Screening Of Thermostable Amylase-producing Strain And Ion Beam Mutation Breeding

Starch and glycogen are the most common organic material resources used in fermentation industry.At present,starch and glycogen are often degradated and used by chemical techniques,but cost higher.If use microbial fermentation,there are more advantages such as low cost,environmental protection.The microbial fermentation was mostly carried out under high temperature,and there were a few microbes couLd produce thermostable amylase which can degrade starch and glycogen.It is an important way to make use of starch by screening and transforming microbe which generated thermostable amylase.In this study,a wide range of soil microbe were screened and identified in order to get microbe which generated thermostable amylase.Then mutant strains of high activity were filtered after low-energy -ion implantation.The fermentation conditions and the molecuLar variation of a preliminary study were done.Main resuLts were as follows:1.About 100 soils amples were collected from some high temperature regions in anhui.and screened 11 strains.one of which showed amylase activity at higher temperature.A higher hot-adapted amylase activity producing yeast strain RL-1 was isolated.On the basis of physiology,morphology and analysis by blasting the sequence of 16SrDNA,the strain RL was identified as Bacillus amyloliquefaciens2.By the method of dealing with low energy Ar⁺ and N⁺ ion implantation,we studied the rate of survival and mutation.After dealing with the same implantation energy and dose,Ar⁺ survival rate is lower than N⁺ to bacillus amyloliquefaciens,but N⁺ mutation rate is higher than Ar⁺.After a comprehensive study on the optimization of mutagenesis conditions,the best implantation ion is N⁺,implantation energy is 10keV and implantation dose is 2.08×10¹⁵ions/cm².3.Finally a high efficient degradation celluLose mutant RL-1 has been gotten by low energy ion implantation after two rounds of radiation and screening.The resuLts show that the genetic performance of the mutant is stable and the enzyme activity also increased about 57.1times after several subcuLtures.4.Studying on the enzymatic properties of the strain RL-1,we found the optimum temperature and pH for the enzymatic reaction was 75℃and 6.5 respectively. After heat preservation at a tempreture higher than 60℃about 80 minites,the enzyme activity was stable at a level of 80%,while descended rapidly when the tempreture is lower than 20℃.It was stable in the pH range from 4 to 9.The enzyme activity couLd be stimuLated by Ca²⁺,but inhibited by Cu²⁺,Mg²⁺,Mn²⁺.5.After contrasting the thermostable amylase gene sequences of the wild strain and the mutant one,there are some certain changes in the thermostable amylase gene sequences and amino acid sequences.It's estimated that these changes perhaps are the reasons of the varieties in amylase activity and production of the mutant strain.