CN108949703A - A kind of thick method of purification of hemicellulose corpusculum analog - Google Patents

Abstract

The invention provides a crude purification method for hemicellulosome analogues, which belongs to the field of industrial biotechnology. The fermentation broth is centrifuged by two-stage tandem centrifugation, the centrifugation condition is greater than 20000g and centrifuged for 20min, and the rotor constant K is greater than 900; 10nm<d<50nm; during the ultrafiltration process, repeatedly add pH 7.5, 50mM Tris-HCl buffer solution for ultrafiltration; use AlCl ₃ solution dropwise to carry out flocculation treatment on the obtained ultrafiltrate, the added AlCl ₃ should be controlled At a final concentration of 3-7g/L, after fully inverting and mixing for 3‑5 minutes, significant flocculation was observed. At this time, the target product was obtained by centrifuging at 6000g for 10 minutes. The method of the invention is used in the fields of industrial biotransformation, bioenergy, preparation of pharmaceutical intermediates and the like.

Description

A kind of crude purification method of hemicellulosome analog

technical field

The invention relates to a crude purification method for hemicellulosome analogs produced by cellulobacteria, belonging to the technical field of industrial biology.

Background technique

Hemicellulosome analogues can be produced by fermentation of a cellulobacterium. The composition of the fermentation broth is complex. In addition to the hemicellulosome analogs, it also contains impurities such as bacteria, protein, pigment, residual sugar and fermentation by-products, which will seriously affect the separation and purification of the hemicellulosome analogues and the quality of the product. At present, the commonly used methods for pretreatment of fermentation broth are: traditional filtration, high-speed centrifugation and membrane separation. Filtration is when the liquid (or gas) in the suspension (or the heating gas containing solid particles) penetrates the medium under the action of driving force or other external force, and the solid particles and other substances are trapped by the filter medium, so that the solid and other substances and the liquid ( or gas) separation operation. However, this separation method has low separation efficiency and high time consumption. Centrifugation is a method of separating substances according to their specific gravity, but it cannot guarantee that the target product will be kept in the supernatant of the fermentation broth while efficiently removing macromolecular impurities. The membrane separation method also has the same problem, and due to the complex composition of the actinomycete fermentation broth, it is easy to cause membrane fouling and membrane pore blockage during membrane separation operation, thereby reducing the membrane flux sharply. Flocculation refers to the aggregation of suspended particles in water or liquid, or the formation of flocs, so as to accelerate the coagulation and sedimentation of particles and achieve the purpose of solid-liquid separation. Usually, flocculation is implemented by adding an appropriate flocculant, whose function is to adsorb particles and "bridge" between particles, thereby promoting agglomeration. Flocculation technology can not only effectively remove solid particles and proteins in fermentation broth, but also components such as dissolved proteins may be partially removed during the flocculation process. At present, the flocculation pretreatment method with low investment and good effect is being applied in the separation of bacteria. Flocculation technology has been successfully reported in the pretreatment of hyaluronic acid fermentation broth, sisomycin fermentation broth, vancomycin fermentation broth, and 2,3-butanediol fermentation broth. Related reports on liquid flocculation technology.

In order to roughly purify the hemicellulosome analogues produced by cellulobacteria, and at the same time achieve the purpose of removing impurities such as solid particles, pigments, residual sugars, and fermentation by-products, more efficient crude purification techniques are needed.

Contents of the invention

In order to solve the above problems, the purpose of the present invention is to provide more efficient technical means for crude purification of hemicellulosome analogs.

Technical scheme of the present invention:

A crude purification method for hemicellulosome analogues, the steps are as follows:

(1) centrifugal

The fermentation broth is centrifuged by two-stage series centrifugation. The centrifugation conditions must be greater than 20000g and centrifuged for 20 minutes, and the rotor constant K must be greater than 900. Centrifugation is a method of separation using different sedimentation coefficients of various substances. In the fermentation broth, most water-insoluble substances such as cells larger than the target product can settle down.

(2) ultrafiltration

After centrifugation, the supernatant fermentation broth was treated by ultrafiltration for concentration. Ultrafiltration adopts inorganic ceramic hollow ultrafiltration membrane, the pore size requirement is 10nm<d<50nm, the main purpose is to intercept the hemifibrillary body analogs >50nm. During the ultrafiltration process, Tris-HCl buffer solution (pH 7.5 50mM) should be added repeatedly for ultrafiltration 6-10 times, and the ultrafiltration fermentation broth will be concentrated to one-tenth of the volume of the stock solution each time. The main purpose of this part is to remove impurities smaller than the target product in the fermentation broth, and to further obtain a relatively pure fermentation broth.

(3) Flocculation

The concentrated solution prepared by ultrafiltration generally has a protein concentration of 20-30 mg/mL. However, there are still a large amount of impurities suspended in colloidal form in the ultrafiltration concentrated solution (ie, the retentate) at this time, and the AlCl ₃ solution can be added dropwise. For flocculation treatment, the added AlCl ₃ should be controlled at a final concentration of 3-7g/L. After fully inverting and mixing, stand for 3-5 minutes to observe significant flocculation. At this time, the target product can be obtained by centrifuging at 6000g for 10 minutes.

Beneficial effects of the present invention: the present invention provides a crude purification method for hemicellulosome analogues, which can remove impurities such as cells, pigments, residual sugar and fermentation by-products in cellulobacteria fermentation broth, while retaining the dissolved state hemifibrillary body analogs. This crude purification method removes 90% of the visible impurities in the stock solution while retaining more than 90% of the enzyme activity, providing an effective technical means for obtaining pure hemicellulosome analogs, which can then be used in industrial biotransformation and bioenergy , Preparation of pharmaceutical intermediates and other fields.

Description of drawings

Figure 1 is a graph of the relative enzyme activity of 10 samples.

Detailed ways

The specific implementation manners of the present invention will be further described below in conjunction with the accompanying drawings and technical solutions.

1. Centrifuge

The cellulosic microbacteria fermentation broth is subjected to centrifugation. The centrifugation condition is 20000g×10min. After centrifugation, discard the precipitate and take the supernatant for the next step.

2. Ultrafiltration

The ultrafiltration adopts 30L industrial laboratory level ultrafiltration equipment, the equipment uses a pneumatic pump, and the filter membrane is an inorganic ceramic hollow ultrafiltration membrane with a pore size of 50nm, buffered by 50mM Tris-HCl with a pH of 7.5 The liquid was ultrafiltered 10 times, and the experimental results are summarized in Table 1. After the ultrafiltration is completed, the intercepted liquid is taken out for use.

Table 1 Summary of ultrafiltration results

3. Flocculation

(1) Dispense 1 mL of the concentrated solution prepared by the above ultrafiltration into ten EP tubes of 2 mL;

(2) According to the experimental sequence, different volumes of 1% (1g/100mL) AlCl3 _were added dropwise to carry out the flocculation experiment;

Table 2 AlCl ₃ addition amount

(3) After flocculation, centrifuge at 6000g for 10min;

(4) After centrifugation, the supernatant was taken for pNPX enzyme activity detection, and the detection results are shown in Table 3, Figure 1.

Table 3 pNPX enzyme activity detection results

4. Result analysis

The experimental results show that when the amount of AlCl ₃ added is 60 μL, that is, when the final concentration of AlCl ₃ is 6g/L, the colloidal state of the enzyme solution is destroyed, and a large amount of dark brown impurities are flocculated and precipitated, while the enzyme activity in the supernatant can still Keep it around 90%.

Claims (1)

1. a kind of thick method of purification of hemicellulose corpusculum analog, which is characterized in that steps are as follows:

(1) it is centrifuged

Fermentation liquid is subjected to centrifugal treating using two-stage tandem centrifugal process, centrifugal condition requires to have to be larger than 20000g centrifugation 20min, rotor constant K have to be larger than 900；

(2) ultrafiltration

Supernatant fermentation liquid hyperfiltration treatment is taken to be concentrated after centrifugation；Ultrafiltration uses inorganic ceramic hollow fiber ultrafiltration film, and aperture requires The hemicellulose corpusculum analog of 10nm<d<50nm, retention>50nm；In ultra-filtration process, to add pH 7.5,50mM Tris- repeatedly HCl buffer, ultrafiltration 6-10 times, it is 1/10th of stoste volume that each ultrafiltration fermentation liquid, which will be concentrated,；

(3) it flocculates

Using AlCl₃The mode that solution is added dropwise carries out flocculation treatment, AlCl be added to the ultrafiltrate that step (2) obtains₃It should control In the final concentration of 3~7g/L, 3-5min is stood after being sufficiently mixed by inversion, that is, observes significant flocculation, and 6000g is centrifuged at this time 10min, i.e. acquisition purpose product.