| In China,a large amount of crop straw was produced in the process of agricultural production every year.At present,most of the measures taken to solve these problems are mainly burning,which causes a lot of pollution to the environment.Straw is rich in nutrients such as cellulose,protein,trace elements and other nutrients needed by animals.In order to convert crop straw into animal feed and increase the source of animal husbandry feed,microorganisms that had the ability to degrade cellulose and starch were isolated and purified from bovine stomach straw fermentation,and the related genes were cloned in this study,and the enzymatic characteristics of proteins were analyzed.The main results are as follows:(1)11 strains with degradability to cellulose and starch were isolated and purified from bovine stomach straw fermentation,of which G7 strain CMC-Na degradation rings was larger and named SCUEC7 strain;A8 strain starch degradation rings were larger and named SCUEC8 strain.Based on the colony morphology,physiological and biochemical characteristics and 16S rDNA sequence analysis,SCUEC7 strain and SCUEC8 strain were preliminarily identified as Bacillus subtilis.(2)The enzyme production conditions of SCUEC7 strain and SCUEC8 strain of Bacillus subtilis were optimized.The OD600 and production of endoglucanase of SCUEC7 strain were higher under the conditions of 12 h,pH of 6.0,temperature of 37?,carbon source 20.0 g/L maltose,nitrogen source 15.0 g/L pancreatic protein,and 1.5 g/L Mn2+.When the culture time was 16 h,pH was 6.0,the culture temperature was 37?,the carbon source was 20.0 g/L malt,the nitrogen source was 15.0 g/L pancreatic protein,and 1.0 g/L Mn2+,the OD600 and production of ?-amylase of SCUEC8 strain were higher.(3)The entire DNA of SCUEC7 strain of Bacillus subtilis was used as template to obtained ba7gA gene fragments by PCR.The length of ba7gA gene was 1482 bp.Amino acid sequence analysis shows that Ba7gA protein consists of 493 amino acids with a theoretical molecular weight of 54.53 kDa,had 98%similarity with Endo-beta-1,4-glucanase(ce15L)which is derived from the strain of Bacillus subtilis,and belongs to alkaline hydrophilic protein;The entire DNA of SCUEC8 strain of Bacillus subtilis was used as template to obtained ba8a gene fragments by PCR.The length of ba8a gene was 1434 bp.Amino acid sequence analysis showed that Ba8a protein consisted of 477 amino acids with theoretical molecular weight of 52.90 kDa,had 98%similarity with ?-amylase which comes from Bacillus subtilis(ACU57501.1)strains,and Ba8a protein was also an alkaline hydrophilic protein(4)The ba7gA gene?ba8a gene and the plasmid pET28a(+)gene were connected,and pET28a(+)-ba7gA and pET28a(+)-ba8a recombinant plasmids were constructed and transformed into E.coli BL21(DE3)strains for heterogenous expression,indicating Ba7gA protein had endoglucanase activity,and Ba8a protein had ?-amylase activity.Ba7gA and Ba8a proteins induced expression of 20 h at IPTG concentrations of 0.5 mmol/L and temperatures of 25?,and purify proteins with nickel columns.The results show that Ba7gA and Ba8a proteins were mainly concentrated in precipitation,and the content in upper liquid was relatively small.Pure Ba7gA protein was obtained by 300 mmol/L imidazole elution solution,and pure Ba8a protein was obtained by 400 mmol/L imidazole elution solution.The Ba7gA protein had a suitable reaction with pH of 6.0 and a reaction temperature of 60?.Cu2+and Fe3+have an inhibitory effect on Ba7gA protease activity.Fe2+ and Mn2+ have a positive effect on Ba7gA protease activity.After processing 2h in buffers with pH of 4.0,5.0,6.0 and 7.0,the relative enzyme activity of Ba7gA protein decreased to 27.82%,48.13%,73.63%and 56.72%,respectively.After 120 min treatment in 0?,20?,37?,50?,60?,70? and 80?,the relative enzyme activity of Ba7gA protein decreased to 95.66%,81.37%,54.03%,46.01%,37.48%,15.5%and 9.96%.The Km of the Ba7gA protein was 11.89 mg/mL,while the Vmax was 0.53 U/mg.The protease study of ?-amylase Ba8a shows:the optimum reaction pH of Ba8a protein was 5.0 and the reaction temperature was 45?.Cu2+,Fe2+and Fe3+have a certain inhibitory effect on Ba8a protease activity,and Mn2+had a strong effect on the promotion of Ba8a protease activity.After processing 2h in buffers with pH of 4.0,5.0,6.0 and 7.0,the relative enzyme activity of Ba8a protein decreased to 48.75%,56.03%,74.63%and 69.25%,respectively.After 120 min treatment in 0?,20?,37?,45?,55?and 65?,the relative enzyme activity of Ba8a protein decreased to 93.76%,70.97%,66.24%,46.15%,35.81%and 10.51%.The Km of Ba8a protein was 2.84 mg/mL,while the Vmax was 2.98×10-1 U/mg. |
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