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A method for immobilizing lipase b from Candida antarctica fermentation supernatant

A technology of Candida Antarctica, fermentation supernatant, applied in microorganism-based methods, immobilized on/in organic carriers, biochemical equipment and methods, etc., can solve the problem of reduced recovery rate of CALB enzyme activity and complex process , low catalytic activity, etc., to achieve the effect of easy to judge the end point, simplify the purification steps, and enhance the catalytic ability

Active Publication Date: 2018-06-29
INNER MONGOLIA KINGDOMWAY PHARMA LTD +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Adsorption methods are divided into aqueous phase adsorption, organic phase adsorption, micro-aqueous phase adsorption, etc. Since the active area of ​​CALB is a hydrophobic area, organic phase adsorption can keep the active area exposed and can better maintain lipase activity, but the cost is high , the amount of organic solvent is large, and the process is relatively complicated; the process of aqueous phase adsorption method is simple and the cost is low, but it will cause the dissolution of metal ions in the active center of CALB, or the formation of multi-layer adsorption on the surface of the carrier will lead to superposition and masking of hydrophobic regions, making the CALB Enzyme activity recovery decreased
[0005] At present, Novozyme 435 produced by Novozymes is the most successful CALB immobilized commercial enzyme. It is prepared by immobilizing CALB fermented by recombinant Aspergillus oryzae and immobilized on macroporous acrylic resin. It has excellent catalytic effect but is expensive, which limits its use in the enzyme catalysis industry. Applications
The price of domestic immobilized lipase is relatively low, but it generally has the disadvantages of low catalytic activity, poor stability or difficulty in large-scale production, making it difficult to occupy a market advantage

Method used

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  • A method for immobilizing lipase b from Candida antarctica fermentation supernatant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The activity of lipase B in the fermentation supernatant of Candida antarctica is 2256U / mL, the volume is 500L, and the temperature is pre-cooled to 20°C in a jacketed stirring tank.

[0036] The diameter of the pre-column is 30cm, and the height is 200cm (diameter-to-height ratio 1:6.67). Take 100L of NKA-II macroporous resin as the pre-column filler (the volume ratio of resin to fermentation supernatant is 1:5), add 50L methanol to soak, and use 50L Carry out wet packing of methanol (column packing volume 0.71BV), use 200L methanol as the mobile phase for cleaning, flow rate 150L / h (flow rate 1.06BV / h), use 300L distilled water to wash out methanol after methanol cleaning, flow rate 150L / h (flow rate 1.06BV / h).

[0037] The diameter of the adsorption column is 20cm, and the height is 200cm (the ratio of diameter to height is 1:10). Take 50L of HPD600 macroporous resin as the filler of the adsorption column (the volume ratio of resin to fermentation supernatant is 1:10...

Embodiment 2

[0052] The activity of lipase B in the fermentation supernatant of Candida antarctica is 2328U / mL, the volume is 5000L, and the temperature is pre-cooled to 15°C in a jacketed stirring tank.

[0053] The pre-columns were prepared and pretreated according to the specifications and methods in Example 1, and a total of 6 pre-columns were prepared and connected in parallel.

[0054] The diameter of the adsorption column is 30cm, and the height is 200cm (diameter-height ratio 1:6.67). A total of 6 columns are prepared, which are respectively connected in series to the pre-column, and 105L of HPD600 macroporous resin is respectively taken as the adsorption column filler (the total volume ratio of the resin to the fermentation supernatant is 1:8), add 50L methanol for soaking, use 50L methanol for wet packing (column packing volume 0.74BV), use 200L methanol as mobile phase for cleaning respectively, flow rate 150L / h (flow rate 1.06BV / h), methanol cleaning Finally, 300L of distilled ...

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Abstract

The invention discloses a process method for immobilizing lipase B from candida antarctica fermented supernatant fluid. According to the method, the immobilized candida antarctica lipase B is prepared by using a cheaper vector, adopting a water-phase immobilization mode and cooperating with a post-treatment process, therefore, the enzyme activity recovery rate under water-phase adsorption is increased, the cost and the using effect are balanced, and the important significance on industrial application of the CALB is achieved.

Description

technical field [0001] The invention relates to a process method for immobilizing lipase B in the fermentation supernatant of Candida antarctica, belonging to the field of enzyme immobilization. Background technique [0002] Candida antarctic lipase B (CALB), derived from Candida antarctica, is an excellent lipase, which has strong catalytic activity for water-soluble and water-insoluble substances, and is used in organic synthesis , chiral compound resolution, pharmaceutical intermediates and other fields have been widely used. [0003] Compared with its liquid enzyme preparation and powder enzyme preparation, immobilized lipase has the advantages of easy separation from the reaction system, continuous reaction in columns, high stability, easy control, etc., and has a wide range of applications. Enzyme immobilization methods are divided into covalent binding, carrier cross-linking, adsorption and embedding methods, among which the adsorption method is the most simple and p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08C12R1/72
CPCC12N9/20C12N11/08C12Y301/01003
Inventor 段然蒋四富廖炜程张斌焉世杰韩雯沈初亮
Owner INNER MONGOLIA KINGDOMWAY PHARMA LTD
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