The Breeding Of Maltotriose-forming Amylase Producing Strain And Fermentation Process Optimization And Preparation Of Maltotriose Optimization

Currently the production process of maltotriose and the enmymes used in which is monopolized by several foreign componies,maltotriose-forming amylase from Microbacterium imperiale is desired to prepare maltritose obtained by hydrolysis of starch.We did some research on Microbacterium imperiale,mainly including three aspects:mutation breeding of the strains,optimization of fermentation process,preparation of maltotriose.(i)First,a mutagenesis of atmospheric and room temperature plasma was used to deal with the starting strain Microbacterium imperiale Mebl-012,and we picked out some single colonies according to the strain colony morphology,after fermentation screening,a high-yielding strain of maltotriose-forming amylase Metp-57 was screened out,its maltotriose-forming amylase production can reach 12.06 U/mL,compared with the yield of the original strain Mebl-012 5.82 U/mL,increased by 107.26%.Then choosing the the high-yielding strain Metp-57 as the starting strain,we conducted the traditional UV mutagenesis on it.After fermentation rescreening,we screened out a high-yielding strain Metu-24.The strain fermentation test showed that after 3 days of fermentation,the maltotriose-forming amylase output can reach 20.42 U/mL,compared with the original strain Mebl-012 yield increased by 257.31%,and its genetic stability is good.(ii)Then we took the high-yielding strain Metu-24 as the research object,and optimized its fermentation process.First of all,the fermentation medium was optimized through the single factor experiment and the method of response surface optimization,and the best medium formula is:soluble starch 17.96 g/L,bran 15.78 g/L,NaNO3 1.51 g/L,K2HPO4 2 g/L,MgSO4·7H2O 1 g/L,Na2HPO4 1 g/L,CaCl2 1 g/L,pH 7.0-7.2.After fermentation verification the maltotriose-forming amylase production can reach 34.71 U/mL,improved 71.07%compared with the yield 20.42 U/mL which obtained before the optimization.Then on the basis of the medium,the shaker fermentation process was optimized,and the optimum culture conditions obtained for fermentation were:liquid volume 80 mL,inoculum size 5%,temperature 30?,the initial pH 7.0,speed of rotary shaker 150 r/min.At last the fermentation conditions of 5 L fermentor were preliminarily explored,on the conditions of liquid filling factor 60%,ventilation ratio 1:1(v/v),tank pressure 0.03 MPa,the following fermentation conditions were preliminarily determined as following,the dissolved oxygen was set at 30%connecting with the rotational speed,during the mid-fermentation,the pH was stabilized at 6.8 when the natural pH dropped to 6.8.Under the optimized fermentation conditions,the yield of maltotriose-forming amylase can reach 56.71 U/mL,although slightly higher than the yield of shaker,it increased by 23.76%,compared with the yield of uncontrolled pH 45.82 U/mL.(iii)The molecular mass of the purified maltotriose-forming amylase was approximately 55 KDa.The optimal reaction temperature was 55? with a complete inactivation at 70? for 4 h,the optimal reaction pH was 6.5,the amylases maintained excellent stability between pH 6.0 and 7.0.The catalytic activity of maltotriose-forming amylase can be promoted by the metal ion of Ca2+but inhibited by Fe3+or Ni2+.The ?-helix,?-helix,?-strand,?-strand,turn,and unordered of the protein showed 0,0,1.4%,9.5%,28.4%and 60.8%,respectively,detected by circular dichroism estimation.After the enzyme contacts substrate,the protein structure had changed,the ?-helix,?-helix,?-strand,?-strand,turn,and unordered of the protein showed 21%,13.5%,4.3%,6.7%,23.1%and 31.3%,respectively.(iv)Then we took the maltotriose-forming amylase as the research object,and optimized preparation of maltotriose.The maltooligosaccharide maltotriose was prepared under the following conditions:substrate,20%soluble starch;pH 6.1;maltotriose-forming amylase,1.5 U/g dry starch;proportion of maltotriose-forming amylase and pullulanase,1.5:1;reaction temperature,57?;and reaction time,48 h.The starch conversion rate was 55.18%,and the maltotriose content in the maltooligosaccharide was 57.25%.