CN105462953A - Preparation of magnetic chitosan microsphere and its application as laccase immobilization carrier - Google Patents

Abstract

The invention relates to preparation of a magnetic chitosan microsphere and its application as a laccase immobilization carrier. The invention adopts glutaraldehyde as the cross-linking agent to prepare magnetic chitosan microsphere and obverves and analyzes the shape and structure, the magnetic chitosan microsphere has good spherical shape, smooth surface, good monodispersity, narrow particle size distribution range, and an average particle size of 019 micrometers. The magnetic chitosan microsphere is taken as the carrier for immobilization of laccase. The optimum temperatures of immobilized laccase are 10 and 55DEG C respectively, which are lower than that of free enzyme by 5DEG C, and the optimum pH value is 3102. After 210min of heat preservation under the condition of 60DEG C, the immobilized enzyme retains 74% of the enzyme activity, while under the same condition, only 1914% of the free enzyme activity is retained. After 10 consecutive operations, the immobilized enzyme still retains over 80% of enzyme activity, therefore the laccase use efficiency is obviously enhanced, and the immobilized laccase has good storage stability.

Description

The preparation of chitosan magnetic micro-sphere and be used as Laccase Immobilization carrier

Technical field

The present invention is applied in the aspects such as wastewater treatment, papermaking, aromatic compound conversion, environmental monitoring, biosensor structure, relates to the preparation of chitosan magnetic micro-sphere specifically and is used as Laccase Immobilization carrier.

Background technology

Laccase be a class cupric polyphenoloxidase (Laccase, p2diphenoloxidase, E.C.1.10.3.2), fungal laccase and the large class of Rhus verniciferalaccase two can be divided into.Because laccase has good Substratspezifitaet and stability, in wastewater treatment, papermaking, aromatic compound conversion, environmental monitoring, biosensor structure etc., there is significant application value.But the free laccase in use easy change deactivation with environment, and be not easily separated from reaction system and reach reusable object, limit the industrial applications of laccase to a certain extent.Enzyme immobilization technology realizes the effective means that enzyme repeats to use continuously and improve stability.

Special porous glass is mainly contained to the fixation support that laccase uses, oxirane acrylic particle and wetting ability microfiltration membrane etc. at present, but not see with chitosan magnetic micro-sphere be the report that carrier fixes laccase.There is good mechanical property, chemical stability, resistance toheat with the carrier of chitosan (Chitosan, β 21,4222 glucosamine) as immobilized enzyme and make enzyme from advantages such as metal ion disturbances.Fix with chitosan microball l2 asparaginases, the activity recovery of immobilized enzyme is higher, and stability is improved; Fix urase with chitosan film, show good operational stability; Peroxidase is fixed on chitosan microball, can be used for micro-H ₂o ₂fluoroscopic examination, and this immobilized enzyme has good reusability.

With glutaraldehyde as cross linker, utilizing inverse suspension method to prepare chitosan magnetic micro-sphere, is carrier immobilized Pycnoporus Sanguineus Laccase with it, have studied the character of immobilization laccase.While raising immobilization laccase stability, also have easily separated, can reuse continuously and improve the advantages such as immobilized enzyme catalysis efficiency, serialization and the industrialization of production technique and checked operation can be realized.Oxygen consumption type optical fiber biosensor based on Laccase Catalyzed has very important application prospect in clinical medicine detection and early stage medical diagnosis on disease etc., uses this immobilization laccase to be expected to improve the performance of this kind of sensor.

Summary of the invention

The present invention is exactly for the problems referred to above, proposes the preparation of chitosan magnetic micro-sphere and is used as Laccase Immobilization carrier.

For realizing above-mentioned purpose of the present invention, the present invention adopts following technical scheme.

The preparation of chitosan magnetic micro-sphere and be used as Laccase Immobilization carrier and comprise the steps:

(1) preparation of chitosan magnetic micro-sphere; Get 5mL magnetic fluid to mix with the chitosan acetic acid solution of 20mL215%, under agitation slowly join in the mixed solvent of 80mL whiteruss and 4mLSpan280, fully 30min is stirred under normal temperature, add the glutaraldehyde of 10mL8%, react 60min in the water-bath of 40 DEG C after, by the NaOH solution of 1mol/L, its pH value is adjusted to 9 ~ 10, reaction 2h is continued in 70 DEG C of water-baths, obtain product magnet to collect, then use sherwood oil successively, acetone, distilled water fully washs, suction filtration, 60 DEG C of vacuum-dryings, obtain chitosan magnetic micro-sphere.

(2) enzyme immobilizatio is painted; Get 50mg chitosan magnetic micro-sphere, add the laccase phosphate buffered saline buffer (011mol/L of 5mL116 × 10-3g/mL, pH=7), 25 DEG C are slowly stirred 1h, at 4 DEG C of standing 2h, outwell supernatant liquor, with pH=7 phosphate buffered saline buffer repetitive scrubbing, suction filtration, the immobilized enzyme obtained is preserved at 4 DEG C.

(3) enzyme activity determination; The mensuration of free laccase activity: get 3mL and contain in the tartaric acid buffer of 0105mmol/LABTS the enzyme liquid adding 8 μ L and suitably dilute, 55 DEG C of changes of the absorbancys with 420nm place in ultraviolet-visible pectrophotometer assaying reaction system 5min, obtain initial velocity of reaction; The mensuration of immobilization laccase vigor: 3mL adds 20mg immobilized enzyme containing in the tartaric acid buffer of 0105mmol/LABTS, 55 DEG C of changes of the absorbancy with 420nm place in ultraviolet-visible pectrophotometer assaying reaction system 5min, obtains initial velocity of reaction; ABTS optical extinction coefficient is ε=36000L/ (molcm), and enzyme amount per minute being oxidized 1 μm of olABTS is defined as 1 enzyme activity unit.

The invention has the beneficial effects as follows.

The present invention has prepared chitosan magnetic micro-sphere with glutaraldehyde as cross linker, and has carried out observation and analysis respectively to its pattern and structure, and result shows, it has good spherical morphology, surface smoothing, and monodispersity is better, particle size distribution range is narrow, and median size is 019 μm.Take chitosan magnetic micro-sphere as carrier fixing laccase.Immobilization laccase optimum temperuture is respectively 10 and 55 DEG C, and equal specific ionization enzyme reduces by 5 DEG C, and optimum pH is 3102.Under 60 DEG C of conditions, be incubated 210min, immobilized enzyme retains enzyme activity 74%, and resolvase enzyme activity only retains 1914% under the same conditions.After immobilized enzyme carries out 10 operations continuously, enzyme activity still keeps more than 80%, and laccase service efficiency significantly improves, and this fixing laccase has good storage stability.

Accompanying drawing explanation

Fig. 1 is the preparation of chitosan magnetic micro-sphere of the present invention and is used as the infrared spectrogram of the magnetic fluid (a) of Laccase Immobilization carrier, chitosan (b) and chitosan magnetic micro-sphere (c).

Embodiment

The preparation of chitosan magnetic micro-sphere and be used as Laccase Immobilization carrier and comprise the steps:

(1) preparation of chitosan magnetic micro-sphere; Get 5mL magnetic fluid to mix with the chitosan acetic acid solution of 20mL215%, under agitation slowly join in the mixed solvent of 80mL whiteruss and 4mLSpan280, fully 30min is stirred under normal temperature, add the glutaraldehyde of 10mL8%, react 60min in the water-bath of 40 DEG C after, by the NaOH solution of 1mol/L, its pH value is adjusted to 9 ~ 10, reaction 2h is continued in 70 DEG C of water-baths, obtain product magnet to collect, then use sherwood oil successively, acetone, distilled water fully washs, suction filtration, 60 DEG C of vacuum-dryings, obtain chitosan magnetic micro-sphere.

(2) enzyme immobilizatio is painted; Get 50mg chitosan magnetic micro-sphere, add the laccase phosphate buffered saline buffer (011mol/L of 5mL116 × 10-3g/mL, pH=7), 25 DEG C are slowly stirred 1h, at 4 DEG C of standing 2h, outwell supernatant liquor, with pH=7 phosphate buffered saline buffer repetitive scrubbing, suction filtration, the immobilized enzyme obtained is preserved at 4 DEG C.

(3) enzyme activity determination; The mensuration of free laccase activity: get 3mL and contain in the tartaric acid buffer of 0105mmol/LABTS the enzyme liquid adding 8 μ L and suitably dilute, 55 DEG C of changes of the absorbancys with 420nm place in ultraviolet-visible pectrophotometer assaying reaction system 5min, obtain initial velocity of reaction; The mensuration of immobilization laccase vigor: 3mL adds 20mg immobilized enzyme containing in the tartaric acid buffer of 0105mmol/LABTS, 55 DEG C of changes of the absorbancy with 420nm place in ultraviolet-visible pectrophotometer assaying reaction system 5min, obtains initial velocity of reaction; ABTS optical extinction coefficient is ε=36000L/ (molcm), and enzyme amount per minute being oxidized 1 μm of olABTS is defined as 1 enzyme activity unit.

Claims (1)

1. chitosan magnetic micro-sphere preparation and be used as Laccase Immobilization carrier, it is characterized in that comprising the steps:

(1) preparation of chitosan magnetic micro-sphere; Get 5mL magnetic fluid to mix with the chitosan acetic acid solution of 20mL215%, under agitation slowly join in the mixed solvent of 80mL whiteruss and 4mLSpan280, fully 30min is stirred under normal temperature, add the glutaraldehyde of 10mL8%, react 60min in the water-bath of 40 DEG C after, by the NaOH solution of 1mol/L, its pH value is adjusted to 9 ~ 10, reaction 2h is continued in 70 DEG C of water-baths, obtain product magnet to collect, then use sherwood oil successively, acetone, distilled water fully washs, suction filtration, 60 DEG C of vacuum-dryings, obtain chitosan magnetic micro-sphere;

(2) enzyme immobilizatio is painted; Get 50mg chitosan magnetic micro-sphere, add the laccase phosphate buffered saline buffer (011mol/L of 5mL116 × 10-3g/mL, pH=7), 25 DEG C are slowly stirred 1h, at 4 DEG C of standing 2h, outwell supernatant liquor, with pH=7 phosphate buffered saline buffer repetitive scrubbing, suction filtration, the immobilized enzyme obtained is preserved at 4 DEG C;

(3) enzyme activity determination; The mensuration of free laccase activity: get 3mL and contain in the tartaric acid buffer of 0105mmol/LABTS the enzyme liquid adding 8 μ L and suitably dilute, 55 DEG C of changes of the absorbancys with 420nm place in ultraviolet-visible pectrophotometer assaying reaction system 5min, obtain initial velocity of reaction; The mensuration of immobilization laccase vigor: 3mL adds 20mg immobilized enzyme containing in the tartaric acid buffer of 0105mmol/LABTS, 55 DEG C of changes of the absorbancy with 420nm place in ultraviolet-visible pectrophotometer assaying reaction system 5min, obtains initial velocity of reaction; ABTS optical extinction coefficient is ε=36000L/ (molcm), and enzyme amount per minute being oxidized 1 μm of olABTS is defined as 1 enzyme activity unit.