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net-immobilization of β -glucosidase on nonwoven fabrics to lower the cost of “cellulosic ethanol” and increase cellulose conversions

by:Ming Yu     2019-11-19
The main limitation hindering the use of enzyme cellulose ethanol in industrial production is its higher cost, which is mainly due to beta-glucosidase (BG).
Here we report a simple strategy.
BG in situ packaging for repeated cellulose ethanol production.
In this strategy, BG
Fixed Poly (Ethylene glycol)(PEG)net-
Cloth layer on PP non-woven fabric through visible light-
Induced surface control/live grafting cross
Link aggregation.
Visible light and mild reaction conditions ensure that BG remains active during fixation, not
Uniform expansion net
Grid formed by the intersection of life
Link aggregation can effectively prevent the leakage of BG (
At a fixed rate of more than 98.
6%, the leakage rate is only 0. 4%). When the BG-
Load fabric with free cellulose (CEL)
The results of the catalytic reaction show that these BG-
The loaded fabric can not only increase the cellulose conversion rate by 40%, but also reuse more than 15 batches without losing activity. These BG-
Load fabrics with easy separation, excellent operational stability, low cost of polymer matrix and simple manufacturing process are particularly interesting for future Biotechnology
Fuel production strategy.
A pp non-woven fabric was ordered from Langfang Huijing paper Plastic Manufacturing Co. , Ltd.
This fabric is washed with excessive acetone and then dried at room temperature.
Get ITX from TH-UNIS Insight, Ltd.
From the National Pharmaceutical chemical reagent company, one-water citric acid and two-water sodium citrate were purchased. , Ltd.
The enzyme is from (EC 3. 2. 1. 4), -
From (EC 3. 2. 1. 21)
Sigma provides peg with a molecular weight of 575-
Aldridge chemical
FUN 1 and Calcofluor are available from Life Technologies Co. Filter paper (
Used as a cellulose model)
It was ordered from Hangzhou special paper industry. , Ltd.
Other chemicals were purchased from Alpha ISA chemical.
ITX can be grafted on the fabric as follows.
First, ITX acetone solution (2 mL, 3 mol/mL)
Evenly coated in an area of 4 X cm of the fabric, after which the fabric is placed between two quartz plates.
Secondly, the system in a high
Mercury lamps (
The wavelength is 254 nm, 9 mW/cm)
It lasts for 6 minutes at room temperature to get the fabric with ITXSP group.
After irradiation reaction, wash the fabric with ITXSP with excessive acetone to remove the residual ITX.
Finally, dry at room temperature.
For free enzyme solution, add 55 mg BG to citric acid buffer in μ l (5u2009mM, pHu2009=u20094. 8)by shaking.
Ready for the cross-
Link BG solution, 0. 25% (v/v)
At 30 °c, GA is added to the free BG solution for BG links for 30 minutes.
First, mix the 800 u2009 μ l BG solution with 600peg μ l peg da by shaking, and then pour the solution onto the fabric-ITXSP.
The fabric is sandwiched between two quartz sheets to evenly disperse the peg da/BG solution.
Then aggregate within 90 minutes under the xenon lamp (
Added a band-
At 380-700 nm, the irradiation intensity is 3 mW cm 420 nm).
Then wash the water three times with Deion and remove the unfixed BG and peg da.
Prepare BG-
This fabric is immersed in 100 Na-for the Study of fluorescenceHEPES buffer (10u2009mM, pHu2009=u20097. 2).
Subsequently, a buffer was added to the 100 u2009 μ L fun space nuclear power source and the 500 u2009 μ L Calcofluor solution.
After 60 minutes at room temperature, BG-
Remove the loaded fabric from the solution and wash it thoroughly with Na-
HEPES buffer to remove fluorescent agents without interaction.
All the steps are done in the darkroom. A Non-
Interfering protein assay kit for detecting the ratio of fixed BG in PEG networkcloth.
All BG and BG absorbed on the quartz board-
The loaded fabric after grafting reaction is thoroughly cleaned and collected to determine the non-
Fixed quantity.
BG concentration was measured by monitoring BG concentration after soaking in citric acid buffer at 480nm using uv-vis spectrometer.
Get the concentration of BG in the solution from the calibration curve, the amount of BG released at t ()
It is calculated by accumulating the total amount of BG released at that time.
The ratio to release BG/can then be calculated.
Here, the amount of BG that was originally fixed.
Activities of BG-
The loaded fabric is measured-
Release p-chlorphenol from NPG using a calibration curve at 405 µnm.
5 ml reaction mixture containing 4mm NPG, 50 ml mm sodium acetate buffer (pHu2009=u20094. 8)
First incubated at 40 °c, then 2 × cm BG-
Add the loaded fabric and the reaction is catalytic for 30 minutes.
Finally, a 10 µml 1 M NaCO was added to stop the reaction and the solution was measured at 405 µnm.
A unit of BG activity is defined as catalytic 1 μ mol-
Phenol sensitivity.
Hydrolysis activity of BG-
The load fabric was tested in several cycles (24u2009h each)
At 50 °c and pH 4. 8.
First 100 u2009 mu L free CEL and 4 u2009 × u2009 4 u2009 length BG-
Mix the loaded fabric in 10 ml citrate buffer (5, pH 4. 8)
After that, add 20 mg of filter paper to the buffer.
Secondly, mix the mixture for 24 hours at 200 rpm and 50 °c.
Finally, BG-
Remove the loaded fabric and thoroughly wash it with citrate buffer at 50 °c for reuse in the next cycle.
Sample the liquid phase after each hydrolysis cycle to determine the concentration of glucose production.
UV-vis spectrometer characterized the UV spectrum (U-
Hitachi 3900 H).
Contact angle data is obtained through the OCA 20 Tension meter from Data Physics company. SEM (JSM-
JEOL Japan electronics company 6701F, Ltd)
The form used for measurement,
Woven fabric before and after grafting reaction.
The structure of this catalytic composite was characterized by a microscope (
BX 53 of Olympus)
And atomic force microscope (AFM, CP-
II from VEECO Co. ).
Surface chemistry is discussed by XPS (
ESCALAB 250 day Saimo feishi Technology Co. , Ltd. )
Use a monochrome. The BG-
Fluorescence spectrum of CLSM for fabric (
TCS SP8 from Leica).
Determination of glucose concentration by a biological sensor (SBA-
Institute of Biology, Shandong Academy of Sciences).
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