降解大豆胰蛋白酶抑制剂的短小芽孢杆菌菌株及其胞外蛋白的鉴定

本研究以大豆胰蛋白酶抑制剂为唯一碳源,从豆类内部筛选获得3株具有降解大豆胰蛋白酶抑制剂的细菌。通过对菌株的16Sr DNA基因的分析,3株细菌分别被鉴定为枯草芽孢杆菌LZ013-1、短小芽孢杆菌LZ013-2和类芽孢杆菌LZ013-3。进一步研究发现LZ013-2菌株的上清液具有高效降解大豆胰蛋白酶抑制剂的活性,2h可将胰蛋白酶抑制剂抑制率降低73.60%。将LZ013-1和LZ013-2菌株应用于豆粕发酵中,两株芽孢杆菌均能高效降解胰蛋白酶抑制剂。原始豆粕的胰蛋白酶抑制剂活性为22.26 mg/g,经过LZ013-1和LZ013-2菌株的液态发酵后分别降低为0.50 mg/g和0.63 mg/g,经过固态发酵后分别降低为1.06 mg/g和1.03 mg/g。通过硫酸铵分级沉淀和凝胶柱层析,分离LZ013-2菌株发酵上清液中具有降解活性的蛋白质,并采用质谱鉴定分离得到的蛋白质,筛选并鉴定出2种活性蛋白,分别为肽酶S8(Uniprot ID:A0A2T0DB16)和肽酶M84(Uniprot ID:A8FIH7)。

Identification of Soybean Trypsin Inhibitor-degrading Bacillus pumilus Strains and Their Extracellular Proteins

Soybean trypsin inhibitor (STI) was used as the sole carbon source, three strains of bacteria capable of degrading soybean trypsin inhibitors were isolated from the interior of soybeans. The 16S rDNA sequence analysis revealed that the strains were Bacillus subtilis LZ013-1, Bacillus pumilus LZ013-2 and Paenibacillus sp. 013-3. Further studies showed that the supernatant of LZ013-2 exhibited a high activity for degrading soybean trypsin inhibitors, and the rate of trypsin inhibitor inhibition was reduced by 73.60% in 2 h. LZ013-1 and LZ013-2 were used in the fermentation of soybean meal, and were s found to degrade trypsin inhibitors efficiently. The trypsin inhibitor activity of the untreated soybean meal was 22.26 mg/g, which was reduced to 0.50 mg/g and 0.63 mg/g, respectively, after liquid fermentation with LZ013-1 and LZ013-2 strains, and decreased to 1.06 mg/g and 1.03 mg/g, respectively, after solid-state fermentation with LZ013-1 and LZ013-2 strains. The proteins with degrading activity were separated from the supernatant of the sample obtained via fermentation with LZ013-2 strain by ammonium sulfate fractionation and gel column chromatography. The separated proteins were identified by mass spectrometry. Two kinds of active proteins were screened and identified as peptidase S8 (Uniprot ID: A0A2T0DB16) and peptidase M84 (Uniprot ID: A8FIH7).