Culture Optimization Of High Density Of Chlorella Sp. And The Extraction Of Metallothioneins-like Proteins From The Algae Under The Stress Of Zinc

Metallothioneins?MTs?are a class of low-molecular-weight proteins which are rich in thiol groups and heavy mental ions.MTs have many biological functions,such as antioxidant,immune modulating function and anti-tumor.However,the application of MTs has always been limited by the high cost because of the rare raw materials?mammalian liver?and complex extraction process.It is very necessary to explore new resources for extending the application of MTs.Recent studies found that Chlorella sp.can produce the metallothionein like?MT-like?proteins if it is induced by heavy metal?zinc,etc?,which providing new insights for MTs research.In this study,culture condition of Chlorella sp.is optimized at first,then the zinc combination metallothionein like?zinc-MTs-like?proteins induced when Chlorella sp.was exposed to Zn²⁺.The structure of zinc-MTs-like was preliminary characterized after separation and purification.This dissertation consists of four chapters.Chapter one details the source,classification,structure,physiological function,separation,purification and detection methods of metallothionein.Besides,the cultivation of Chlorella sp.and the status of Chlorella sp.MTs proteins research were summarized.In chapter two,the pH and salinity of the medium was optimized first for production of high-density Chlorella sp.culture.Based on this,Plackett-Burman method is used to screen three main factors influencing the growth of Chlorella sp.,sodium bicarbonate,potassium nitrate and Vitamin B₁₂.Box-Behnken method is then employed to determine the optimal concentration.The results show that the optimal culture condition is as follows:pH 5,40 salinity,0.93 g L^-1sodium bicarbonate,0.40 g L^-1 magnesium sulphate,0.46 g L^-1 potassium nitrate,0.02 g L^-1potassium phosphate dibasic,0.60 mg L Vitamin B1,1.8?g L Vitamin B12,and 2.0?g L^-1 biotin.The Chlorella sp.biomass reached 4.5×10⁷ cell/mL after 1:1 72 h expanding cultivation,which was increased by 32.5%than before.Chapter three mainly focus on the effect of zinc ion on the growth of Chlorella sp.and the induction of metallothionein.This chapter studied the changes of biomass,heat stable proteins and zinc-binding proteins when Chlorella sp.was exposed to different concentrations of Zn²⁺(5,10,20,50 and 100?mol L^-1).According to the results,the growth of Chlorella sp.had no significant effect at low concentrations(<10?mol L^-1)of Zn²⁺?P>0.5?,while,it was significantly inhibited at 50?mol L^-1 or higher of Zn²⁺?P<0.5?.All Zn²⁺at different concentrations significantly inhibited the growth of Chlorella sp.when extended to 4 days or more.When Chlorella sp.was exposed to zinc gluconate at 50?mol L^-1 for three days,the Zinc binding protein induced the most and the heat stable proteins reached the maximum of 34.5 mg/g,which was 3.3 times compared to control.Therefore,the best stress condition is exposure to zinc gluconate at 50?mol L^-1 for three days.In chapter four,Chlorella sp.was exposed to 50?mol L^-1 of zinc gluconate and was cultivated for 3 days.After being harvested by centrifugation,the washed algal cells were homogenized by a supersonic cell disintegrator.Chlorella sp.Zn-MT-like proteins were separated with a Sephadex G-75 gel filtration column and purified with a desalting G-25 gel filtration column,and then characterized its structure features.The results showed that the breakage rate can reach 95%and heat resistant protein can reach more than 46 mg/g after 3 times of freeze-thaw cycles,37^oC heating for five minutes,ultrasonic cell disruption for nine minutes.Moreover,the target peak components were collected and subjected after vacuum freeze drying for three days.Preliminary characterization results indicated that the protein's ultraviolet absorption spectroscopy and infrared absorption spectra characteristics had typical MTs spectral characteristics.