Preparation method of nano immobilized cellulase

A cellulase and cellulose technology, which is applied in the direction of being fixed on or in an inorganic carrier, can solve the problems of low enzymatic activity retention rate, incapable of industrialized production, complicated preparation process, etc., and achieves a simple and environmentally friendly method for repeated use. , the effect of compensating for production costs

Inactive Publication Date: 2013-11-20
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Problems solved by technology

[0007] The currently applied immobilized cellulase method has the following problems: use of surfactants: glutaraldehyde, ammonia water, acetic acid, polyvinyl alcohol, span80, etc.; low temperature freezing or high temperature is required; repeated freezing and thawing is required; enzyme activity is retained The rate is low, about 40%; these deficiencies lead to the preparation of immobilized cellulase, the preparation process is complicated, the environment is not friendly, and industrial production cannot

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  • Preparation method of nano immobilized cellulase
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  • Preparation method of nano immobilized cellulase

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Embodiment 1

[0035] Embodiment 1: Preparation of nano-immobilized cellulase according to the present invention

[0036] Materials Orthoethyl silicate (TEOS), Sinopharm Reagent Group, purity > 99%

[0037] Cellulase, Sigma Aldrich, 10000U / g

[0038] D-Glucose, Aladdin's reagent, >99%

[0039]Accurately weigh 32.0g of tetraethyl orthosilicate (TEOS) into a 100ml beaker, add 2.5ml of 0.5M HCl solution under stirring, after vigorous stirring, the tetraethyl orthosilicate can be hydrolyzed after about 15min to obtain transparent solution, the transparent solution obtained after hydrolysis was cooled in a 4°C refrigerator, and then transferred to a conical flask with a high vacuum system to recover the ethanol obtained in the hydrolysis process. When the quality of the entire system dropped to 50% of its original value (at this time ethanol content in the solution <1%), stop vacuuming, transfer the solution into a 100ml beaker, add glucose solution (2.0g glucose dissolved in 10ml water) under ...

Embodiment 2

[0040] Example 2: Influence of residual ethanol in the system on the activity of nano-immobilized cellulase in the preparation method of the present invention

[0041] With reference to Example 1, keeping other conditions unchanged, the ethanol content in the transparent solution was controlled to be 0.5%, 1.0%, 2.0%, 5.0%, and 10% respectively after recovering ethanol, and the prepared nano-cellulase was measured for enzyme activity, and the results See table below.

[0042] System residual ethanol%

[0043] The above results show that when the ethanol content in the transparent solution is less than 1% after controlling the recovery of ethanol, the relative free enzyme activity of the nanozyme is above 85%.

Embodiment 3

[0044] Embodiment 3: Nano-immobilized cellulase activity test of the present invention

[0045] Usually, filter paper activity (FPA) represents the total activity of cellulase, which is measured according to the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC), and expressed in IU. Put a piece of 1×6cm whatman No.1 filter paper (about 50mg) into the test tube, add 0.5mL of appropriately diluted cellulase solution and 1mL of citric acid buffer (pH4.8), and incubate and shake at 50°C for 30min. In the blank test, except that the enzyme solution was inactivated in advance, the other conditions were unchanged. After the reaction, the resulting reducing sugar content was determined by the DNS method.

[0046] One filter paper enzyme activity international unit (FPIU) is equal to the amount of enzyme required to generate 1.0umol of glucose (expressed as reducing sugar) per minute in the enzymatic reaction. Then there...

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Abstract

The invention relates to the field of biochemical engineering, and discloses a preparation method of nano immobilized cellulase, which comprises the following steps: adding a HCl solution into ethyl orthosilicate while stirring, and hydrolyzing to obtain a transparent solution; cooling the transparent solution, recovering ethanol, and controlling the ethanol volume percent of the transparent solution to be less than 1%; adding a glucose solution into the transparent solution while stirring, then adding a cellulase citric acid-sodium citrate buffer solution, finally regulating the pH value to approximate neutral, transferring to 4-10 DEG C, and gelatinizing; and grinding the wet gel obtained after gelatinizing, washing with water to remove the glucose, performing vacuum filtration on the mixed solution after water washing to obtain a white gel, drying the gel, grinding, and screening for later use. Experiment shows that the enzyme activity is almost kept unchanged after the nano immobilized cellulase prepared by the method is repeatedly used multiple times; and the method can be used for large-scale production of cellulosic ethanol.

Description

technical field [0001] The invention relates to the field of biochemical industry, in particular to a preparation method of nano-immobilized cellulase. Background technique [0002] With rising international crude oil prices and worsening global pollution problems, countries all over the world are sparing no effort to develop new energy sources, and bioethanol is a new type of fuel that is generally favored. In the 21st century, the use of cellulase to convert cellulose to produce glucose and then ferment to obtain bioethanol can avoid a large loss of food crops, which has attracted the attention of governments and research institutions in various countries. The key to this is the cost of cellulase . [0003] Due to the low fermentation activity of cellulase, its application cost is also high. Compared with other glycoside hydrolases, the specific activity of cellulase is at least 1 to 2 orders of magnitude lower, resulting in lower enzyme efficiency. In addition, because...

Claims

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Application Information

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IPC IPC(8): C12N11/14
Inventor 李书润廖文强危岩
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