated in point a, however, use 5 ml of the solution obtained in accordance with example 3 which contains 3.3 srvc. units p -xylosidase and 60 sm. units an enzyme that cleaves the urban acid. . The resulting preparation 2 contains in a bound state per gram of approximately 33 conv. units L-xylozidase and 60 srvc. units an enzyme that removes uronic acid. Example 5. Hydrolysis of beech wood xylan. 2 ml of the xylan solution obtained in Example 1 by washing with beech wood water after cooking (this solution contains 1.3% xylan), is incubated with 60 mg of the preparation 1 L С 60 MG of preparation 2, obtained as in the example moreover, during incubation in water, the contents of it are stirred by shaking, and the temperature in it is kept equal. After 4 hours, as a result of the hydrolysis of the xylan of beechwood, the monomeric components of xylose and -0-methyl-gl10curonic acid are formed. Figure 1 shows the chromatogram after k h incubation. It shows that in solution, the complete destruction of xylan to xylose occurs. The solution does not contain xylobiosis. PRI me R 6. Silica gel (mercogel SI 1000) was chosen as the carrier for the enzyme. The operation is carried out exactly as indicated in the example, wherein both of the following enzyme preparations are obtained, which are associated with the carrier: a) 1 g of activated carrier containing 59 units. bound xylan; b) 1 g of an activated carrier containing about 2 units of p-xyloside and 59 units of an enzyme in a bonded form that cleaves uronic acid, Example 7, 7. It is operated as described in Example 4 ,. and diatomaceous earth (infusorial earth) is used as the carrier material. In this case, the following two enzymatic preparations are obtained, coupled with the carrier: 50 a) 1 g of activated carrier contains 52 units. coupled xylanase; b 1 g of activated carrier contains 30 units of L-xylosidase and b7 units. bound uronic acid cleaving enzyme. Example Hydrolysis of beech wood xylan. 2 ml of a xylan solution prepared according to Example T was washed with water from a beechwood translated into a soluble form (the solution contained 1.3 xylan), and 60 mg of preparation 1 and 60 mg of preparation 2, prepared according to example, were incubated on a shaking water bath. The xylan is hydrolyzed analytically by column chromatography using an ion exchange resin. After h, the xylan of beech tree is hydrolyzed to its monomeric constituents; FIG. Figure 2 shows the chromatogram after four hours incubation. From this, it is obvious that the xylan is completely cleaved to xylose in solution. The solution does not contain xylobiosis. Comparative experience. Each 2 ml of xylan rastvor obtained according to example 1 (the solution contains 13 mg of xylan in 1 ml) is treated for 4 hours with the three enzyme preparations described below: a) 60 mg of the enzyme (3.8 units of active xylanase, respectively, relative activity ) associated with the carrier, prepared according to example k} b) 60 mg of the preparation associated with the carrier, obtained as follows: 1 g of aerated active glass; ruted according to example C. 1 g of the carrier so activated is stirred overnight with 5 ml crude enzyme used Example 3 was then washed according to a pri- measure. Relative activity the product thus obtained is not determined, since subsequent experience has shown that with this enzymatic preparation xylan cannot be broken down to xylose; c) A mixture of 60 mg of the preparation obtained according to example Aa (3.8 units of xylanase, respectively, relative activity) and 60 mg of prepe7101 rata, obtained according to example kb (2 units of | -xylosidase and 3.6 units of enzyme, removing uronic acid, respectively, relative activity 100%). 10508 The splitting of xylans in three solutions is monitored by 3 m column chromatography according to Example 5. The xylobiosis and xylose content in the solutions is shown as. Fig 3. T a b l and c a 1
    T a b l and c a 3
    Ima (0 S 2568 7968 1290 1996 gA480 13 52 - 1011 1817 21173 19730 -
    权利要求:
    Claims (1)
    [1]
    METHOD FOR PRODUCING XYLOSIS BY ENZYMATIC HYDROLYSIS OF WATER. XYLANE SOLUTIONS by means of an enzyme: enzyme system including xylanase ir-xylosidase, characterized in that, in order to increase the yield of the target product, enzymatic hydrolysis is carried out at 40 ° C for 4 hours, and IR-xylosidase is used as the enzyme system. and, if necessary, an enzyme that cleaves uronic acid, separately immobilized on an inorganic carrier selected from the group: porous glass, silica gel or kieselguhr.
    BoMin
    4 i I bi §4
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    Water hydrolysis
    Fig!
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
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