Improved Laccase Production From Trametes Versicolor By Copper Ion Induction And Ultrasound Intensification And Its Application In Juice Clarification

Laccases（EC 1.10.3.2）are belonging to a family of copper-containing oxidases,which exist in a variety of bacteria,fungi,insects,and plants.Laccase-based biocatalysts are well suited to the development of efficient,sustainable,and environmentally friendly industries.Every laccase application required the enzyme to be generated on a large scale ideally in a cost-effective manner.The current study focused on improving laccase production from Trametes versicolor and its application in juice clarification.For this purpose,laccase production from T.versicolor was enhanced by adding copper in a novel manner and the effect of ultrasound on the intensified laccase production from T.versicolor was explored.In addition,the immobilized laccase on magnetic metal-chelated nanoparticle was prepared and applied for apple juice clarification in the magnetically stabilized fluidized bed.The experimental results have been summarized as follows.1.First,the experimental results have shown that it was not that the better laccase production was achieved if the more Cu²⁺was added.Excessive Cu²⁺addition exhibited an adverse effect.Appropriate Cu²⁺addition could shorten the fermentation time and increase the production of laccase.Consequently,the Cu²⁺addition amount for promoting the growth of T.versicolor was 200μM,and the Cu²⁺addition amount for the best laccase production was 400μM.In addition,the results showed that the multiple addition of Cu²⁺in the fermentation process provided a promoting effect on the production of laccase,and it was best to add low Cu²⁺dosage in the early culture stage to improve cell growth and then to increase to high Cu²⁺dosage to promote laccase production and laccase activity.Therefore,to obtain the best laccase production,Cu²⁺needed to be added step by step in the early stage of fermentation.2.Optimal conditions for low-frequency ultrasound-enhanced laccase production by T.versicolor fermentation were:once at 48 h and once at 60 h for 5 min,respectively,during the incubation process.The best sonication effect was obtained at a sonication frequency of 28 k Hz with a laccase activity of 1273.1 U/L,which was 2.5times higher than that of the control group,significantly increasing the level of laccase production by T.versicolor.Furthermore,the optimal ultrasound intensification strategy was high-frequency ultrasound combined with low-frequency ultrasound.Ultrasound was performed once at 70 k Hz for 7 min at 24 h and 36 h of the culture period,and once at 28 k Hz for 5 min at 48 h and 60 h of the culture period.The fermentation period was shortened to 5 days,and the laccase enzyme activity and protein content in the fermentation broth reached 2237.6 U/L and 468.2 mg/L,which were both 4-fold higher compared to the control group.In this case,the mycelial morphology was changed by ultrasound treatment.Ultrasonication intensification promoted the increase of rho A gene expression and the decrease of rho B gene expression,resulted in a hyperbranched mycelial morphology and a bulb-like top mycelial morphology that were favorable for laccase secretion.These could be the possible mechanism of ultrasonic intensification strategy to improve the laccase production level of T.versicolor.3.Laccase was immobilized on metal-chelated magnetic silica nanoparticles and used for continuous juice clarification in a magnetically stabilized fluidized bed（MSFB）assisted by alternating magnetic field.Furthermore,a new combination of laccase catalysis and microfiltration was developed for juice clarification.Immobilized laccase provided high relative activity within broader ranges of p H and temperature compared to the free enzyme.Magnetically immobilized laccase exhibited the best reaction rate of 12.1μmol g^-1min^-1for catechol oxidation under the alternating magnetic field of 400 Hz,60 Gs.No activity loss occurred in immobilized laccase after 20 h continuous operation of juice treatment in MSFB under alternating magnetic field.Combined with microfiltration after treatment with immobilized laccase,the color of apple juice was decreased by 33.7%and the light transmittance was enhanced by 20.2%.Furthermore,only 16.3%of phenolic compounds and 15.1%of antioxidant activity were reduced for apple juice after the clarification.By this combination strategy,the apple juice possessed good freezing-thaw and thermal stability.