AU2019203362B2

AU2019203362B2 - Monolithic cellulose acetate column, its enzyme reactor, preparation method and application

Info

Publication number: AU2019203362B2
Application number: AU2019203362A
Authority: AU
Inventors: Qianchun DENG; Fenghong HUANG; Jie Shi; Hu Tang; Yuqin Xiao; Mingming ZHENG
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2018-07-27
Filing date: 2019-05-14
Publication date: 2024-06-13
Anticipated expiration: 2039-05-14
Also published as: CN109022272A; CN109022272B; AU2019203362A1

Abstract

Monolithic cellulose acetate column, its enzyme reactor, preparation method and application ABSTRACT OF THE DISCLOSURE A monolithic cellulose acetate colum, which is characterized in having a diameter of 1 to 10 cm and a specific surface area of 60 to 80 m2 /g, and being a macroporous-division hierarchical pore structure; wherein a pore size of a mesopore is at a range of 5 to 30 nm, and a pore size of the a macropore is at a range of t to 3pm. An enzyme reactor for a monolithic colunm, includes an enzyme, a monolithic cellulose acetate colunm, a heat shrinkable tube and a sealing head made of tetrafluoroethylene. The monolithic immobilized enzyme reactor of the present invention is applied to an enzymatic reaction, and a fractional pore structure and the continuous flow reaction form can improve mass transfer efficiency greatly, facilitate the exposure of the active sites of the enzyme. 19 161 300 0.012 I 20.010 250 0.008 200 10.006 0.004- 0150 o 0.002 100- 0 1 2 4 16 32 64 Pore Width (nm) 50 0 I I * I * * I * 0.0 0.2 0.4 0.6 0.8 1.0 Relative Pressure (P/Po) 2

Description

300 0.012 I 20.010 250 0.008 200 10.006 0.004- 0150 o 0.002

100- 0 1 2 4 16 32 64 Pore Width (nm)

50

0 I I * I * * I *

0.0 0.2 0.4 0.6 0.8 1.0 Relative Pressure (P/Po)

TITLE

Monolithic cellulose acetate column, its enzyme reactor, preparation method and application

BACKGROUND OF THE PRESENT INVENTION

Field of Invention

The present invention relates to the technical field of biocatalysis, and more particularly to a monolithic cellulose acetate column, its enzyme reactor, preparation method and application.

Description of Related Arts

Enzymatic catalysis is an excellent method for environmental protection chemical synthesis due to the high chemical, stereo and region-selectivity of enzymes and mild conditions during operation. In use process, the free enzyme loses activity under certain conditions, such as organic solvents, extreme temperature and pH, and is not easily recovered and reused. The use of immobilized enzymes has become an alternative method which not only reduces enzyme contamination, but also improves the stability and recovery of enzyme in the reaction medium. In fact, most immobilized enzymes are used in batch agitation reactions. However, there are still plenty of problems as follows. (1) The presence of frictional shear forces makes the enzymes more susceptible to inactivation. (2) The process of repeatedly using, cleaning and recycling is troublesome. (3) The batch stirring reaction cannot be continuously produced, and the industrialization process is slow.

The use of immobilized enzymes in packed bed reactors enables continuous production and can be reused over long periods of time. However, the packing material of the packed bed type immobilized enzyme reactor is the main factor affecting its performance. The commonly used enzyme immobilized carrier often requires complex surface modification; the particle-filled immobilized enzyme reactor has an additional filling step and high requirements on the equipment. The above problem can be solved by using the monolithic column as a filling material. Monolithic colums are generally classified into types made of organic materials and inorganic materials. The preparation of the inorganic monolithic column is difficult, the operation is cumbersome, and the controllability is poor. The preparation usually requires a long aging time and is easy to crack. Among them, silica monolithic colum is the most common, but a maximum diameter of the silica monolithic column is usually within 500 pm due to mechanical strength. The silica monolithic colum can only be used for separation and in-line hydrolysis, and it is only used in micro-reactors, which limits the application scale in the enzyme reactor. The organic polymer monolithic column has poor permeability and high column pressure, and is generally applied to high-pressure liquid chromatography, and is not suitable for large-scale enzyme reactors.

A patent with a publication number of 101845430A disclosed an organic inorganic hybrid monolithic material and a method thereof for applying in an immobilized enzyme reactor, using tetramethoxy or tetraethoxysilane, 2 glycidoxypropyltrimethoxysilane as a front molecular molecule, iminodiacetic acid as a metal ion chelating agent, cetyltrimethylammonium bromide as a template molecule, and octylamine as a catalyst, the metal surface of the monolithic column was modified, the preparation process is very cumbersome. A patent with a publication number of

102,391,947 disclosed a method for preparing a porous monolithic colum immobilized enzyme microreactor, but requires modification of the cysteamine and gold nanoparticles

on the surface of the monolithic colum, the preparation process is cumbersome, and the

size is usually limited to the micron order due to mechanical strength.

Any discussion of the prior art throughout the specification should in no way be

considered as an admission that such prior art is widely known or forms part of common

general knowledge in the field.

SUMMARY OF THE PRESENT INVENTION

According to an aspect, the present invention provides a monolithic cellulose acetate column having a diameter of I to 10 cm and a specific surface area of 60 to 80 m2 /g, and being a macroporous-division hierarchical pore structure; wherein a pore size of a mesopore is at a range of 5 to 30 nm, and a pore size of the macropore is at a range of1 to 3 im; wherein a preparation method of the monolithic cellulose acetate column comprises steps of: dissolving cellulose acetate powder in a solvent of N, N-dimethyl acetamide, adding n-hexanol, heating and stirring, pouring a transparent solution obtained into a glass tube, and performing phase separation in a constant temperature environment to obtain a solid, performing Soxhlet extraction on the solid obtained by the phase separation, replacing the solvent with ethanol, and wherein the monolithic cellulose acetate column is obtained after drying under a vacuum.

According to an aspect, the present invention provides an enzyme reactor for a monolithic column, comprising an enzyme, a monolithic cellulose acetate column serving as a carrier for immobilizing the enzyme, a heat shrinkable tube and a sealing head made of tetrafluoroethylene; wherein a diameter of the monolithic cellulose acetate column is at a range of 1 to 10 cm, a specific surface area is at a range of 60 to 80 m 2 /g, and the monolithic cellulose acetate column is a macroporous-division hierarchical pore structure, a pore size of a mesopore is at a range of 5 to 30 nm, and a pore size of the macropore is at a range of I to 3 pm.

According to an aspect, the present invention provides a method for preparing the enzyme reactor of the invention comprising the steps of:

step (1): preparing monolithic cellulose acetate column, comprising: dissolving cellulose acetate powder in a solvent of N, N-dimethyl acetamide, adding n-hexanol, heating and stirring; pouring a transparent solution obtained into a glass tube and placing the glass tube at a environment with constant temperature for performing phase separation to obtain a solid; performing Soxhlet extraction on the solid obtained by the phase separation, replacing a solvent with ethanol, and wherein a monolithic cellulose acetate column is obtained after drying under a vacuum, wherein a diameter of the monolithic cellulose acetate column is at a range of 1 to 10 cm, a specific surface area is at a range of 60 to 80 m2 /g, and the monolithic cellulose acetate column is a macroporous-division hierarchical pore structure, a pore size of a mesopore is at a range of 5 to 30 nm, and a pore size of the macropore is at a range of I to 3 m; step (2): preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate column with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene; step (3): immobilizing the enzyme in the reactor for the monolithic cellulose acetate column, comprising: dispersing the enzyme into a buffer solution to prepare an enzyme solution; driving the enzyme solution by a peristaltic pump to flow at a certain rate through the reactor for the monolithic cellulose acetate column for immobilizing the enzyme, so as to obtain the enzyme reactor for the monolithic cellulose acetate column.

According to an aspect, the present invention provides a method for applying the enzyme reactor of the invention in enzymatic reaction, comprising: placing the enzyme reactor for the monolithic cellulose acetate column reactor in a column oven to maintain a certain temperature, wherein reaction solution is subjected to an enzymatic

reaction through a cellulose acetate monolithic column reactor at a certain flow rate under a driving flow of a peristaltic pump.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

The present invention relates to a monolithic cellulose acetate column with good mechanical strength and good permeability, and is suitable as a carrier for an enzyme reactor without cumbersome filling. The process of adsorption and immobilization of the

4a enzyme can be achieved without surface modification, and the monolithic column can be easily enlarged, and is capable of performing continuously production for a long time.

The present invention relates to the following.

A monolithic cellulose acetate colum, has a diameter of 1to 10 cm and is a macroporous-division hierarchical pore structure; wherein a pore size of a mesopore is at a range of 5 to 30 nm, and a pore size of the a macropore is at a range of 1 to 3 pm; and a specific surface area of 60 to 80 m2/g.

A preparation method of the monolithic cellulose acetate column comprises steps of: dissolving cellulose acetate powder in a solvent of N, N-dimethyl acetamide, adding n-hexanol, heating and stirring, pouring a transparent solution obtained into a glass tube, and performing phase separation in a constant temperature environment to obtain a solid, performing Soxhlet extraction on the solid obtained by the phase separation, replacing the solvent with ethanol, and drying under vacuum to obtain the monolithic cellulose acetate column (CA-MIN).

According to the above, a ratio of the N, N-dimethyl acetamide, the cellulose acetate, and the n-hexanol is 1 mL: (0.15-0.2) g: (1.25-1.50) mL. A stirring temperature is at a range of 65-70 °C; the constant temperature is at a range of 15-25 °C, a time of the phase separation is at a range of 36-48 hours; a temperature of the Soxhlet extraction is at a range of 110-125 °C, and a time of the Soxhlet extraction is at a range of 48-72h; a temperature of the vacuum drying is 40 °C, and a time of the vacuum drying is 6 to 24

hours.

Based on the monolithic cellulose acetate colum, the present invention further

provides an enzyme reactor for the monolithic column, which comprises an enzyme, a

monolithic cellulose acetate column serving as a carrier for immobilizing the enzyme, a heat shrinkable tube and a sealing head made of tetrafluoroethylene.

A method for preparing the enzyme reactor for the monolithic column, comprises steps of:

step (1): preparing monolithic cellulose acetate colum, comprising: dissolving cellulose acetate powder in a solvent of N, N-dimethyl acetamide, adding n-hexanol, heating and stirring ; pouring a transparent solution obtained into a glass tube and placing the glass tube at a environment with constant temperature for performing phase separation to obtain a solid; performing Soxhlet extraction on the solid obtained by the phase separation, replacing a solvent with ethanol, and drying under vacuum to obtain a monolithic cellulose acetate column;

step (2): preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate column with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene to obtain the reactor for the monolithic cellulose acetate column;

step (3): immobilizing the enzyme in the reactor for the monolithic cellulose acetate colum, comprising: dispersing the enzyme into a buffer solution to prepare an enzyme solution; driving the enzyme solution by a peristaltic pump to flow at a certain rate through the reactor for the monolithic cellulose acetate colum for immobilizing the enzyme, so as to obtain the enzyme reactor for the monolithic cellulose acetate column.

Preferably, in the step (3), an immobilized amount of the enzyme on the monolithic cellulose acetate column is at a range of 50 to 400 mg/g.

Preferably, in the step (3), an immobilization temperature of the enzyme is at a range of 10 to 37 °C, and a flow rate is at a range of 0.15 to 2.0 mL/min.

Preferably, in the solution mentioned above, the enzyme comprises: porcine pancreatic enzyme, Candida rugosa lipase, Candida lipolytica lipase, Candida antarcticalipase B, Pseudomonas cepacia lipase, Rhizopus oryzae, phospholipase, pepsin, trypsin, cathepsin papain, pectinase.

A method for applying the enzyme reactor for the monolithic column mentioned above in enzymatic reaction, comprises: placing enzyme reactor for the monolithic cellulose acetate colum reactor in a column oven to maintain a certain temperature, wherein reaction solution is subjected to an enzymatic reaction through a cellulose acetate monolithic colum reactor at a certain flow rate under a driving flow of a peristaltic pump, wherein a reaction temperature is at a range of 30 to 70 °C, and a flow rate is at a range of (0.10 to 10.0) m/min depending on a diameter of the monolithic column.

According to the solution mentioned above, the enzymatic reaction comprises: esterification, transesterification, ester hydrolysis, protein hydrolysis, phospholipids, pectin hydrolysis reaction.

Compared with the conventional art, the beneficial effects of the present invention are as follows.

1. The present invention firstly proposes a monolithic cellulose acetate colum as a carrier to immobilize an enzyme and carry out a continuous catalytic reaction, and by two steps of liquid phase separation of the liquid-liquid and solid-liquid polymer crystal phase, a cellulose acetate macroporous-mesopores hierarchical pore structure is produced. A centimeter-level monolithic column produced has good mechanical strength and good permeability, and is suitable as a carrier for the enzyme reactor.

2. The monolithic column reactor of the present invention provides relatively sufficient enzyme catalytic sites, and the mesopores in the monolithic colum provide space for the immobilization of the enzyme, and the macropores facilitate the passage of the reaction solution and enhance the interaction between the substrate and the enzyme,. The convective mass transfer efficiency makes the reaction faster and the conversion rate is high.

3. Compared with the granular immobilized enzyme, the monolithic colum enzyme reactor of the present invention avoids the damage of the immobilized enzyme during the batch stirring reaction, the damage of the shearing force and the loss of the washing and recycling process, thereby greatly increasing the stability and reusability of enzyme.

4. Compared with the granular filling reaction, the monolithic colum enzyme reactor of the invention has the advantages of simple preparation method, no complicated filling process, long service life of the enzyme, and continuous production for a long time.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a cross-sectional scanning electron micrograph of a monolithic cellulose acetate column obtained in Example 2.

Fig. 2 is a diagram showing the nitrogen elution attachment and mesoporous pore size distribution ofthe monolithic cellulose acetate column obtained in Example 2.

Fig. 3 is a diagram of an enzyme reactor apparatus for the monolithic cellulose acetate column.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following examples are only intended to illustrate the present invention in detail, and in order to facilitate a better understanding of the present invention, the examples are intended to be included within the scope of the invention and not intended to be limiting.

Example 1

A method for preparing monolithic cellulose acetate colum and application thereof, comprises following steps of: step (1) preparing a monolithic cellulose acetate colum comprising: completely dissolving 2 g of cellulose acetate powder in 10 mL solvent of N, N-dimethyl acetamide (DMF), and slowly adding 12.5mL of n-hexanol at 65 °C, stirring until transparent; pouring a transparent solution obtained into a glass tube, sending in a 25°C incubator for 48 h to perform phase separation to obtain a solid; dissociating the solid obtained by phase separation with ethanol, performing Soxhlet extraction at 120 °C for 72 h, and finally drying in vacuum under 40°C for 12h, a monolithic cellulose acetate column (CA MN) with a length of 5 cm and a diameter of lcm was obtained; step (2) preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate column with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene; step (3) immobilizing the lipase in the reactor for the monolithic cellulose acetate column, comprising: dispersing 3g of candida antarctica lipase B into a buffer solution of phosphas phosphate with a volume of 30mL, an amount of substance of 50mmol and a pH of 7.0 to prepare an enzyme solution; under a temperature below 20°C driving the enzyme solution by a peristaltic pump to flow at a rate of 0.70ml/min through the reactor for the monolithic cellulose acetate colum for immobilizing the enzyme, cleaning with buffer solution, the enzyme reactor for the monolithic cellulose acetate column was obtained after drying, wherein an enzyme loading amount is 50mg/g; step (4) applying the enzyme reactor for the monolithic cellulose acetate colum for combining fatty acids and sterols to synthesize sterol esters comprising: weighing lg of p-sitosterol, and weighing a- linolenic acid according to a molar ratio of the a-linolenic acid to p-sitosterol of 6:1, adding 50 mL of isooctane, heating to completely dissolve a reaction solution; placing the monolithic cellulose acetate column reactor into a colum oven to maintain at 55 °C, wherein the reaction solution was driven by a peristaltic pump to pass through the enzyme reactor for the monolithic cellulose acetate column with a flow rate of 0.1 m/mmin to process a synthesis reaction of the sterol esters, wherein an esterification conversion is 93.0%, and after continuous utilizing for 100h, the esterification conversion is still greater than 83%.

Example 2

A method for preparing monolithic cellulose acetate colum and application thereof, comprises following steps of:

step (1) preparing a monolithic cellulose acetate colum comprising: completely dissolving 2 g of cellulose acetate powder in 12 mL solvent of N, N-dimethyl acetamide (DMF), and slowly adding 18 mL of n-hexanol at 70 °C, stirring until the mixture becomes transparent; pouring a transparent solution obtained into a glass tube, sending in a 20 °C incubator for 48 h to perform phase separation to obtain a solid; dissociating the solid obtained by phase separation with ethanol, performing Soxhlet extraction at 120 °C for 72 h, and finally drying in vacuum under 40°C for 12h, a monolithic cellulose acetate column (CA-MN) with a length of 15 cm and a diameter of 5cm was obtained;

wherein it can be seen from Fig. 1 and Fig. 2 that the monolithic cellulose acetate column obtained has a macropore-mesopores grading pore structure inside, a pore size of the macropore is at a range of 1 to 3 pm, a pore size of the mesopores is at a range of 5 to 30 nm, and a specific surface area is 60-80m 2/g;

step (2) preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate column with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene;

step (3) immobilizing the lipase in the reactor for the monolithic cellulose acetate column, comprising: dispersing 2g of pseudomonas cepacia lipase into a buffer solution of phosphas phosphate with a volume of 40mL, an amount of substance of 20mmol and a pH of 4.0 to prepare an enzyme solution; under a temperature of 20°C driving the enzyme solution by a peristaltic pump to flow at a rate of 0.50ml/min through the reactor for the monolithic cellulose acetate colum for immobilizing the enzyme, cleaning with buffer solution, the enzyme reactor for the monolithic cellulose acetate column was obtained after drying, wherein an enzyme loading amount is 100mg/g; step (4) applying the enzyme reactor for the monolithic cellulose acetate colum for chiral separation of phenylethyl alcohol comprising: weighing 2.5g of 1 phenylethanol and weighing vinyl acetate by a molar ratio of vinyl acetate to 1 phenylethanol of 4:1; adding 50 mL of isooctane until a reaction solution is completely dissolved; placing the monolithic cellulose acetate column reactor into a column oven to maintain at 50 °C, wherein the reaction solution was driven by a peristaltic pump to pass through the enzyme reactor for the monolithic cellulose acetate column with a flow rate of 2.5 m/min to perform chiral separation on the phenylethyl alcohol, distilling solvent off under reduced pressure from the solution in the reactor, and wherein a phenylethanol conversion is 50%, after 150 hours of continuous use, the conversion remains at around 50%.

wherein an esterification conversion is 93.0%, and after continuous utilizing for 100h, the esterification conversion is still greater than 83%.

Example 3

A method for preparing monolithic cellulose acetate column and application thereof, comprises following steps of:

step (1) preparing a monolithic cellulose acetate colum comprising: completely dissolving 3 g of cellulose acetate powder in 15 mL solvent of N, N-dimethyl acetamide (DMF), and slowly adding 21 mL of n-hexanol at 70 °C, stirring until the mixture becomes transparent; pouring a transparent solution obtained into a glass tube, sending in a 25°C incubator for 48 h to perform phase separation to obtain a solid; dissociating the solid obtained by phase separation with ethanol, performing Soxhlet extraction at 120 °C for 72 h, and finally drying in vacuum under 45°C for 18h, a monolithic cellulose acetate column (CA-MN) with a length of 20 cm and a diameter of10cm was obtained;

step (2) preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate column with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene; step (3) immobilizing the lipase in the reactor for the monolithic cellulose acetate colum, comprising: dispersing 2 g of phospholipase A into a buffer solution of phosphas phosphate with a volume of 20 mL, an amount of substance of 100 mmol and a pH of 5.0 to prepare an enzyme solution; under a temperature of 37°C driving the enzyme solution by a peristaltic pump to flow at a rate of 0.30ml/min through the reactor for the monolithic cellulose acetate column for immobilizing the enzyme, cleaning with buffer solution, the enzyme reactor for the monolithic cellulose acetate colum was obtained after drying, wherein an enzyme loading amount is 200 mg/g; step (4) applying the enzyme reactor for the monolithic cellulose acetate colum for synthesis of functional phospholipids comprising: weighing 3.0 g of soy lecithin, and weighing ethyl linolenate according to a mass ratio of ethyl linolenate to soy lecithin of 5:1, adding 3.0 mL of deionized water and adding 30 mL of n-hexane, until a reaction solution is completely dissolved; placing the monolithic cellulose acetate colum reactor into a column oven to maintain at 55 °C, wherein the reaction solution was driven by a peristaltic pump to pass through the enzyme reactor for the monolithic cellulose acetate column with a flow rate of 10.0 m/min to process a synthesis reaction of the functional phospholipids, wherein an esterification conversion is 92.0%, and after continuous utilizing for 200 h, the esterification conversion is still greater than 90%.

Example 4

step (1) preparing a monolithic cellulose acetate colum comprising: completely dissolving 1 g of cellulose acetate powder in 5 mL solvent of N, N-dimethyl acetamide (DMF), and slowly adding 7.5mL of n-hexanol at 65 °C, stirring until transparent; pouring a transparent solution obtained into a glass tube, sending in a 25°C incubator for 48 h to perform phase separation to obtain a solid; dissociating the solid obtained by

phase separation with ethanol, performing Soxhlet extraction at 120 °C for 72 h, and finally drying in vacuum under 40°C for 8 h, a monolithic cellulose acetate column (CA MN) with a length of 12 cm and a diameter of 5 cm was obtained; step (2) preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate column with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene; step (3) immobilizing the lipase in the reactor for the monolithic cellulose acetate column, comprising: dispersing 4g of candida lipalytica Lipase into a buffer solution of phosphas phosphate with a volume of 20 mL, an amount of substance of 100 mmol and a pH of 6.0 to prepare an enzyme solution; under a temperature of 37 °C driving the enzyme solution by a peristaltic pump to flow at a rate of 0.30ml/min through the reactor for the monolithic cellulose acetate colum for immobilizing the enzyme, cleaning with buffer solution, the enzyme reactor for the monolithic cellulose acetate column was obtained after drying, wherein an enzyme loading amount is 300mg/g; step (4) applying the enzyme reactor for the monolithic cellulose acetate colum for synthesis of human milk substitute comprising: weighing 1 g of tripalmitin, weighing oleic acid according to a molar ratio of tripalmitin to oleic acid of 1:6, adding 0.6 mL of deionized water, adding 30 mL of n-hexane, until a reaction solution is completely dissolved; placing the monolithic cellulose acetate column reactor into a column oven to maintain at 55 °C, wherein the reaction solution was driven by a peristaltic pump to pass through the enzyme reactor for the monolithic cellulose acetate column with a flow rate of 2.5 mL/min to synthesis of human milk substitute, distilling a solvent off under reduced pressure from a solution passing through the reactor, and an esterification rate was 75.6%, after 100 hours of continuous use, the esterification conversion is still greater than 70%.

wherein an esterification conversion is 93.0%, and after continuous utilizing for

100h, the esterification conversion is still greater than 83 %.

Example 5

step (1) preparing a monolithic cellulose acetate colum comprising: completely dissolving 2 g of cellulose acetate powder in 10 mL solvent of N, N-dimethyl acetamide (DMF), and slowly adding 14 mL of n-hexanol at 75 °C, stirring until transparent; pouring a transparent solution obtained into a glass tube, sending in a 25°C incubator for 48 h to perform phase separation to obtain a solid; dissociating the solid obtained by phase separation with ethanol, performing Soxhlet extraction at 120 °C for 72 h, and finally drying in vacuum under 40°C for 15 h, a monolithic cellulose acetate colum (CA-MN) with a length of 12 cm and a diameter of 8 cm was obtained;

step (2) preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate colunm with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene;

step (3) immobilizing the protease in the reactor for the monolithic cellulose acetate colum, comprising: dispersing 2 g of candida antarctica lipase B into a buffer solution of phosphas phosphate with a volume of 30mL, an amount of substance of 80 mmol and a pH of 6.0 to prepare an enzyme solution; under a temperature of10°C driving the enzyme solution by a peristaltic pump to flow at a rate of 2.0 ml/min through the reactor for the monolithic cellulose acetate colum for immobilizing the enzyme, cleaning with buffer solution, the enzyme reactor for the monolithic cellulose acetate colunm was obtained after drying, wherein an enzyme loading amount is 400 mg/g;

step (4) applying the enzyme reactor for the monolithic cellulose acetate column for catalytic hydrolysis of casein protein, comprising: weighing 0.5g of casein protein, adding 0.6 mL of deionized water, adding a buffer solution of phosphas phosphate with a volume of 50mL, an amount of substance of 100 mmol and a pH of 6.0, heating to dissolve the solution completely; placing the monolithic cellulose acetate column reactor into a colunm oven to keep in a temperature of 40 °C, and driving a reaction solution by a peristaltic pump at a flow rate of 6.4 mL/min through a cellulose acetate monolithic column reactor for catalytic hydrolysis of the casein protein, and measuring an absorbance of the solution passing through the reactor by a spectrophotometer at a wavelength of 275 nm; wherein one enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze the substrate casein protein to produce 1 pmol of L tyrosine in 1 min of immobilized enzyme at 40 °C, pH 6.0; wherein the results showed that the enzyme activity of the cellulose acetate monolithic column reactor is 182.8 U/g carrier, and the enzyme activity was still above 160 U/g for continuous use of 200 h.

In summary, the cellulose-based monolithic colum enzyme reactor of the present invention can improve the immobilization stability and catalytic activity of the enzyme with good reusability in many application field, and thus is a kind of application-oriented immobilization enzyme carrier. The prepared immobilized enzyme has excellent stability, high catalytic activity, with an esterification conversion maintained above 80%, and has good reusability in continuous use time up to 100 hours or more.

The raw materials, such as the specific raw materials of the lipase, enumerated in the present invention, as well as the upper and lower limits of the raw materials of the present invention, the values of the intervals, and the upper and lower limits of the process parameters, such as temperature, time, etc. The above is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and changes without departing from the inventive concept all belong to the scope of protection.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing an enzyme reactor for a monolithic column comprising the steps of:

step (1): preparing monolithic cellulose acetate column, comprising: dissolving cellulose acetate powder in a solvent of N, N-dimethyl acetamide, adding n-hexanol, heating and stirring; pouring a transparent solution obtained into a glass tube and placing the glass tube at a environment with constant temperature for performing phase separation to obtain a solid; performing Soxhlet extraction on the solid obtained by the phase separation, replacing a solvent with ethanol, and wherein a monolithic cellulose acetate column is obtained after drying under a vacuum, wherein a diameter of the monolithic cellulose acetate column is at a range of 1 to 10 cm, a specific surface area is at a range of 60 to 80 m2 /g, and the monolithic cellulose acetate column is a macroporous-division hierarchical pore structure, a pore size of a mesopore is at a range of 5 to 30 nm, and a pore size of the macropore is at a range of I to 3 m;

step (2): preparing a reactor for the monolithic cellulose acetate column, comprising: coating the monolithic cellulose acetate column with a heat shrinkable tube, and sealing with the sealing head made of tetrafluoroethylene;

step (3): immobilizing the enzyme in the reactor for the monolithic cellulose acetate column, comprising: dispersing the enzyme into a buffer solution to prepare an enzyme solution; driving the enzyme solution by a peristaltic pump to flow at a certain rate through the reactor for the monolithic cellulose acetate column for immobilizing the enzyme, so as to obtain the enzyme reactor for the monolithic cellulose acetate column.

2. The method of claim 1 wherein in step (1), a ratio of the N, N-dimethyl acetamide, the cellulose acetate, and the n-hexanol is 1 mL: (0.15-0.2) g: (1.25-1.50) mL.

3. The method of claim 1 wherein in step (1), a stirring temperature is at a range of 65-70°C; the constant temperature is at a range of 15-25 0 C, a time of the phase separation is at a range of 36-48 hours; a temperature of the Soxhlet extraction is at a range of110 125 °C, and a time of the Soxhlet extraction is at a range of 48-72h; a temperature of the vacuum drying is 40 °C, and a time of the vacuum drying is 6 to 24 hours.

4. The method of claim 1 wherein in step (3), an immobilized amount of the enzyme on the monolithic cellulose acetate column is at a range of 50 to 400 mg/g; an immobilization temperature of the enzyme is at a range of 10 to 37 °C, and a flow rate is at a range of 0.15 to 2.0 mL/min.

5. The method of claim 1 wherein in step (3), the enzyme comprises: porcine pancreatic enzyme, Candida rugosa lipase, Candida lipolytica lipase, Candida antarctica lipase B, Pseudomonas cepacia lipase, Rhizopus oryzae, phospholipase, pepsin, trypsin, cathepsin papain or pectinase.

6. A method for applying the enzyme reactor of claim 1 in enzymatic reaction, comprising: placing the enzyme reactor for the monolithic cellulose acetate column reactor in a column oven to maintain a certain temperature, wherein reaction solution is subjected to an enzymatic reaction through a cellulose acetate monolithic column reactor at a certain flow rate under a driving flow of a peristaltic pump.

7. The method of claim 6 wherein a reaction temperature is at a range of 300 C to 70 °C, and a flow rate is at a range of 0.10 to 10.0 mL/min depending on a diameter of the monolithic column.

8. The method of claim 6 wherein the enzymatic reaction comprises: esterification, transesterification, ester hydrolysis, protein hydrolysis, phospholipids or pectin hydrolysis reaction.