CN103232988A - Agarase immobilization method - Google Patents

Abstract

The invention belongs to the field of enzyme engineering research and development technology. The invention relates to an agarase immobilization method research. The invention comprises the following steps: preparing a 1:20 (W/V) of chitosan solution with 1.5-2% (V/V) of acetate for fully dissolving; dropping the dissolved chitosan solution drop by drop into a 1 mol/L sodium hydroxide solution, and filtering and collecting a chitosan bead; washing to neutral with distilled water, and then immersing with a 0.2 mol/L phosphate buffer solution of pH7.6 overnight; fetching the immersed chitosan bead carrier 2.5-5 g, and adding 2.5% of glutaraldehyde 20-25 ml, carrying out water-bath oscillation for 0.5 hours and room temperature crosslinking for 5-6 hours, and washing the chitosan bead cross-linked carrier with a phosphoric acid buffer; adding diluted agarase liquid 10-15 ml into the chitosan bead cross-linked carrier, stirring uniformly, fixing at 4 DEG C for 2-3 hours, washing with the phosphoric acid buffer; obtaining the immobilized agarase by vacuum-pumping. The agarase immobilization method provided by the invention has the advantages of cheap and easily available carrier, simple technology, mild condition and small loss of enzyme activity, and the enzyme activity recovery rate reaches to 77.6%.

Description

A kind of process for fixation of gelase

Technical field

Enzyme engineering research and development technical field under the present invention.The present invention relates to the method research of chitin microspheric immobilized gelase, be fit to suitability for industrialized production.

Background technology

Agar-agar is a kind of important Sargassum polysaccharides, mainly is made up of agarose and agaropectin.Agarase degraded agarose according to its mode of action difference, can be divided into two classes to agarase: α-agarase and β-agarase.The α-1 of α-agarase cracking agarose, the 3-glycosidic link generates with β-D-semi-lactosi and is non reducing end and is the fine jade oligosaccharides row of reducing end under neutral with 3,6-inner ether-α-L-semi-lactosi; The β-1 of β-agarase cracking agarose, the 4-glycosidic link generates with β-D-semi-lactosi and is reducing end under neutral and is the new fine jade oligosaccharides series of non reducing end with 3,6-inner ether-α-L-semi-lactosi.The agarase degradation process has the yield height, pollutes the advantages such as security height of little and product, is the most potential method in degraded agarose.

Agarase degraded agarose produces fine jade oligosaccharides product and has been widely used in grocery trade, and cosmetic industry and medicine industry are for example as natural sanitas, age of starch inhibitor, functional food ingredient, antioxidant etc.Simultaneously agarase prepares process such as protoplastis and has important use value at unicellular separation, the enzymolysis broken wall of marine alga, is a kind of toolenzyme of marine alga genetic engineering; Aspect molecular biology, utilize agarase degraded agarose from the gel of agarose, to reclaim DNA and RNA, be proved to be one of the best way; Agarase also is used for the structural research of Sargassum polysaccharides, and with the polysaccharide of some marine alga of agarase hydrolysis, measure the structure of its hydrolysate, and then infer the structure of polysaccharide, be efficient ways.

But the applied research of this enzyme at present mainly is to resolvase, but the resolvase poor stability, and the quality guaranteed period is short, and repeating utilization factor is low, application cost is increased and has limited its application.So in the applied research of enzyme, the research of immobilized enzyme is a special kind of skill emerging in the biological technical field.

The immobilization of enzyme refers to solid support material enzyme is limited to carries out catalyzed reaction in the certain space, immobilized enzyme one class technology recyclable and that utilize continuously repeatedly.Immobilized enzyme has kept efficiently single-minded, the gentle catalytic property of resolvase, has overcome simultaneously the shortcoming of resolvase again, has high stability, easily separated purifying, reclaims and advantage such as use, easy and simple to handle.Yet the immobilized enzyme that extensively drops into industrial applications is few.Therefore, further develop more economical suitable process for fixation, make more enzyme obtain industrial widespread use, be still the target that the scientific worker pursues.

What the process for fixation of enzyme was commonly used is: absorption method, crosslinking, covalent attachment and method entrapping method.The combined techniques of aforesaid method etc. has also appearred on this basis

In the enzyme immobilization research, good carrier is the key of immobilized enzyme success.So in the enzyme immobilization field, just become main direction of studying as the screening of fixation support and preparation.

Chitin be by acetamido glucose with the natural linear polymer that β-1,4 chain is formed by connecting, be that occurring in nature is only second to cellulosic second renewable resources.Chitosan is the product that chitin obtains through deacetylated reaction, contains abundant free amine group in the molecular structure.It is abundant to select chitosan to be based on its source as fixation support, tool wetting ability, biocompatibility and antimicrobial resolution characteristic etc.Amino of chitosan and glutaraldehyde base form schiff alkali, form one deck compact protective film in capsule surface, can increase the intensity of microcapsule ball, Here it is absorption-crosslinking immobilized enzyme.

Because the immobilization of gelase at present is also no one's research both at home and abroad, thus utilize cheap chitosan as fixation support fixedly gelase have bigger theory and practice meaning.And the purpose of present method research is the Application Areas of exploitation chitosan, and can strengthen gelase in the application in fields such as industry, medical science, food, environment protection.

Summary of the invention

1. the present invention relates to the method with chitosan-immobilized gelase, its feature comprises following process in the method for chitosan-immobilized gelase:

Step (one): with the chitosan solution of 1.5-2% (V/V) acetic acid preparation 1: 20 (W/V), fully dissolving;

Step (two): the chitosan solution of step () is dropwise splashed in the 1mol/L sodium hydroxide solution, and chitosan will be condensed into white particle, filter and collect the chitosan pearl;

Step (three): the chitosan pearl of step (two) is washed with distilled water to neutrality;

Step (four): the phosphate buffer solution soaked overnight of the chitosan pearl of step (three) being used 0.2mol/L, pH7.6;

Step (five): get the chitosan bead carrier 2.5-5g in the step (four), add 2.5% glutaraldehyde 20-25ml, water-bath vibration 0.5 hour, normal temperature crosslinked 5-6 hour, the crosslinked carrier of phosphoric acid buffer flushing chitosan pearl;

Step (six): add the gelase liquid of 10-15mL dilution in the crosslinked carrier of chitosan pearl in step (five), stir, 4 ℃ fixedly 2-3 hour, wash with phosphoric acid buffer;

Step (seven): the immobilized enzyme in the step (six) is vacuumized being fixed gelase.

2. the carrier according to preparation immobilization gelase of the present invention is chitosan microball.

Description of drawings

Fig. 1 be the pH value to the figure that influences of enzymic activity, Fig. 2 be temperature to the figure that influences of enzymic activity, Fig. 3 is the thermostability figure of resolvase and immobilized enzyme.

Embodiment

Of the present inventionly relate to chitin microspheric immobilized gelase method and comprise following examples, the following examples can further specify the present invention, but do not limit the present invention in any way.

Embodiment 1:pH value is to the influence of enzymic activity

Parallel chitin immobilized enzyme 0.5g and the corresponding resolvase for preparing of getting adds substrate and resolvase and immobilized enzyme effect respectively under different pH (4.0-9.0) condition, measure its enzyme activity respectively, and the result as shown in Figure 1.

Fig. 1 shows that the optimum pH of resolvase is 8.0, along with the rising enzymic activity decline of pH value; For immobilized enzyme, under this experiment condition, enzymic activity reaches maximum value when the pH value reaches 8.5, the optimal pH that immobilization makes enzyme 0.5 unit that raises toward alkaline aspect, meet enzyme and carrier in conjunction with after basic law.

Embodiment 2: temperature is to the influence of enzymic activity

Parallel chitin immobilized enzyme 0.5g and the corresponding resolvase for preparing of getting adds substrate and resolvase and immobilized enzyme effect respectively under differing temps (30 ℃-80 ℃) condition, measure its enzyme activity respectively, and the result as shown in Figure 2.

As shown in Figure 2, free agarase shows the highest vigor under 40 ℃, raise with temperature, vigor sharply descends, and enzyme begins inactivation, but for immobilized enzyme, all have big enzyme to live in 40-60 ℃ of scope, enzyme activity reaches maximum value in the time of 50 ℃, and the immobilized enzyme thermotolerance significantly improves.

Embodiment 3: the thermostability of resolvase and immobilized enzyme

Parallel chitin immobilized enzyme 0.5g and the corresponding resolvase for preparing of getting is incubated 0.5h under differing temps (20 ℃-80 ℃) condition, add substrate and its effect then, measures its enzyme activity respectively, and the result as shown in Figure 3.

As shown in Figure 3, enzyme liquid is less to enzyme influence alive in of short duration insulation below 40 ℃, when temperature further is promoted to 50 ℃ the resolvase activity influence is increased, and loss is about 27.36%, and still less to the immobilized enzyme effect of vigor.In the time of 60 ℃, the activity of resolvase is very low, and immobilized enzyme work when being about 50%, 70 ℃ enzyme substantially all do not have activity fully, illustrate that the thermostability of immobilized enzyme is higher than resolvase.

Embodiment 4: the operational stability of immobilized enzyme

Get immobilized enzyme 0.5g, add 0.5% agarose substrate 1.5ml and react with it, operate continuously 6 times is measured the enzyme activity size behind 6 secondary responses respectively, result such as table 1.

The operational stability of table 1 immobilization agarase

The stability of enzyme can be weighed from product (reducing sugar) yield of reaction.And the yield of product can be indirect the size from enzyme activity judge.Therefore, as can be seen from Table 2, immobilized enzyme has higher vigor when using the 1st time, illustrates that the amount of reducing sugar is higher, that is to say that the yield of product is higher; The immobilized enzyme vigor slightly reduces after the 2nd time, along with the increase of access times, though enzyme activity reduce gradually, use 6 times after enzyme activity still have about 83.8.Illustrate that the immobilization agarase has the distinguishing feature of immobilized enzyme, gets final product reuse.

Embodiment 5: the immobilization agarase is to conversion and the transformation efficiency of agarose

Accurately take by weighing and get immobilization agarase 0.5g, add 0.5% agarose substrate 1.5ml and react with it, add the 2ml damping fluid, at 50 ℃ of following enzymolysis 1h, survey its transformation efficiency, its average conversion can reach 92.6%.

Embodiment 6: the stability of immobilization gelase

Gained immobilized enzyme under top condition, its vigor is recovered as 77.6%, at room temperature stores 4 months, and as seen the vigor that records reclaims 72.8%., the excellent in stability of immobilization gelase.

Claims (2)

1. the present invention relates to the method with chitosan-immobilized gelase, its feature comprises following process in the method for chitosan-immobilized gelase:

Step (one): with the chitosan solution of 1.5-2% (V/V) acetic acid preparation 1: 20 (W/V), fully dissolving;

Step (two): the chitosan solution of step () is dropwise splashed in the 1mol/L sodium hydroxide solution, and chitosan will be condensed into white particle, filter and collect the chitosan pearl;

Step (three): the chitosan pearl of step (two) is washed with distilled water to neutrality;

Step (four): the phosphate buffer solution soaked overnight of the chitosan pearl of step (three) being used 0.2mol/L, pH7.6;

Step (five): get the chitosan bead carrier 2.5-5g in the step (four), add 2.5% glutaraldehyde 20-25ml, water-bath vibration 0.5 hour, normal temperature crosslinked 5-6 hour, the crosslinked carrier of phosphoric acid buffer flushing chitosan pearl;

Step (six): add the gelase liquid of 10-15mL dilution in the crosslinked carrier of chitosan pearl in step (five), stir, 4 ℃ fixedly 2-3 hour, wash with phosphoric acid buffer;

Step (seven): the immobilized enzyme in the step (six) is vacuumized being fixed gelase.

2. the carrier according to preparation immobilization gelase of the present invention is chitosan microball.

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