CN103451119A - Alteromonas and method thereby for producing gel-type enteromorpha polysaccharide degrading enzyme by using Alteromonas - Google Patents
Alteromonas and method thereby for producing gel-type enteromorpha polysaccharide degrading enzyme by using Alteromonas Download PDFInfo
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Abstract
The invention relates to a marine bacterium, wherein the bacterium is characterized in that the bacterium is Alteromonas cdwelliana A321 separated in seawater in Qingdao sea area. The preserving unit is CCTCC (China Center For Type Culture Collection) for short and the preserving number is M 2012132. The culture provided by the invention has the characteristics of simplicity in nutritional requirement, easiness in cultivation and short generation time. The growth temperature range of the bacterium is 10-40 DEG C, most preferably 28 DEG C. The growth pH range is 4-11, most preferably 7. The invention further discloses a method for producing a gel-type enteromorpha polysaccharide degrading enzyme by the Alteromonas and the gel-type enteromorpha polysaccharide degrading enzyme obtained by the method. The gel-type enteromorpha polysaccharide degrading enzyme produced by the method provided by the invention can efficiently degrade enteromorpha polysaccharide and can be used to preparation of enteromorpha polysaccharides. Compared with chemical methods, the method provided by the invention is simple in preparation process, high in product yield and stable in quality, active groups are not damaged in the preparation process, and activity study and development of polysaccharides are guaranteed.
Description
Technical field
The present invention relates to a kind of alternately Zymomonas mobilis, particularly a kind of separation is from the alternately Zymomonas mobilis A321 (Alteromonas cdwelliana A321) of Qingdao sea area, on April 23rd, 2012, be deposited in Chinese Typical Representative culture collection center C CTCC, deposit number is CCTCC NO:M 2012132; The invention still further relates to this bacterial strain and produce method of gel-type sea grass polysaccharide degrading enzyme and products thereof.
Background technology
Enteromorpha (Entermorpha) is a kind of large-scale green alga, and aboundresources in the wild algae of China, all have distribution in southeastern coast.Due to Global warming effect and sea pollution, the carbon in seawater, nitrogen, phosphorus source continue to increase in recent years, cause the Enteromorpha overpreading and spread.The Enteromorpha outburst causes " green tide ", also may bring a series of secondary environment harm, so the recycling of Enteromorpha becomes the focus of research.At present the comprehensive utilization of Enteromorpha is studied carefully to scope also very little, produce chain and mainly be divided into low side and high-end two aspects.Low side is produced the exploitation that chain mainly concentrates on biomass fertilizers and biomass energy; High-end production chain is mainly around several aspects such as food and additive, feed, medicine, Enteromorpha biologically active substance and engineering materialss.
In Enteromorpha, each component content (butt) is: crude protein content is 22.0%, crude fat 1.24%, food fibre 61.36%.Seaweed diet fiber is that the anti-human body small intestine of energy extracted from marine plant is digested and assimilated, and the summation of edible plant composition, carbohydrate and the similar substance that can partly or entirely ferment at the human body large intestine, comprise polysaccharide, oligosaccharides, xylogen and corresponding plants material, be divided into water-soluble dietary fibre and water insoluble dietary fiber.What wherein be widely studied is the polysaccharide part in water-soluble dietary fibre, and this part is called as sea grass polysaccharide.
At present the research of sea grass polysaccharide is mainly concentrated on the research aspect of extraction, purifying, evaluation and the physiological function thereof of sea grass polysaccharide.According to pertinent literature, sea grass polysaccharide have reducing blood-fat, anti-ageing, to skin carcinoma restraining effect etc.But, than oligosaccharides, polysaccharide has the deficiency of himself as poor than oligosaccharides as solvability, the physiologically active shown is also than a little less than oligosaccharides etc.Therefore want more to further investigate the Enteromorpha biological activity, need to improve the degradation technique of sea grass polysaccharide.
At present the degraded of sea grass polysaccharide mainly is hydrolyzed by dilute sulphuric acid, hydrolyzed solution mainly contains glucose, rhamnosyl and wood sugar through liquid-phase chromatographic analysis, and contains a small amount of seminose and semi-lactosi.Dilute sulphuric acid degraded sea grass polysaccharide will be in high-pressure sterilizing pot 121 ℃ process 90min, process like this biological activity that intensity can be destroyed polysaccharide and oligosaccharides.Therefore the hydrolyzed solution that degraded obtains through dilute sulphuric acid is mainly used in ethanol fermentation.Obtain having the oligosaccharides of better activity, enzymolysis is optimal degradation method.Brown alga oligose, fucose and oligochitosan all prepare and obtain by enzyme process comparatively widely in research at present.Enzyme, as a kind of special catalyzer, has extremely strong substrate specificity and product specificity.Enzyme process prepares oligosaccharides and has that catalytic efficiency is high, product specificity good, the molecular weight of degradation process and product thereof or the advantage such as the polymerization degree is easy to control.Use enzyme process to prepare oligosaccharides and can easily to reaction, implement monitoring in real time, make in time the adjustment of condition, obtain the oligose of the polymerization degree of wanting.And compare with the physics and chemistry method, the method is to carry out in comparatively gentle condition, does not need to add much other chemical reagent, therefore environmental pollution is corresponding also just less.Yet, consult pertinent literature and books according to the inventor, also find no at present the report that closes the sea grass polysaccharide degrading enzyme both at home and abroad.
The present invention be take the gel-type sea grass polysaccharide as raw material, separate the temporary transient called after Alteromonas of strain bacterial strain cdwelliana A321 from Qingdao sea area, this bacterial strain can be secreted a kind of gel-type sea grass polysaccharide degrading enzyme, and the recycling that this endonuclease capable is Enteromorpha provides corresponding technical director.
Summary of the invention
An object of the present invention is for the problem that it is difficult that current sea grass polysaccharide is degraded, the bacterial strain that provides a strain can produce gel-type sea grass polysaccharide degrading enzyme, utilize the enzyme of its secretion to improve enzyme process and prepare system.
Another object of the present invention is to provide a kind of method for preparing gel-type sea grass polysaccharide degrading enzyme and products thereof, can be used for the preparation of oligose, and it can overcome existing acidolysis, oxidative degradation, the technical defect of mechanical degradation sea grass polysaccharide.
The present invention reaches two purposes of the present invention by the following technical programs.
Feature of the present invention comprises alternately Zymomonas mobilis A321 (Alteromonas cdwelliana A321) itself, and utilize this bacterial strain to produce the method for gel-type sea grass polysaccharide degrading enzyme, and the gel-type sea grass polysaccharide degrading enzyme product that utilizes the method to produce.
The bacterial strain of mentioning in the present invention is the alternately Zymomonas mobilis A321 (Alteromonas cdwelliana A321) separated in the seawater of Qingdao sea area, alternately Zymomonas mobilis A321 was deposited in the Chinese Typical Representative culture collection center that is positioned at Wuhan, China Wuhan University on April 23rd, 2012, and deposit number is CCTCC NO:M2012132.
One, the morphological specificity of bacterial strain of the present invention and physiological and biochemical property
(1) bacterial strain be the Gram-negative coryneform bacteria, without pod membrane, without gemma, have end to give birth to flagellum, size is about 0.3-0.7 μ m * 1.8-2.4 μ m, morphological specificity is as shown in Figure 1.This bacterial strain on gel-type sea grass polysaccharide substratum 28 ℃ cultivate 48h after, colony diameter 2-3mm, circular milk yellow, the smooth of the edge, median rise, moistening, easy picking.Flat board after cultivation drips iodine liquid, can produce obvious gel-type sea grass polysaccharide degraded transparent circle (Fig. 2).
(2) physiological and biochemical property of bacterial strain
The physiological and biochemical property of strain A32 1 is in Table 1.Strain A32 1 can hydrolyzed starch, oxidase negative, the indole test feminine gender, chromogenesis, can not Oxidation of Alcohol to acetic acid, can not liquefy gelatin, do not produce H
2s, can not utilize Citrate trianion, malonate, can not utilize cellobiose, sucrose, D-Glucose, D-Maltose, D-MANNOSE, L-arabitol, ancient sugar, D-Tag, D-trehalose, PEARLITOL 25C, α-glucose can be utilized, SODIUMNITRATE, ammonium chloride, ammonium nitrate, ammonium sulfate can be utilized.
The physiological and biochemical property of table 1Alteromonas cdwelliana A321
(3) molecular biology identification of bacterial strain
Pcr amplification and sequential analysis: adopt the chelex-100 genome to extract DNA.Forward primer 27F:5 ＇-AGAGTTTGATCMTGCTCAG-3 ＇, reverse primer 1492R:5 ＇-ACGGCTACCTTGTTACGACTT-3 ＇.Reaction system 50u1.
Reaction conditions is 94 ℃ of denaturation 2min, 94 ℃ of sex change 30s, and 55 ℃ of annealing 40s, 72 ℃ are extended 1min, and last 72 ℃ are extended 10min.The purifying of PCR product, clone, order-checking are completed by Shanghai biotechnology company limited, the row that check order carry out homology relatively with Genbank, the similarity of this bacterial strain 16S rRNA sequence and Alteromonas sp.BG20 is 98%.This bacterial strain 16S rRNA sequence is shown in specification sheets final section.
Two, the growth characteristics of bacterial classification of the present invention
(1) about the substratum in the present invention
2216E substratum: yeast extract paste 1g, peptone 5g, high ferric phosphate 0.01g, agar 18g, sodium-chlor 15g, tap water 1000m1, pH7.6.
Gel-type sea grass polysaccharide substratum: agar 10g, gel-type sea grass polysaccharide 5g, yeast extract paste 5g, dipotassium hydrogen phosphate 0.5g, sal epsom 0.5g, calcium chloride 0.1g, sodium-chlor 15g, ammonium chloride 1g, high ferric phosphate 0.02g, ferric sulfate 0.01g, tap water 1000ml, pH7.0.
Fermention medium: gel-type sea grass polysaccharide 5g, yeast extract paste 5g, dipotassium hydrogen phosphate 0.5g, sal epsom 0.5g, calcium chloride 0.1g, sodium-chlor 15g, ammonium chloride 1g, high ferric phosphate 0.02g, ferric sulfate 0.01g, tap water 1000m1, pH7.0.
(2) preparation of seed liquor; The bacterium be kept on gel-type sea grass polysaccharide substratum is inoculated in fermention medium, and rotating speed 170r/min, cultivate 24h for 28 ℃.
(3) impact of temperature on growth: 8% seed liquor is inoculated in to fermention medium, pH7.0, rotating speed 170r/min cultivates respectively under differing temps, measures cell concn.The growth temperature range of this bacterium is 10-40 ℃, and wherein 28 ℃ is optimum growth temperature.
(4) impact of pH on growth: adjust medium pH with 2M hydrochloric acid and 1M sodium hydroxide after the fermention medium sterilizing, the pH of sterilizing post-fermentation and culture base is respectively between 3-12, at 28 ℃, cultivate 24h, other conditions, with (three), are measured cell concn.The growth pH scope of this bacterium is 4-11, and the most suitable growth pH is 7.
Three, bacterial classification produces the method for gel-type sea grass polysaccharide degrading enzyme
Seed liquor is inoculated in fermention medium by 8% inoculum size, and 170r/min cultivates 12-18h for 28 ℃; By the centrifugal 5min of fermented liquid 10000r/min, get supernatant liquor, add 60% ammonium sulfate, 4 ℃ are spent the night, and 10000r/min is centrifugal, with isopyknic distilled water, will precipitate redissolution, obtains crude enzyme liquid.
Four, the character of gel-type sea grass polysaccharide degrading enzyme
(1) impact that the enzyme operative temperature is lived on enzyme: the crude enzyme liquid obtained is measured to enzyme activity respectively under 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ conditions, find that the optimal reactive temperature of enzyme is 35 ℃.
(2) impact that the enzyme action pH is lived on enzyme: the crude enzyme liquid obtained is measured to enzyme activity respectively under pH2,3,4,5,6,7,8,9,10 conditions, find that the optimal reaction pH of enzyme is 5-8.
(3) thermostability of enzyme: crude enzyme liquid is incubated respectively 3h, 6h, 12h, 24h, 36h respectively under 30 ℃, 35 ℃, 40 ℃ conditions, and 48h measures the residual enzyme vigor at 40 ℃.Find that this enzyme is best 30 ℃ of stability, along with the rising of temperature, stability descends gradually, and the transformation period of 30 ℃ is 36h, and the transformation period of 35 ℃ is 24h, and the transformation period of 40 ℃ is 6h.
(4) the pH stability of enzyme: measure the residual enzyme vigor after crude enzyme liquid being incubated under 30 ℃ of condition of different pH to 1h under the optimal reactive temperature condition, this enzyme stability when pH 6-8 is better, and in neutral environment, enzyme activity is more stable.
Five, the mensuration of gel-type sea grass polysaccharide degrading enzyme
(1) gel-type sea grass polysaccharide degrading enzyme measuring method alive: gel-type sea grass polysaccharide degrading enzyme is lived and adopted 5 ‰ gel-type sea grass polysaccharides is substrate, 35 ℃ of reaction certain hours, reducing sugar amount after adopting 3,5-dinitrosalicylic acid method assaying reaction to finish in system.
(2) enzyme activity unit definition: at 35 ℃, under the condition of pH5.03, every min degraded gel-type sea grass polysaccharide produces the needed enzyme amount of 1ug reducing sugar (with glucose meter) and is defined as 1 enzyme activity unit (u).
The accompanying drawing explanation
Fig. 1 is the bacterial strain electromicroscopic photograph.
Fig. 2 is the transparent loop graph of bacterial strain on gel-type sea grass polysaccharide solid medium.
Fig. 3 is the affect figure of temperature on gel-type sea grass polysaccharide degrading enzyme vigor.
Fig. 4 is the affect figure of temperature on gel-type sea grass polysaccharide degrading enzyme stability, 30 ℃ (◆), 35 ℃ (■), 40 ℃ (▲).
Fig. 5 is the affect figure of pH on gel-type sea grass polysaccharide degrading enzyme vigor.
Fig. 6 is the affect figure of pH on gel-type sea grass polysaccharide degrading enzyme stability.
Specific embodiments
Embodiment 1: a kind of alternately Zymomonas mobilis A321 (Alteromonas cdwelliana A321), this bacterial strain has following characteristics: bacterial strain is the Gram-negative coryneform bacteria, without pod membrane, without gemma, have end to give birth to flagellum, size is about 0.3-0.7 μ m * 1.8-2.4 μ m; This bacterial strain on gel-type sea grass polysaccharide substratum 28 ℃ cultivate 48h after, colony diameter 2-3mm, circular milk yellow, the smooth of the edge, median rise, moistening, easy picking; Flat board after cultivation drips iodine liquid, can produce obvious gel-type sea grass polysaccharide degraded transparent circle; Strain A32 1 can hydrolyzed starch, oxidase negative, can not Oxidation of Alcohol to acetic acid, do not produce H
2s, chromogenesis, can not utilize Citrate trianion, malonate, the indole test feminine gender, can utilize α-glucose, cellobiose, sucrose, D-Glucose, D-Maltose, D-MANNOSE, L-arabitol, ancient sugar, D-Tag, D-trehalose, PEARLITOL 25C can not be utilized, SODIUMNITRATE, ammonium chloride, ammonium nitrate, ammonium sulfate can be utilized.
Embodiment 2: described a kind of alternately its growth characteristics of Zymomonas mobilis A321 (Alteromonas cdwelliana A321) of embodiment 1 are: the growth temperature range of this bacterium is 10-40 ℃, and optimum growth temperature is 28 ℃; Growth pH scope is 3-11, and the most suitable growth pH scope is 7.
Embodiment 3: a kind of embodiment 1 or 2 any one described method that alternately Zymomonas mobilis A321 (Alteromonas cdwellianaA321) produces gel-type sea grass polysaccharide degrading enzyme, and its step is as follows;
(1) bacterium that will be kept on gel-type sea grass polysaccharide substratum is inoculated in fermention medium, and rotating speed 170r/min cultivates 24h, obtains seed liquor for 28 ℃;
(2) seed liquor is inoculated in fermention medium by 8% inoculum size, and 170r/min cultivates 12-18h for 28 ℃; By the centrifugal 5min of fermented liquid 10000r/min, get supernatant liquor, add 60% ammonium sulfate, 4 ℃ are spent the night, and 10000r/min is centrifugal, with isopyknic distilled water, will precipitate redissolution, obtains crude enzyme liquid.
Embodiment 4: the gel-type sea grass polysaccharide degrading enzyme that a kind of described method of embodiment 3 is produced, and this enzyme has following characteristics:
(1) optimal reactive temperature of this enzyme is 35 ℃, and 47% remnant enzyme activity is arranged in the time of 20 ℃, and 19% remnant enzyme activity is arranged in the time of 70 ℃; This enzyme is best 30 ℃ of stability, and along with the rising of temperature, stability descends gradually, and the transformation period of 30 ℃ is 36h, and the transformation period of 35 ℃ is 24h, and the transformation period of 40 ℃ is 6h.
(2) the optimal reaction pH of this enzyme is 5-8, and this enzyme has 14% remnant enzyme activity when pH 2, and 72% remnant enzyme activity is arranged when pH 10; This enzyme stability when pH 6-8 is better, and in neutral environment, enzyme activity is more stable.
(3) Ba
2+this enzyme work is had to promoter action, Ca
2+, Mg
2+enzyme is lived and do not affected, Co
2+, Zn
2+, Fe
3+, Cu
2+, EDTA can be strong inhibitory enzyme live.
Claims (5)
1. a bacterium, is characterized in that this bacterium is for replacing Zymomonas mobilis, called after Alteromonas cdwelliana A321, and depositary institution is called for short CCTCC, and preserving number is M2012132.
2. one kind replaces Zymomonas mobilis A321 (Alteromonas cdwelliana A321), it is characterized in that producing gel-type sea grass polysaccharide degrading enzyme with it.
3. one kind by the method that alternately Zymomonas mobilis is produced gel-type sea grass polysaccharide degrading enzyme, comprises bacterium is put into to gel-type sea grass polysaccharide fermention medium, in certain culture temperature, cultivate certain hour, with the ammonium sulfate precipitation collecting precipitation protein, then separated last lyophilize.
4. a gel-type sea grass polysaccharide degrading enzyme, is characterized in that its gel-type sea grass polysaccharide of degrading, to prepare oligose.
5. a kind of use as claimed in claim 3 replaces the method that Zymomonas mobilis is produced gel-type sea grass polysaccharide degrading enzyme, it is characterized in that described culture temperature is 28 ℃, and described incubation time is 12-18h, the ammonium sulfate that described enzyme extracting agent is 60%.
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| CN103352016A (en) * | 2013-06-27 | 2013-10-16 | 中国海洋大学 | Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha |
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| CN105602995A (en) * | 2016-01-21 | 2016-05-25 | 青岛海洋生物医药研究院股份有限公司 | Method for preparing Enteromorpha bio-fertilizer through liquid deep quick fermentation |
| CN105624067A (en) * | 2016-03-02 | 2016-06-01 | 青岛海洋生物医药研究院股份有限公司 | Marine bacterium Pseudoalteromonas sp SC127 and ulva sulfuric acid bhamnosan enzyme prepared from same |
| CN106578379A (en) * | 2016-12-09 | 2017-04-26 | 中国海洋大学 | Method for preparing functional feed additive through biological glycolysis of enteromorpha |
| CN108239176A (en) * | 2016-12-27 | 2018-07-03 | 中国海洋大学 | Low molecular weight sea grass polysaccharide and preparation method thereof, sulphation low molecular weight sea grass polysaccharide and preparation method thereof and application |
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| CN103352016A (en) * | 2013-06-27 | 2013-10-16 | 中国海洋大学 | Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha |
| CN104560774B (en) * | 2014-06-30 | 2017-04-26 | 中国海洋大学 | Method for preparing block oligosaccharide containing rich rhamnose sulfate from Enteromorpha polysaccharide |
| CN104560774A (en) * | 2014-06-30 | 2015-04-29 | 中国海洋大学 | Method for preparing block oligosaccharide containing rich rhamnose sulfate from Enteromorpha polysaccharide |
| CN105602995A (en) * | 2016-01-21 | 2016-05-25 | 青岛海洋生物医药研究院股份有限公司 | Method for preparing Enteromorpha bio-fertilizer through liquid deep quick fermentation |
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| CN106578379A (en) * | 2016-12-09 | 2017-04-26 | 中国海洋大学 | Method for preparing functional feed additive through biological glycolysis of enteromorpha |
| CN108239176A (en) * | 2016-12-27 | 2018-07-03 | 中国海洋大学 | Low molecular weight sea grass polysaccharide and preparation method thereof, sulphation low molecular weight sea grass polysaccharide and preparation method thereof and application |
| CN108239613A (en) * | 2016-12-27 | 2018-07-03 | 中国海洋大学 | A kind of feed series bacillus, its culture medium and the application in sea grass polysaccharide degrading enzyme is prepared |
| CN108239613B (en) * | 2016-12-27 | 2021-07-06 | 中国海洋大学 | Paenibacillus feed, culture medium thereof and application of Paenibacillus feed in preparation of enteromorpha polysaccharide degrading enzyme |
| CN108239176B (en) * | 2016-12-27 | 2021-08-17 | 中国海洋大学 | Low-molecular-weight enteromorpha polysaccharide and preparation method thereof, sulfated low-molecular-weight enteromorpha polysaccharide and preparation method and application thereof |
| WO2020244031A1 (en) * | 2019-06-03 | 2020-12-10 | 中国海洋大学 | Ulva lactuca polysaccharide lyase, encoding gene thereof, and application thereof |
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