Breeding Of High-yield Raw-Starch-digesting-glucoamylase Producer And Study On Its Mechanism For Producing This Enzyme

Raw starch digesting glucoamylase(RSDG)can directly hydrolyze raw ungelatinized crystal starch below the gelatinization temperature and release glucose as the sole product.The ulization of RSDG in hydrolyzing starch process will omit the high temperature.Therefore the process will be simplified and a lot of energy will be saved.A strain which can secret RSDG was screened from decayed cassava root in this study.The strain was identified as Aspergillus niger by molecular and morphology analysis and named as Aspergillus niger F-01(F-01).Aspergillus niger G-11,a high-producing mutant from F-O1 after ultraviolet and nitrosoguanidine mutagenesis,produced RSDG activity of 77.8 U/mL when starch was used as carbon source,which was 3.4-fold of that from F-01.To investigate which mutations resulted in the improved RSDG activity from F-01 to G-11,the genes encoded RSDG,its promotor and creA(carbon catabolite repression)in F-01 and G-11 were cloned and sequenced.The functional difference between the genes encoding RSDG and their promotors from F-01 and G-11 was analyzed.Analyzing the sequences revealed that the full length of DNA and cDNA open reading frame of the gene encoded RSDG from F-01(rsdgL)were 2169 bp and 1920 bp,respectively.The gene contains 4 introns and encodes the protein containing 639 amino acids with a signal peptite of 18 amino acids.The full length of DNA and cDNA open reading frame of the gene(rsdgH)encoded RSDG from G-11 were 2172 bp and 1923 bp,respectively.The gene contains 4 introns and encodes the protein containing 640 amino acids with a signal peptite of 18 amino acids.Th homology of the two genes was 99.86%.The difference between the two genes was that rsdgH contains extra 3 bp(CGG)which lies in 1768-1770 bp whereas rsdgL does not have.The rest sequences of the two genes were the same.The length of the promotors of rsdgL F-011 and rsdgH are 653 bp and 615 bp,respectively.The two promotors both contain the conserved sequences in eukaryotic promotors.The promotor from G-11 lacks a fragment of 38 bp which lies in the region from 415-452 bp in the promotor of F-01 and the rest sequences of the two promotors were the same.The sequences of creA from F-01 and G-11 were identical.The gene contains no intron and encodes the protein containing 427 amino acids.It was observed that Pichia pastoris expressing rsdgL and rsdgH produced almostly the same RSDG activity.It can be concluded that the mutation in the gene encoding RSDG cannot result the improved enzyme activity from F-0 to G-11.But the comparison of the functions of the two promotors revealed that the mutation in the promotor brought the increased activity of the promotor.The result indicated that the mutation in the promotor accounts for the increased RSDG activity from F-01 to G-11.When cassava starch,dextrin,maltohexaose,maltose and glucose were used as carbon source for RSDG production by F-01 and G-11,respectively,it was found that the more the carbon source easily was utilized by the strain,the less RSDG was produced.Therefor production of RSDG in F-01 and G-11 was effected by the kind of carbon sources and under the control of carbon catabolite repression.RNAi with creA in F-01 or G-11 resulted in RNAi mutants,FR-06 and GR-6.RNAi relieved the carbon catabolite repression and improved RSDG production.In the glucose carbon source based medium,RSDG acvtivity form FR-06,a strain with RNAi with creA from F-01,was 16.2-fold of that from F-01.RSDG activity form GR-6,a strain with RNAi with creA from G-11,was 11.9-fold of that from G-11.In the starch carbon source based medium,RSDG acvtivity form FR-06 was 11.4-fold of that from F-01,and RSDG acvtivity form GR-6 was 5.2-fold of that from G-11.It was found that maltose stearic acid ester,an analog of maltose,could trigger the transcript of RSDG gene and therefore improve RSDG production obviously.In the glucose carbon source based medium,8 g/L maltose stearic acid ester improved RSDG from F-01 by 8.1-fold and 5 g/L maltose stearic acid ester improved RSDG from G-11 by 8.3-fold.In the starch carbon source based medium,6 g/L maltose stearic acid esterimproved RSDG from F-01 by 6.1-fold and 4 g/L maltose stearic acid ester,improved RSDG from G-11 by 4.5-fold.RSDG activity improved from 408 U/mL to 687.3 U/mL after optimization the medium composition and cultural conditions by single factor,Plackett-Burman and response surface experiment.The RSDG activity was greatly higher than similar results from other researchers.The RSDG was purification by salting out from ammonium sulphate,DEAE Sepharose FF ion exchange chromatography and Sephadex G-75 gel chromatography,with a purify fold of 45.0 and recovery rate of 6.35%.The optimum temperature of RSDG in this study was 50 ? and the enzyme was stable below 50 ?.The optimum pH of the enzyme was 6.5 and with a pH sable range of pH5.0-8.0.Mn2+,Ca2+ and Fe2+ active the enzyme activity.Zn2+ and Ni2+ inhibit enzyme activity obviously.The molecular weight of the enzyme was about 70 kDa.The product from cassava starch hydrolyzed by the enzyme was only glucose,which indicated the enzyme was RSDG.RSDG from this study was used in the fermentation of raw cassava meal to produce ethanol.18.10%(v/v)ethanol was obtained at 72 h while 16.22%(v/v)ethanol was obtained from liquefied cassava meal under the same conditions.Therefore,RSDG has a potential use in production of ethanol from raw material fermentation.