CN102660533A - Preparation method of graphene oxide immobilized alkali protease - Google Patents
Preparation method of graphene oxide immobilized alkali protease Download PDFInfo
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- CN102660533A CN102660533A CN2012101343349A CN201210134334A CN102660533A CN 102660533 A CN102660533 A CN 102660533A CN 2012101343349 A CN2012101343349 A CN 2012101343349A CN 201210134334 A CN201210134334 A CN 201210134334A CN 102660533 A CN102660533 A CN 102660533A
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Abstract
The invention relates to a preparation method of a graphene oxide immobilized alkali protease. The preparation method comprises the following steps of: (1), using a graphene oxide as a raw material, dissolving the graphene oxide by using a phosphate buffer solution, and then, carrying out magnetic agitation and ultrasonic treatment, and standing overnight to obtain a graphene oxide solution; (2), adding glutaraldehyde in the graphene oxide solution, crosslinking for 1-12 hours in conditions that a pH value is 7.0-8.0 and a temperature is 4-37 DEG C, centrifuging, and then adding 100-1000 mul of alkali protease, adsorbing for 0.5-8 hours at 4-37 DEG C, centrifuging, and finally, washing by the phosphate buffer solution to prepare the graphene oxide immobilized alkali protease. The preparation method is simple in process and low in cost; the shape of the prepared graphene oxide immobilized protease is uniform; a surface has abundant active groups; the dispersibility is favorable; the surface area is large; the recovery rate of enzyme activity averagely reaches 70.1%; and compared with a solution enzyme, the affinity of an immobilized enzyme towards a substrate is improved.
Description
Technical field
The invention belongs to ankyrin enzyme field, particularly a kind of preparation method of graphene oxide immobilization Sumizyme MP.
Background technology
Graphene (Graphene) is the carbon atomic layer of monatomic thickness, just found in recent years two-dimentional carbon atom crystal.It is considered to the basic structural unit of soccerballene, carbon nanotube (CNT), graphite, and is special because of its mechanics, quantum and electrical properties, paid attention to by physics and material educational circles.(Graphite Oxide is to add the compound that water decomposition obtains, the same compound between graphite layers that can classify as covalent linkage with fluorographite after the powerful oxidation of the high graphite of crystallinity GO) to graphene oxide.It is generally acknowledged that the graphene oxide two-dimensional layered structure that is as the criterion, interlayer contain a large amount of hydroxyls and the acid reactive group of carboxyl.Its loading capacity is big, and long-chain fat hydrocarbon, transition metal ion, hydrophilic molecule and polymkeric substance etc. are easy to form intercalation compound through between effect interposed layer such as interlayer hydrogen bond, ionic linkage and covalent linkage.Therefore the character such as structure and reactive group that can use graphene oxide fully are used for the immobilization technology of enzyme.Though graphene oxide has good dispersiveness in water; And the surface possesses a large amount of reactive groups; Think in theory and can modify and directly carry out immobilized material; But experiment showed, with it and carry out direct immobilized poor, still will add linking agent (like LUTARALDEHYDE) and modify.
Proteolytic enzyme is defined as the enzyme of peptide chain hydrolysis in the catalytic proteins, extensively is present in pluck, plant stem-leaf, fruit and the mikrobe, and microbial protease mainly by mould, bacterium, is secondly produced by yeast, actinomycetes.Proteolytic enzyme has strict selectivity to the reaction substrate that is acted on, and a kind of proteolytic enzyme only can act on peptide bond certain in the protein molecule, like the formed peptide bond of trypsinase catalytic hydrolysis basic aminoacids.At present proteolytic enzyme has been widely used in leather, fur, silk, medicine, food, the aspect such as has brewageed, and like the depilation of leather industry with softeningly utilize proteolytic enzyme in a large number, has both saved time, and improves labour health condition again; Can do medicinal clinically; As use the pepsin treatment maldigestion; With acidic protein enzyme treatment bronchitis, with fear property proteolytic enzyme treatment vasculitis and with trypsinase, Quimotrase to the treatment of serous coat adhesion between the purification of the suppurative wound of surgery and thoracic cavity etc.In order to improve the service efficiency of proteolytic enzyme; Reduce production costs; The researchist has carried out a lot of research to the immobilization of proteolytic enzyme both at home and abroad; Set up many process for fixation, as with chitin or chitosan, FM, modified high-molecular film etc. as fixation support, adopt absorption method or crosslinking immobilization proteinase.And with graphene oxide as fixed enzyme vector, also see relevant report with crosslinked-absorption method ankyrin enzyme.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of graphene oxide immobilization Sumizyme MP, and this method technology is simple, and cost is low; The graphene oxide immobilization proteinase shape homogeneous that makes, the surface possesses a large amount of reactive groups, good dispersibility, surface-area is big.
The preparation method of a kind of graphene oxide immobilization Sumizyme MP of the present invention comprises:
(1) be raw material with the graphite oxide, with phosphate buffered saline buffer it dissolved that magnetic agitation is ultrasonic then, hold over night obtains graphene oxide solution;
(2) in above-mentioned graphene oxide solution, add LUTARALDEHYDE, the pH value is 7.0-8.0, crosslinked 1-12h under 4-37 ℃; Centrifugal; Add 100-1000 μ l Sumizyme MP afterwards, absorption 0.5-8h is centrifugal under 4-37 ℃; After the phosphate buffered saline buffer flushing makes graphene oxide immobilization Sumizyme MP; Wherein, the add-on of LUTARALDEHYDE is the 0.1-4% of solution total volume.
The graphite oxide in the said step (1) and the mass volume ratio of phosphate buffered saline buffer are 1g: 1000ml.
The time of magnetic agitation is 0.5-3 hour in the said step (1), and the ultransonic time is 0.5-3 hour.
The pH value of phosphate buffered saline buffer is 7.0-8.0 in said step (1) and (2).
Centrifugal speed in the said step (2) is 15000rpm/min, and centrifugation time is 10 minutes, and centrifugal three times, each centrifugal finishing adds the phosphate buffered saline buffer flushing.
The object of the invention be to make full use of graphene oxide character, widen its range of application, excavate its application potential, improve as enzyme immobilization carrier proteolytic enzyme service efficiency, reduce immobilization proteinase production cost, improve the using value of proteolytic enzyme and a kind of novel immobilization proteinase, i.e. graphene oxide immobilization proteinase be provided.
Fig. 1 has shown the particle size range of graphene oxide immobilization proteinase;
Fig. 2 shows: in the preparation process, if constant other condition, when the enzyme concentration of every milligram of graphene oxide was 500 μ l, the enzyme activity of immobilized enzyme reached 86U/mg, and the enzymatic activity recovery of immobilized enzyme reaches 68.5% for the highest;
Fig. 3 can find out: when adsorption time reaches 1 hour, the enzyme activity of immobilized enzyme and enzymatic activity recovery all reach peak, are respectively 115.4U/mg and 92%;
Fig. 4 shows: when temperature was 25 ℃, the enzyme activity of immobilized enzyme and enzymatic activity recovery were respectively 61.8U/mg and 49.2%, all reached peak;
Fig. 5 shows: when the ultimate density of glutaraldehyde solution was 1%, the enzyme activity of immobilized enzyme and enzymatic activity recovery all reached peak, were respectively 67.02U/mg and 53.4%;
Fig. 6 shows: when the crosslinking time of LUTARALDEHYDE is 4 hours, the enzyme activity of immobilized enzyme and enzymatic activity recovery all reach peak, are respectively 60.0U/mg and 47.8%; In sum, preparation graphene oxide immobilization proteinase under above optimal fixation condition, its enzyme activity on average reaches 70.4U/mg, and enzymatic activity recovery on average reaches 70.1%;
Fig. 7 shows: the apparent Michaelis-Menton constant of graphene oxide immobilization proteinase is K
m=8.57mg/ml, V
Max=6.10 μ g/min;
Fig. 8 shows: the ph optimum of graphene oxide immobilization proteinase is 8;
Fig. 9 shows: the graphene oxide immobilization proteinase is reused 3 recovery more than 40%;
Figure 10 shows: the thermostability of graphene oxide immobilization proteinase will be got well than resolvase.
Beneficial effect
(1) the present invention utilizes the superperformance of this fixed enzyme vector of graphene oxide; Particularly the shape homogeneous, in water, have good dispersiveness, surface and possess that a large amount of reactive groups, surface-area are big, good adsorption performance etc.; With crosslinked-absorption method immobilization proteinase, thereby the good premise condition is provided for inventing the graphene oxide immobilization proteinase;
(2) technology of the present invention is simple, and cost is low; The graphene oxide immobilization proteinase shape homogeneous that makes, the surface possesses a large amount of reactive groups, good dispersibility, surface-area is big; Enzymatic activity recovery on average reaches 70.1%; Compare with the solution enzyme, immobilized enzyme improves the avidity of substrate.
Description of drawings
Fig. 1 is electromicroscopic photograph (a, the 5nm of graphene oxide immobilization proteinase under the different size; B, 50nm);
Fig. 2 is an enzyme concentration to the fixing Sumizyme MP influential effect of graphene oxide;
Fig. 3 is an adsorption time to the fixing Sumizyme MP influential effect of graphene oxide;
Fig. 4 is a temperature to the fixing Sumizyme MP influential effect of graphene oxide;
Fig. 5 is a glutaraldehyde concentration to the fixing Sumizyme MP influential effect of graphene oxide;
Fig. 6 is a crosslinking time to the fixing Sumizyme MP influential effect of graphene oxide;
Fig. 7 is the fixing Sumizyme MP double reciprocal curve of graphene oxide;
Fig. 8 is the pH stability of immobilization Sumizyme MP of the present invention;
Fig. 9 is the repetition practicality of immobilization Sumizyme MP of the present invention;
Figure 10 is the thermostability of immobilization Sumizyme MP of the present invention.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
With the graphite oxide is raw material, and use phosphate buffered saline buffer to be 1g by mass volume ratio: 1000ml dissolves it, and magnetic agitation is 0.5 hour then, and ultrasonic 3 hours, hold over night until not stratified, obtained graphene oxide solution;
Liquid-transfering gun is accurately measured 5ml 1mg/ml GO solution, adds 400 μ l LUTARALDEHYDEs (final concentration is 1%), 25 ℃ of following crosslinked 12h of temperature of reaction; Centrifugal (15000rpm/min, 10min), pH8 phosphate buffered saline buffer flushing three times; Add 100 μ l 1mg/ml proteolytic enzyme; Uniform temp is absorption 4h down, and is centrifugal, the damping fluid flushing.The residual collection at night of supernatant (adopting the Coomassie brilliant blue method to survey protein concentration).Preserve in 4 ℃ of refrigerator damping fluids of immobilized material.Enzyme activity and the enzymatic activity recovery of measuring immobilized enzyme at last are respectively 67.02U/mg and 53.4%.
With the graphite oxide is raw material, and use phosphate buffered saline buffer to be 1g by mass volume ratio: 1000ml dissolves it, and magnetic agitation is 3 hours then, and ultrasonic 0.5 hour, hold over night until not stratified, obtained graphene oxide solution;
Liquid-transfering gun is accurately measured 5ml 1mg/ml GO solution, adds 400 μ l LUTARALDEHYDEs (final concentration is 1%), 37 ℃ of following crosslinked 12h of temperature of reaction; Centrifugal (15000rpm/min, 10min), pH8 phosphate buffered saline buffer flushing three times; Add 100 μ l 1mg/ml proteolytic enzyme; Uniform temp is absorption 8h down, and is centrifugal, the damping fluid flushing.The residual collection at night of supernatant (adopting the Coomassie brilliant blue method to survey protein concentration).Preserve in 4 ℃ of refrigerator damping fluids of immobilized material.Enzyme activity and the enzymatic activity recovery of measuring immobilized enzyme at last are respectively 60.0U/mg and 47.8%.
Embodiment 3
With the graphite oxide is raw material, and use phosphate buffered saline buffer to be 1g by mass volume ratio: 1000ml dissolves it, and magnetic agitation is 2 hours then, and ultrasonic 2 hours, hold over night until not stratified, obtained graphene oxide solution;
Liquid-transfering gun is accurately measured 5ml 1mg/ml GO solution, adds 400 μ l LUTARALDEHYDEs (final concentration is 1%), 11 ℃ of following crosslinked 1h of temperature of reaction; Centrifugal (15000rpm/min, 10min), pH8 phosphate buffered saline buffer flushing three times; Add 500 μ l 1mg/ml proteolytic enzyme; Uniform temp is absorption 0.5h down, and is centrifugal, the damping fluid flushing.The residual collection at night of supernatant (adopting the Coomassie brilliant blue method to survey protein concentration).Preserve in 4 ℃ of refrigerator damping fluids of immobilized material.Enzyme activity and the enzymatic activity recovery of measuring immobilized enzyme at last are respectively 86U/mg and 68.5%.
Embodiment 4
With the graphite oxide is raw material, and use phosphate buffered saline buffer to be 1g by mass volume ratio: 1000ml dissolves it, and magnetic agitation is 1 hour then, and ultrasonic 3 hours, hold over night until not stratified, obtained graphene oxide solution;
Liquid-transfering gun is accurately measured 5ml 1mg/ml GO solution, adds 400 μ l LUTARALDEHYDEs (final concentration is 1%), 25 ℃ of following crosslinked 4h of temperature of reaction; Centrifugal (15000rpm/min, 10min), pH8 phosphate buffered saline buffer flushing three times; Add 500 μ l 1mg/ml proteolytic enzyme; Uniform temp is absorption 1h down, and is centrifugal, the damping fluid flushing.The residual collection at night of supernatant (adopting the Coomassie brilliant blue method to survey protein concentration).Preserve in 4 ℃ of refrigerator damping fluids of immobilized material.Enzyme activity and the enzymatic activity recovery of measuring immobilized enzyme at last are respectively 115.4U/mg and 92%.
Claims (5)
1. the preparation method of a graphene oxide immobilization Sumizyme MP comprises:
(1) be raw material with the graphite oxide, with phosphate buffered saline buffer it dissolved that magnetic agitation is ultrasonic then, hold over night obtains graphene oxide solution;
(2) in above-mentioned graphene oxide solution, add LUTARALDEHYDE, the pH value is 7.0-8.0, crosslinked 1-12h under 4-37 ℃; Centrifugal; Add 100-1000 μ l Sumizyme MP afterwards, absorption 0.5-8h is centrifugal under 4-37 ℃; After the phosphate buffered saline buffer flushing makes graphene oxide immobilization Sumizyme MP; Wherein, the add-on of LUTARALDEHYDE is the 0.1-4% of solution total volume.
2. the preparation method of a kind of graphene oxide immobilization Sumizyme MP according to claim 1 is characterized in that: the graphite oxide in the said step (1) and the mass volume ratio of phosphate buffered saline buffer are 1g: 1000ml.
3. the preparation method of a kind of graphene oxide immobilization Sumizyme MP according to claim 1 is characterized in that: the time of magnetic agitation is 0.5-3 hour in the said step (1), and the ultransonic time is 0.5-3 hour.
4. the preparation method of a kind of graphene oxide immobilization Sumizyme MP according to claim 1 is characterized in that: the pH value of phosphate buffered saline buffer is 7.0-8.0 in said step (1) and (2).
5. the preparation method of a kind of graphene oxide immobilization Sumizyme MP according to claim 1; It is characterized in that: the centrifugal speed in the said step (2) is 15000rpm/min; Centrifugation time is 10 minutes, and centrifugal three times, each centrifugal finishing adds the phosphate buffered saline buffer flushing.
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Cited By (9)
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| CN102876656A (en) * | 2012-10-16 | 2013-01-16 | 河北工业大学 | Process method of oxidized graphene directional immobilization glucose oxidase |
| CN103013824A (en) * | 2012-12-21 | 2013-04-03 | 复旦大学 | Proteolysis micro-fluidic chip based on silica gel oxidized graphene composite membrane and fabrication method of proteolysis micro-fluidic chip |
| CN104237351A (en) * | 2014-09-12 | 2014-12-24 | 西北师范大学 | Preparation method and application of glucose sensor |
| CN104650240A (en) * | 2015-01-26 | 2015-05-27 | 江苏大学 | Preparation method and application of functionalized graphene oxide carrier |
| CN106582810A (en) * | 2016-11-28 | 2017-04-26 | 江南大学 | Preparation method of graphene immobilized enzyme catalyst |
| CN106755150A (en) * | 2016-12-30 | 2017-05-31 | 天津百利食品有限公司 | A kind of preparation method of diglyceride |
| CN108315318A (en) * | 2018-04-24 | 2018-07-24 | 中国船舶重工集团公司第七二五研究所 | A kind of process for fixation of antifouling enzyme in surface of graphene oxide |
| CN109266641A (en) * | 2018-09-27 | 2019-01-25 | 福建海峡石墨烯产业技术研究院有限公司 | A kind of method and detecting electrode that enzyme is fixed on graphene based on glutaraldehyde |
| CN112480458A (en) * | 2020-11-05 | 2021-03-12 | 武汉轻工大学 | High-enzyme-activity modified membrane and preparation method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876656A (en) * | 2012-10-16 | 2013-01-16 | 河北工业大学 | Process method of oxidized graphene directional immobilization glucose oxidase |
| CN103013824A (en) * | 2012-12-21 | 2013-04-03 | 复旦大学 | Proteolysis micro-fluidic chip based on silica gel oxidized graphene composite membrane and fabrication method of proteolysis micro-fluidic chip |
| CN104237351A (en) * | 2014-09-12 | 2014-12-24 | 西北师范大学 | Preparation method and application of glucose sensor |
| CN104650240A (en) * | 2015-01-26 | 2015-05-27 | 江苏大学 | Preparation method and application of functionalized graphene oxide carrier |
| CN106582810A (en) * | 2016-11-28 | 2017-04-26 | 江南大学 | Preparation method of graphene immobilized enzyme catalyst |
| CN106755150A (en) * | 2016-12-30 | 2017-05-31 | 天津百利食品有限公司 | A kind of preparation method of diglyceride |
| CN108315318A (en) * | 2018-04-24 | 2018-07-24 | 中国船舶重工集团公司第七二五研究所 | A kind of process for fixation of antifouling enzyme in surface of graphene oxide |
| CN109266641A (en) * | 2018-09-27 | 2019-01-25 | 福建海峡石墨烯产业技术研究院有限公司 | A kind of method and detecting electrode that enzyme is fixed on graphene based on glutaraldehyde |
| CN112480458A (en) * | 2020-11-05 | 2021-03-12 | 武汉轻工大学 | High-enzyme-activity modified membrane and preparation method thereof |
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Inventor after: Li Dengxin Inventor after: Duan Yuandong Inventor after: Lv Wei Inventor after: Lu Junhao Inventor after: Li Chunjiang Inventor after: Ou Yanggan Inventor after: Wang Jin Inventor after: Cai Wenbei Inventor after: Ji Hao Inventor after: Gao Fupeng Inventor after: Hou Xiaopeng Inventor after: Su Ruijing Inventor after: Shi Zhentao Inventor after: Chen Yinchuan Inventor after: Huang Na Inventor after: Zhang Wenfeng Inventor after: Shi Penghui Inventor after: Bi Defu Inventor after: Tan Dongdong Inventor after: Li Yanhong Inventor after: Guo Guanglin Inventor after: Zhu Shaobo Inventor after: Yi Yu Inventor after: Zhu Zhenxin Inventor after: Chen Minghua Inventor after: Guo Miao Inventor after: Wang Qian Inventor after: Yin Jiayin Inventor after: Zhou Wanyuan Inventor after: Shao Xiantao Inventor after: Sun Xiuzhi Inventor after: Li Jiebing Inventor before: Li Dengxin Inventor before: Shao Xiantao Inventor before: Li Jiebing Inventor before: Sun Xiuzhi Inventor before: Zhu Zhenxin Inventor before: Chen Minghua Inventor before: Guo Miao Inventor before: Su Ruijing Inventor before: Zhang Wenfeng Inventor before: Shi Penghui Inventor before: Bi Defu Inventor before: Tan Dongdong Inventor before: Li Yanhong Inventor before: Guo Guanglin Inventor before: Zhu Shaobo |
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Free format text: CORRECT: INVENTOR; FROM: LI DENGXIN SU RUIJING ZHANG WENFENG SHI PENGHUI BI DEFU TAN DONGDONG LI YANHONG GUO GUANGLIN ZHU SHAOBO SHAO XIANTAO LI JIEBING SUN XIUZHI ZHU ZHENXIN CHEN MINGHUA GUO MIAO TO: LI DENGXIN SU RUIJING YI YU WANG QIAN YIN JIAYIN ZHOU WANYUAN SHAO XIANTAO SUN XIUZHI LI JIEBING DUAN YUANDONG LV WEI LU JUNHAO LI CHUNJIANG OU YANGGAN WANG JIN CAI WENBEI JI HAO GAO FUPENG HOU XIAOPENG SHI ZHENTAO CHEN YINCHUAN HUANG NA ZHANG WENFENG SHI PENGHUI BI DEFU TAN DONGDONG LI YANHONG GUO GUANGLIN ZHU SHAOBO ZHU ZHENXIN CHEN MINGHUA GUO MIAO |
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Application publication date: 20120912 |