| As an important industrial enzyme,cellulase can convert cellulose to the energy and other industrial raw materials hunman needed environmentally and friendly.So it has been widely used in industrial fields such as livestock,paper,food and so on.However,its application is still restricted by the low enzyme activity and high production costs.So it is significant to improve the yield of cellulase.In the paper,a high-yielding mutant Rhizopus stolonifer TZ-03 was obtained by mutation screening.And then the fermentation of cellulase media and culture conditions was studied.Subsequently,the amplification experiment was carried out in 10 L fermentor with the control of conditions in the fermentation process.Finally,the FPA of TZ-03 was peaked at 21.32 IU/m L at 84 h when microcrystalline cellulose as the sole carbon source.During the experiment,it was found that ?-glycosidase activity(BG)increased significantly after mutagenesis,so molecular mechanism and structure and function of which was discussed to analysis of reasons.Main research results are as follows:(1)The parent strain Rhizopus stolonifer TP-02 was mutagenized by two different mutantion ways of UV and ethyl methanesulfonate(EMS),and some high-yielding mutants was obtained by the first-screening and second-screening.Then the two ways were combined to do compound mutation,five most high-yielding mutants are as research object.A highyielding strain TZ-03 was obtained,the filter paper activity(FPA)of which was 4.96 IU/m L(37.8% higher than that of TP-02).(2)Filter paper activity as index,the effect of carbon sources,nitrogen sources,amino acid addition,Polyethylene glycols(PEGs),tempertature and initial fermentation p H on the cellulase production was studied.Orthogonal experiment method was applied to confirmed optimized medium composition are as follows: Avicel 20 g/L,peptone 10 g/L,wheat bran extract 2.5%,Ca Cl2 2 g/L,Mg SO4·7H2O 4 g/L,KH2PO4 3 g/L,glutamine 1 g/L,Tween 80 200 ?L/L,PEG-4000 0.25 g/L,trace element solution 1 m L/L,the temperature 30?,the initial p H 5.0.In the above conditions,the FPA of TZ-03 was peaked at 12.75 IU/m L at 108 h.The highest ?-glycosidase activity,endoglucanase activity(EG)and cellobiohydrolase activity(CBH)were 24.10,15.98 and 19.70 IU/m L,respectively.(3)Under optimum conditions previously optimized in shake flask,enlarge experiment was carried out in 10 L fementation tank.The culture conditions are as follows: 10 Lfermentation tank are installed in the liquid amount of 5 L,culture temperature 30 ?,initial pressure 0.08 MPa,ventilation 0.6 L/min and rotational speed 300 rpm.By controling dissolved oxygen is about 30%,p H value is about 4.8 and reducing sugar is about 1.2 mg/m L after 24 h.The results showed that the maximum of FPA was 21.32 IU/m L peaked at 84 h(89.8% higher than that in the shake flask culture conditions).The highest BG,EG and CBH were 41.06,26.76 and 16.94 IU/m L,respectively.(4)The BG activity and transcription levels was compared between TP-02 and TZ-03.The results show that BG is about 1.51 times and transcription levels is 90.19% higher than that of TP-02.A ?-glycosidase gene bgl4 was obtained and the sequence of BGL and BGL IV were compared.Then their structural models was built by DS 3.0 and regard cellobiose as the substrate molecule to dock.The results show that BGL and BGL IV all have a classical structure of(?/?)8-TIM barrel.For BGL,a tunnel-shaped pocket is formed inside the enzyme molecule and cellobiose deep into the binding pocket to interaction with substrate.For BGL IV,the position of active sites shifted a lot and interacted with cellobiose rather surface than closed to the binding pocket wall deeply.This structure can broaden the active site,which help to solve many problems,such as inhomogenous and substrate accumulation.In addition,the amount of hydrogen bonds formed by amino acids and ligands have been increased after mutagenesis.These are possible factor affect the change of ?-glycosidase activity. |
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