Enzymatic Properties,Immobilization And Application Of Laccase Produced By Penicillium Rubens JD3-1

In nature,microorganisms are diverse and abundant,and play an important role in fermentation production,biological transformation and environmental protection.As the"home"for many microorganisms,soil is extremely rich in strains of bacteria.Screening microorganisms from soil and other environments for human needs and applying them to practical work can provide solutions for environmental management,biomass conversion,renewable energy development and promote sustainable development.Laccase,a copper-containing polyphenol oxidase widely found in nature,can use molecular oxygen as an electron acceptor to oxidize various types of substrates such as arylamines,polyphenols and polyamines.It is widely used in various fields such as natural polymer modification and environmental protection.Although laccase is widely available,it is essentially a biological macromolecule that needs to function in a mild environment and is costly and not easily recovered,resulting in many constraints in its practical application.With the improvement of domestic living standards in recent years,the use of various types of antibiotics in the farming industry and other fields is also increasing year by year.Among them,the use of tetracycline antibiotics is the most widespread,at the same time this also inevitably brings environmental pollution problems.Traditional treatment methods are often harsh or prone to cause new pollution,therefore,the use of greener and more natural methods to transform antibiotics in the environment has received more and more attention.In this paper,we screened microorganism JD3-1,which can produce laccase from natural environment,and identified it as Penicillium rubens after morphological and molecular biological identification.After optimizing the enzyme-producing conditions of this strain,it was concluded that its optimal enzyme-producing time was 5 days,its optimal enzyme-producing p H was 5,and its optimal enzyme-producing inoculum was6%.After ammonium sulfate graded precipitation and DEAE-Sepharose FF anion-exchange column chromatography of the laccase produced by P.rubens JD3-1,the enzyme solution was obtained with a specific activity of 35.75 U/mg and a purification factor of 6.64 times.The enzyme properties of the laccase showed that the optimum temperature of the laccase produced by P.rubens JD3-1 was 40℃and the optimum p H was 4.In terms of stability,the optimum tolerance temperature was 30℃and the optimum tolerance p H was 4.Among the common metal ions,the lower concentration of Cu²⁺promoted the activity of the laccase to a certain extent,and the other metal ions,such as Ca²⁺and Mg²⁺,inhibited the activity of the laccase.ions all inhibit laccase activity.At the same time,the surfactant Triton X-100 also produces a more obvious inhibitory effect on the activity of laccase.In terms of storage stability,the enzyme was stored well at 4°C,and 93.7%enzyme activity was still present after 7 days.However,there was only 28.3%enzyme activity after 7 days of storage at 28℃.In order to be able to reduce the actual application cost of laccase and improve its recycling efficiency.In this study,we used cheap corn kernel as the raw material,and after the activation of corn kernel powder by KOH,the magnetic biochar carrier of corn kernel was prepared by high temperature carbonization and chemical co-precipitation,and characterized by scanning electron microscopy,Fourier infrared spectroscopy and thermogravimetric analysis.It was found that the magnetic biochar carrier of corn cob had a very well-developed pore structure with many-OH groups on the surface.The obtained laccase was immobilized using this carrier,and the optimal immobilization time was obtained to be 3 h,and the optimal loading was about 60μg/mg.Finally,the enzymatic properties of the immobilized laccase were investigated.The optimum temperature and p H of the immobilized enzyme were the same as those of the free enzyme.The immobilized laccase was able to adapt to a wider range of environmental conditions,such as temperature and p H,and showed good tolerance to several common metal ions and organic reagents.In terms of storage stability,immobilized laccase still has 73.2%enzyme activity after 7 days of storage at 28°C.In terms of reusability,the immobilized laccase still has 72%enzyme activity after 5 times of reuse,which indicates that the immobilized laccase has good practicality.The preliminary practical application of laccase produced by P.rubens JD3-1 was explored using two tetracycline antibiotics,gentamycin and doxycycline,as substrates.The results showed that the free laccase produced by P.rubens JD3-1 exhibited certain degradation effects on both antibiotics.In the process of removing the two antibiotics by using the immobilized enzyme,it was found that the removal effect of the immobilized enzyme could reach 78.1%and 89%for gentamycin and doxycycline after3 h of reaction.From the above,it can be seen that P.rubens JD3-1 strain can produce laccase under milder environmental conditions,and both its free laccase and immobilized laccase are effective in the removal of tetracycline antibiotics.This study broadens the sources of fungal laccase to a certain extent and may provide ideas for subsequent wider applications.