CN101781640A - Enzyme preparation using chlorphyllase as tobacco processing accessories, preparation method and application method - Google Patents
Enzyme preparation using chlorphyllase as tobacco processing accessories, preparation method and application method Download PDFInfo
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Abstract
The invention provides an enzyme preparation using chlorphyllase as tobacco processing accessories, a preparation method and an application method, which belong to an enzyme preparation added before the tobacco redrying. The invention uses treated later stage vertex-weighted waste materials in the tobacco planting process as extracting raw materials of the chlorphyllase, 250 U/mL to 450 U/mL chlorphyllase raw liquid or concentrated liquid is prepared in multiple batches, the activity of prolease is inhabited by a buffer solution with PMSF in the extraction process, and metal ions are removed by an EDTA complexing agent. The invention also provides a convenient chlorphyllase enzyme activity measuring method. Diluent of the invention is used for replacing secondary leaf wetting water during sheet tobacco redrying, chlorophyll remained in cured tobacco can be converted, and the tobacco fragrance and the quality can be improved.
Description
Technical field
The invention belongs to multienzyme compound formulation and the preparation and the using method of the raising tobacco grade of adding before the tobacco redrying.
Background technology
Chlorphyllase (E.C.3.1.1.14) is a plant physiology important physical sexual function enzyme, and as most important photosynthetic pigments on the earth, plant physiology phenomenons such as chlorophyll degradation and farm crop aging, fruit maturation and trees fallen leaves are closely related.After reporting chlorphyllase in 1912, in higher plant blade and algae, concern between chlorphyllase and green plain enzyme and the chlorophyll degradation and extensively studied.Can get higher chlorphyllase activity by 30%, 35% acetone and 60%, 75% ammonium sulfate precipitation component; After processing such as 40%～60% ammonium sulfate precipitation, dialysis, dextran G-100 gel-filtration, can get the chlorphyllase of 86 times of purifying.The chlorphyllase of having determined in recent years is a key enzyme in the chlorophyll degradation, the chlorphyllase gene that clones has 4 kinds and all successful at expression in escherichia coli, is respectively multitude's chlorphyllase CaCLH, citrus chlorphyllase CHLASE1 and arabidopsis ' chlorophyll enzyme AtCLH1, AtCLH2.Chlorphyllase has distribution widely in plant and algae, molecular weight does not wait from 27～65, and this species diversity derives from the existence of floristic difference or isozyme.Have at least a kind of chlorphyllase to be present in the plastid.CaCLH and AtCLH1 are present in organoid but not in the chloroplast(id).Plant senescence promotor methyl jasmonic (methyl jasmonate) can increase the concentration of AtCLH1 mRNA, and respective egg white matter content and chlorphyllase vigor [Tang Lei.The new development of chlorphyllase research.The chemistry of life, 2002,22 (4): 373～374].As the enzyme that plant physiology relates to, close day strong a grade and studied the influence of Permanone cucumber photosynthetic pigments and relevant enzymic activity.Handle cucumber seedling 48h with Permanone (PER), the contrast of chlorphyllase specific activity raises 4.72%～22.61%, the chlorophyll total amount is compared to shine and is descended 5.55%～46.43%, and the carotenoid content that PER handles behind the 6d descends 36.47%[pass day respectively by force, etc.Permanone is to the influence of cucumber photosynthetic pigments and relevant enzymic activity.Agricultural University Of South China's journal 2001,22 (2): 52～55].In many research of tobacco quality, Gao Chunliang etc. think that the degraded of plamochromic pigment and polyphenol obviously improves the tobacco leaf visual appearance, this be since polyphenol as a kind of dark-brown pigment, can degrade under the effect of polyphenoloxidase generates a kind of iron-protein-chlorogenic acid violaguercitrin mixture, and pale yellow, true yellow before the degraded of polyphenol impels the leaf look by ageing change to golden yellow, deep yellow and pale brown look; The degraded of plamochromic pigment causes tobacco leaf color to shoal, and color and luster is [atmospheric moisture is to the Chinese tobacco science that influences of Shandong and Guizhou smoked sheet quality, 2008,29 (5): 32～36 between aging period such as Gao Chunliang] evenly.
On the mensuration of chlorophyll content and enzyme, because the absorption spectrum of chlorophyll in different solvents is variant, extract pigment with different solvents, it is also different to measure calculation formula.The wavelength of chlorophyll a, b maximum absorption band in 95% ethanol is respectively 665nm and Lee's 649nm[symphysis, etc.Plant physiology and biochemistry experimental principle and technology.Beijing: Higher Education Publishing House, 2000, p130～137].Chlorophyll content of plant is measured and is broadly divided into spectrophotometer and live body chlorophyll instrument two big classes, the first kind, and spectrophotometer method comprises: 1. be widely used in the world, as the acetone method of Arnon method.Have that extracted amount is big, chlorophyll is subject to photoxidation and destroys troubles such as a large amount of samples are not suitable for; 2. the more ether method (also being the Smith-Benitoz method) of trouble that on the basis of acetone method, forms; 3. dimethyl sulfoxide method and 4. dimethyl formamide method; Also has 5. dehydrated alcohol method, 6. the acetone ethanol hybrid system and 7. acetone dehydrated alcohol balanced mix form extracting solution, directly soak the chlorophyllous acetone ethanol mixed solution method of extracting, this method 1985 is proposed by Zhang Xianzheng, chlorophyll extracting solution than acetone ethanol water mixed liquid method, dimethyl sulfoxide method is stable, be not subject to photoxidation and destroy, the formula of pressing acetone method calculates chlorophyll content.Second class is mainly used in the 8. transmission-type live body chlorophyll instrument method that has of live body chlorophyll measuring, has one to absorb the peak at the light of 670～675nm wavelength, and its absorption value becomes positive correlation with chlorophyll content.Because of the different live body blade of plant specific absorbance difference, need calibrated instrument just can carry out the nondestructive rapid determination to the blade of growth conditions; 9. reflection-type live body chlorophyll instrument, be develop after 80 generations mainly with the ratio (ρ 801/ ρ 550, x or y) of 800nm and 550nm two wavelength light reflectivity chlorophyll content (μ g/cm, y or x) to unit leaf area, research y=ax+b and dependency [Su Zhengshu, etc.The method of several mensuration chlorophyll content of plant relatively.Plant Physiology Communications, 1989, (5): 77～78].
For many with spectrophotometric acetone ethanol hybrid system in the mensuration of chlorophyll content of plant, different sample acetone and ethanol ratio of mixture are different.To chlorphyllase is sample is ground after, with acetone repeatedly wash-out remove the determination of activity that chlorophyllous method is carried out chlorphyllase as far as possible: after getting a certain amount of plant tissue sample and grinding, with acetone repeatedly wash-out remove chlorophyll, drying at room temperature is pulverized.Add 2mL substrate and 1mL phosphoric acid buffer (pH8.0) insulation 12h under 25 ℃, add the 3mL sherwood oil immediately, fully the centrifugal 15min of vibration back 3500r/min.The volume of metering supernatant liquor, and get supernatant liquid and under 667nm, measure absorbancy.Whole mensuration process carry out in the dark [Yu Zhifang, etc.The variation of chlorophyll, Mierocrystalline cellulose and relevant enzyme in the fresh-cut reed wormwood artemisia storage.China's wild plant resource, 2003,22 (4): 61～64; Gan Zhijun, etc.Wheat chlorphyllase biochemical kinetics characteristic research.The northwest Botany Gazette, 2003,23 (5): 750～754].
The difference of the survey live body system of chlorphyllase and other enzyme is that chlorphyllase gets final product show activity under the room temperature in organic solvent or washing agent, and is being in the reaction system of microenvironment with water, the temperature of its show activity higher (65 ℃～75 ℃).Chlorphyllase is the catalytic activity height in slight alkalinity (7.5～8.0) environment, and the optimum pH of olive (Oleae Uropaea L.) chlorphyllase is 8.5, and citrus leaves chlorphyllase optimum pH is 7.8.To studies show that of the Km of chlorphyllase, Vmax value, the Km value of the chlorphyllase in different plant species source is 3.1～278 μ mol/L, with the chlorophyll b is substrate, Km value when the Km value of chlorphyllase is substrate than with the chlorophyll a is little, this shows that chlorphyllase is higher to the affinity of chlorophyll b, and when being substrate with the pheophytin a, the speed ratio of its reaction of chlorphyllase catalysis with phoeophytin b be substrate fast [Gan Zhijun, etc.The progress of chlorphyllase.Life science, 2002, (1): 21～24].As the enzyme that physiology relates to, employing method for potted such as Wei Fengjie have been studied cake fertilizer pigment degraded and relevant physiological in the flue-cured tobacco blade growth course have been changed.Show that chlorophyll, chlorphyllase activity, total carotinoid whole growing content that cake fertilizer handles are all higher, cake fertilizer is handled the metabolism that promotes chlorophyll a, and the Neoxanthine prometaphase is played the promotion summation, the middle and later periods rise promote metabolism [Wei Fengjie, etc.Cake fertilizer is to plamochromic pigment degraded and the active influence of relevant enzymes in the flue-cured tobacco blade growth course.Acta Agronomica Sinica, 2006,32 (5): 766～771].
Summary of the invention
The object of the invention at first is to take the lead in proposing a kind of method of extracting chlorphyllase and turning waste into wealth, be raw material promptly with plant refuse tobacco top wooden fork, extracting chlorphyllase is used for and will residues in the plamochromic pigment of tobacco leaf, mainly be that plamochromic pigment such as chlorophyll is converted into the important as precursors thing that the tobacco grade improves and improves, positive assisted modulation effect is played in the alcoholization after the redrying of sheet cigarette.
Another purpose of the present invention is to propose a kind of comparatively fast simple chlorphyllase vigour-testing method, and with the detection method of this method as raw material sampling and leaching process and the auxiliary chlorphyllase zymin of adding of tobacco processing.
Purpose of the present invention realizes in the following manner:
(1) as the chlorphyllase preparation of the present invention of product
Said preparation has chlorphyllase, and its enzyme activity is 250～450U/mL.
The best enzyme activity of described preparation chlorphyllase is 370U/mL.
Contain PMSF protease activity inhibitor in the described preparation and remove metal ion EDTA complexing agent.
PMSF is the biochemical reagents commonly used of arrestin enzymic activity performance.
EDTA chemistry disodium ethylene diamine tetraacetate by name is the complexing of metal ion agent of using always.
(2) chlorphyllase vigour-testing method of the present invention
(1) the fresh and alive tobacco leaf sheet of adding 1.0g diameter D≤1mm in the 10mL dehydrated alcohol being added a cover vibration and soaking 60min, gets supernatant liquor 2.5mL and mixes as the chlorophyll substrate solution with 2.5mL distilled water; Or the dry product tobacco leaf powder 2.0g that stores, with 30mL 95% ethanol lixiviate 120min, gained filters deep green clear liquid 2.5mL and 2.5mL distilled water is mixed into the chlorophyll substrate solution;
(2) in the cuvette of 8mL, add 1mL enzyme liquid to be measured, add the chlorophyll substrate solution that 5.0mL extracts preparation, under spectrophotometer 720nm wavelength, read initial A=OD at once behind the mixing
720nmValue when reading the accurate timing 20min of A value, reads B=OD
720nmValue;
Definition per minute reaction system changes 0.001 OD under the 720nm wavelength
720nmValue is 1 enzyme activity unit 1U;
The sample enzyme activity passes through calculating formula: E (U)=N (A-B)/20 obtains, and wherein N is the diluted sample multiple.
The used chlorophyll substrate of described measuring method can be to get fresh tobacco leaf, completes under 105 ℃, again in 80 ℃ of oven dry down, the airtight storage of grinding powder; During use, take by weighing 2.0g in small beaker, add 30mL 95% ethanol lixiviate 120min, soaking and stirring treats that ethanol liquid is deep green, collects filtered liquid and promptly gets the chlorophyll substrate solution.
During enzyme activity determination, need sample product are diluted to the extension rate of suitable enzymatic determination, have added 0.3g Ca (NO among the every 100mL of used damping fluid
3)
2, 0.3g MgSO
4With 0.1g ZnSO
4Stable and activator detects with releasing enzyme activity as basic enzyme, and detected result is calculated as product concentrated solution enzyme and lives.
Chlorophyll substrate in the above-mentioned detection method produce with preparation be according to [Li Hesheng, etc.Plant physiology and biochemistry experimental principle and technology.Beijing: Higher Education Publishing House, 2000, p131] fresh spinach raw material be revised as fresh tobacco leaf.Detect used chlorophyll substrate for chlorphyllase in the product zymin of the present invention, ensure the quality of products for the product of producing in a long time detects stable, we produce the dry powder of some amount in advance.
(3) preparation is with the method for chlorphyllase as the auxiliary enzymes preparation of tobacco processing
(1) with the raw material of tobacco top wooden fork leaf as the extraction chlorphyllase, sampling Detection chlorphyllase enzyme activity merges the close top wooden fork leaf of enzyme activity;
(2) with pH8.0 damping fluid dissolving PMSF proteinase inhibitor, making its concentration is to add top wooden fork leaf behind the 0.01mol/L, and top wooden fork leaf is 1g: 4mL with the ratio of the damping fluid that contains PMSF, smashes homogenate, filters, and collects filtrate, leaves standstill 1h;
(3) leave standstill liquid with the centrifugal 20min of 3500r/min, get supernatant liquor, add (NH
4)
2SO
4, making the ammonium sulfate final concentration is 25%, slow stirring and dissolving is added (NH after leaving standstill 2h
4)
2SO
4Making final concentration is 80%, standing over night, and 4 ℃ of centrifugal 30min of 15000r/min get supernatant liquor again, add concentration (W/V) and be 10% (NH
4)
2SO
4Spend the night 4 ℃ of centrifugal 30min of 15000r/min again, collecting precipitation, merging;
(4) will precipitate fully dissolve with half of initial buffer liquid total amount after, leaves standstill 2h with 0.1%EDTA solution complement is long-pending to former damping fluid volume, centrifugal, detect the enzyme activity of supernatant liquor, convert and mix clear liquid, making enzyme activity is 250～450U/mL.
Add the sucrose oligopolymer of concentration 0.01% in the supernatant liquor that described step (4) obtains, supernatant liquor and sucrose oligopolymer volume ratio are 100: 1, concentrate 8～10 times through-8 ℃ of lyophilizes and must concentrate the proenzyme liquid formulation.
Described step (2) is 1g: 5mL with initial top wooden fork leaf quality with the damping fluid volume ratio that contains PMSF further for to add damping fluid in filter residue, stirs 2h, filters shingle, and filtrate and last time filtrate merging are left standstill 1h.
According to enzyme activity determination method provided by the invention, above-mentioned concentrated enzyme liquid chlorphyllase enzyme activity in concentrating stoste after recovering activity is between 250～450U/mL, and the best is 370U/mL.
(4) using method of zymin of the present invention
The chlorphyllase preparation is diluted 250 times as the leaves moisting second time water before the redrying of sheet cigarette, and its enzyme activity scope is 1～1.8U/mL.
Described using method further is that the chlorphyllase preparation is diluted the back as the leaves moisting second time water before the redrying of sheet cigarette, and its best enzyme activity is 1.46U/mL.
More than used damping fluid can select the pH8.0Britton-Robinsion damping fluid, see [Yin Yongjia for details.The university chemistry handbook.Jinan: Shandong Science Press, 1985] compound method.The method of improving and be modulated into pH8.0 with reference to [Liu Shiqing, etc.Basal enzyme technical study method is built discussion in the agro-ecology environment.Agricultural research, 2008, (10): 162～166,175] use.
The present invention has following positively effect:
1. chlorphyllase is the ubiquitous physiological enzyme of plant.Though the height that the chlorphyllase that other plant contains in the Solanaceae contains than tobacco leaf, its material quantity can not satisfy the production demand.On the other hand, in tobacco leaf planting, the ripe tobacco leaf in top can produce a large amount of depleted top wooden fork leaf after pruning binding, and stack retting rots.This part tobacco leaf comprises that two canopy leaves and top account for 40% of single plant yield, it is the raw tobacco material valuable source, but generally visual appearance is loose inadequately after baking, and blade is stiff, and variegated cigarette and dust cigarette are many, it is big that few its inner quality of oil content shows as the higher pungency of nicotine content, assorted gas is heavier, perfume quantity deficiency, not convenient usefulness in cigarette composition, even can't utilize, thereby how the part of pruning that binds throws away as waste.[permitted to have of one's own, etc.Different picking methods is to the influence of flue-cured tobacco upper leaf interior quality.Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's journal (natural science edition), 2005,33 (11): 13～17].For utilizing tobacco leaf top wooden fork leaf, the present invention takes the lead in it is extracted raw material as chlorphyllase, turns waste into wealth, and has improved the biomass utilization ratio of tobacco.
The present invention take the lead in having proposed with chlorphyllase as tobacco flue-curing after, auxiliary zymin of adding in the course of processing before redrying, further catalyzed oxidation promotes to residue in the conversions such as chlorophyll in the tobacco leaf, becomes the tobacco grade and improves and improve the reaction important as precursors thing that required fragrance note material produces.
3. extract the chlorophyll substrate and measure the chlorphyllase enzyme activity with easy method.In order to guarantee the detection data stability and the reliability of formulation products in a long time, the detection method of specific determination formulation products has been proposed.
4. the present invention is that raw material extracts the preparation chlorphyllase with handling tobacco top wooden fork, need not the technology of purifying chlorphyllase, extract enzyme and can be used as one of applicant's of the present invention " a kind of cigarette quality improves and the tobacco processing active complex enzyme preparation that improves " allotment compound enzymic preparation basis or enzyme component.
5. tobacco top wooden fork can not only prepare the chlorphyllase preparation, can be used for extracting other enzyme.As, be retained in the part of remaining 3～5 upper leafs on the plant, be as preparation carotenoid oxydase (lipoxygenase, extraction raw material LOX) (seeing applicant's " with the auxiliary enzymes preparation of lipoxygenase ") as tobacco processing.
6. the present invention is not only applicable to tobacco processing, can be used for the research and development and the application in fields such as biomass yet.
The present invention also can be used with other tobacco processing active zymin, or becomes applicant's " a kind of cigarette quality improves and the tobacco processing active complex enzyme preparation that improves " the enzyme component or the basic thing of modulation compound enzymic preparation.
Description of drawings
Fig. 1 is preparation technology's schematic flow sheet of zymin of the present invention.
Be described further below in conjunction with embodiment, but protection domain of the present invention includes but not limited to the content that embodiment is enumerated, also should comprise the interim mixing liquid or the solid preparation of enzyme related to the present invention, and interim proportioning adjustment.
Embodiment
(1) preparation chlorphyllase preparation of the present invention and mensuration chlorphyllase enzyme activity method
Clip tobacco top wooden fork leaf is collected respectively by the field piece and to be deposited sampling Detection chlorphyllase enzyme activity after shady and cool 3 days, merges the close top of enzyme activity wooden fork leaf as starting raw material; After being 0.01mol/L with pH8.0 damping fluid dissolving PMSF proteinase inhibitor concentration, it is added top wooden fork leaf, top wooden fork leaf quality is 1g: 4mL with the ratio of the damping fluid volume that contains PMSF, fully smashes homogenate, filters collection filtrate.Than 1g: 5mL, add damping fluid to filter residue with the damping fluid volume of top wooden fork leaf quality and PMSF once more, stir 2h, filter shingle, filtrate merges with filtrate last time, leave standstill 1h after, with the centrifugal 20min of 3500r/min, get supernatant liquor, add (NH
4)
2SO
4, making the ammonium sulfate final concentration is 25%, slowly stirring and dissolving leaves standstill 2h, adds (NH
4)
2SO
4Making final concentration is 80%, standing over night, and 4 ℃ of centrifugal 30min of 15000r/min get supernatant liquor, add concentration (W/V) and be 10% (NH
4)
2SO
4Spend the night 4 ℃ of centrifugal 30min of 15000r/min again, collecting precipitation, merging; With half abundant dissolution precipitation of initial buffer liquid total amount, leave standstill 2h with 0.1%EDTA solution complement is long-pending to former damping fluid volume, centrifugal, obtain removing metal ion enzyme supernatant liquor.Supernatant liquor adds the sucrose oligopolymer of concentration 0.01%, be that supernatant liquor and sucrose oligopolymer volume ratio are 100: 1, concentrate 8～10 times through-8 ℃ of lyophilizes and get concentrated enzyme preparation, detect enzyme activity, convert mutually with multiple batches of zymin and to join, adjusting the preparation enzyme activity is 250～450U/mL, and best enzyme activity is 370U/mL.
Above chlorphyllase enzyme activity determination method is:
Get fresh tobacco leaf, blue or green under 105 ℃, in 80 ℃ of oven dry down, grinding powder is airtight to be stored in the brown bottle again.Time spent takes by weighing 2.0g in small beaker, adds 30mL 95% ethanol lixiviate 120min, stirs at any time in the immersion process, and final ethanol liquid should be deep green, filters and promptly gets the chlorophyll substrate solution.
To be measured with enzyme liquid pH8.0 damping fluid, and added 0.3gCa (NO among the every 100mL of used damping fluid
3)
2, 0.3g MgSO
4With 0.1g ZnSO
4Stable and the activator as basic enzyme.With this damping fluid with diluted sample to proper concn, releasing enzyme activity and detecting simultaneously.
In the cuvette of 8mL, add 1mL enzyme liquid to be measured, add the chlorophyll substrate solution that 5.0mL extracts preparation, at 721 spectrophotometers, read initial A=OD under the 720nm wavelength at once behind the mixing
720nmValue when reading the accurate timing 20min of A value, reads B=OD
720nmValue;
Definition per minute reaction system changes 0.001OD under the 720nm wavelength
720nmValue is 1 enzyme activity unit 1U;
The sample enzyme activity passes through calculating formula: E (U)=N (A-B)/20 obtains, and wherein N is the diluted sample multiple.
The concentrated solution zymin that obtains according to above preparation method is equivalent to the chlorphyllase vigor of 370U/mL.This concentrated solution zymin also contains lipoxygenase, proteolytic enzyme, glucose oxidase and nicotine dehydrogenase simultaneously based on chlorphyllase.
(2) use of the present invention and effect
Processing mode: with the representational cloud 85 kind B2F in Hongta District, Yuxi, Yunnan (going up tangerine two) grade is process object, and simulation tobacco leaf sheet cigarette redrying alcoholization process is investigated concentrated enzyme preparation to alcoholization (spontaneous fermentation) influence in early stage under laboratory condition.The concentrated chlorphyllase preparation of 370U/mL is diluted 250 times as the leaves moisting second time water before the redrying of sheet cigarette with tap water, and the best enzyme activity of chlorphyllase is 1.46U/mL.Contrast uses the tap water of same amount as leaves moisting water.After playing leaf, redrying, each packing 500g tobacco leaf is handled and is parallelly repeated to be no less than 3 times, and natural alcoholization is 60 days under the room temperature of relative humidity 65%, makes the pipe tobacco mixing respectively, sampling analysis be rolled into cigarette and smoke panel test.Each handles the tobacco leaf routine analysis:
Through Yunnan tobacco scientific research " tobacco causes fragrant analysis of components table " result to sample: with to compare in the same old way, quantitatively detecting in the treatment samples has increased by 29 kinds of compositions to non-existent material in the same old way; Handling in the sample and compare according to having reduced by 5 kinds of compositions, is respectively 3,8,8-trimethylammonium naphthane, Cis-3,5-dimethylphenylcarbinol, vinylbenzene, two ring [7.1.0] diine in the last of the ten Heavenly stems, aromadendrene and valencenes 2.Treatment samples and the composition that contrast contains jointly except that nicotine descends significantly, all have the increase of different amplitudes, and particularly neophytadiene increases more than 2 times.Isolating protein does not have outside the considerable change in the routine analysis, and the amount of other composition all has decline in various degree.In specialty was smoked panel test, blue foreign smell and pungency had obtained control preferably compared with the control.
Claims (10)
1. process auxiliary zymin with chlorphyllase as tobacco for one kind, it is characterized in that said preparation has chlorphyllase, its enzyme activity is 250～450U/mL.
2. according to claim 1ly process auxiliary zymin with chlorphyllase as tobacco, the best enzyme activity that it is characterized in that the said preparation chlorphyllase is 370U/mL.
3. describedly process auxiliary zymin as tobacco according to claim 1,2, it is characterized in that containing in the said preparation PMSF protease activity inhibitor and remove metal ion EDTA complexing agent with chlorphyllase.
4. the measuring method of a chlorphyllase enzyme activity is characterized in that:
(1) the fresh and alive tobacco leaf sheet of adding 1.0g diameter D≤1mm in the 10mL dehydrated alcohol being added a cover vibration and soaking 60min, gets supernatant liquor 2.5mL and mixes as the chlorophyll substrate solution with 2.5mL distilled water; Or will prepare the 2.0g dry product tobacco leaf powder of storage in advance, clear filtrate 2.5mL is got in usefulness 30mL 95% ethanol lixiviate, filtration and distilled water 2.5mL is mixed into the chlorophyll substrate solution;
(2) in the cuvette of 8mL, add 1mL enzyme liquid to be measured, add the chlorophyll substrate solution that 5.0mL extracts preparation, under spectrophotometer 720nm wavelength, read initial A=OD at once behind the mixing
720nmValue when reading the accurate timing 20min of A value, reads B=OD
720nmValue;
Definition per minute reaction system changes 0.001 OD under the 720nm wavelength
720nmValue is 1 enzyme activity unit 1U;
The sample enzyme activity passes through calculating formula: E (U)=N (A-B)/20 obtains, and wherein N is the diluted sample multiple.
5. the measuring method of chlorphyllase enzyme activity according to claim 4 is characterized in that the used chlorophyll substrate of this measuring method is to get fresh tobacco leaf sheet, completes under 105 ℃, again in 80 ℃ of oven dry down, the airtight storage of grinding powder; During use, take by weighing 2.0g in small beaker, add 30mL 95% ethanol lixiviate 120min, soaking and stirring treats that ethanol liquid is deep green, collects filtered liquid and gets the chlorophyll substrate solution.
6. one kind prepares as claim 1～3 is described and processes the method for auxiliary zymin with chlorphyllase as tobacco, it is characterized in that:
(1) with the raw material of tobacco top wooden fork leaf as the extraction chlorphyllase, sampling Detection chlorphyllase enzyme activity merges the close top wooden fork leaf of enzyme activity;
(2) with pH8.0 damping fluid dissolving PMSF proteinase inhibitor, making its concentration is to add top wooden fork leaf behind the 0.01mol/L, and top wooden fork leaf is 1g: 4mL with the ratio of the damping fluid that contains PMSF, smashes homogenate, filters, and collects filtrate, leaves standstill 1h;
(3) leave standstill liquid with the centrifugal 20min of 3500r/min, get supernatant liquor, add (NH
4)
2SO
4, making the ammonium sulfate final concentration is 25%, slow stirring and dissolving is added (NH after leaving standstill 2h
4)
2SO
4Making final concentration is 80%, spends the night, and at 4 ℃ of centrifugal 30min of 15000r/min, gets supernatant liquor, adds concentration (W/V) and be 10% (NH
4)
2SO
4Spend the night, in 4 ℃ of centrifugal 30min of 15000r/min, collecting precipitation, merging;
(4) will precipitate fully dissolve with half of initial buffer liquid total amount after, leaves standstill 2h with 0.1%EDTA solution complement is long-pending to former damping fluid volume, centrifugal, detect the enzyme activity of supernatant liquor, convert and mix clear liquid, making enzyme activity is 250～450U/mL.
7. the method for preparing zymin according to claim 6, it is characterized in that in the supernatant liquor that step (4) obtains, adding the sucrose oligopolymer of concentration 0.01%, supernatant liquor and sucrose oligopolymer volume ratio are 100: 1, concentrate 8～10 times through-8 ℃ of lyophilizes and get concentrated enzyme preparation.
8. the method for preparing zymin according to claim 6 is characterized in that step (2) further adds damping fluid in filter residue, and top wooden fork leaf quality is 1g: 5mL with the damping fluid volume ratio that contains PMSF, stir 2h, filter shingle, filtrate and last time filtrate merging are left standstill 1h.
9. use as the described chlorphyllase of claim 1～3 and process the method for auxiliary zymin as tobacco, it is characterized in that diluting 250 times in chlorphyllase preparation as the leaves moisting second time water before the redrying of sheet cigarette, its enzyme activity scope is 1～1.8U/mL.
10. the method for use zymin according to claim 9 is characterized in that diluting the second time leaves moisting water of chlorphyllase preparation before as the redrying of sheet cigarette, and its best enzyme activity is 1.46U/mL.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115736A (en) * | 2010-12-07 | 2011-07-06 | 江南大学 | Purification method of plant chlorophyllase |
| CN105647889A (en) * | 2016-02-25 | 2016-06-08 | 云南万芳生物技术有限公司 | Auxiliary enzyme preparation prepared from commercially available acidic xylanase and used for tobacco processing and application |
| CN106318895A (en) * | 2016-09-30 | 2017-01-11 | 河南中烟工业有限责任公司 | Method for degrading chlorophyll in tobacco leaves |
| CN114027547A (en) * | 2021-11-05 | 2022-02-11 | 上海烟草集团有限责任公司 | Method for solving precipitation generated in reconstituted tobacco coating liquid preparation process |
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2010
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Cited By (6)
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| CN102115736A (en) * | 2010-12-07 | 2011-07-06 | 江南大学 | Purification method of plant chlorophyllase |
| CN102115736B (en) * | 2010-12-07 | 2012-12-05 | 江南大学 | Purification method of plant chlorophyllase |
| CN105647889A (en) * | 2016-02-25 | 2016-06-08 | 云南万芳生物技术有限公司 | Auxiliary enzyme preparation prepared from commercially available acidic xylanase and used for tobacco processing and application |
| CN106318895A (en) * | 2016-09-30 | 2017-01-11 | 河南中烟工业有限责任公司 | Method for degrading chlorophyll in tobacco leaves |
| CN114027547A (en) * | 2021-11-05 | 2022-02-11 | 上海烟草集团有限责任公司 | Method for solving precipitation generated in reconstituted tobacco coating liquid preparation process |
| CN114027547B (en) * | 2021-11-05 | 2023-02-17 | 上海烟草集团有限责任公司 | Method for solving precipitation generated in preparation process of reconstituted tobacco coating liquid |
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| CN101781640B (en) | 2012-10-10 |
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