Purification and characterization of a trypsin inhibitor from the seeds of Artocarpus heterophyllus Lam  

作　　者：Junchen Lyu Yuan Liu Tianchen An Yujun Liu Manchuriga Wang Yanting Song Feifei Zheng Dan Wu Yingxia Zhang Shiming Deng 

机构地区：[1]Key Laboratory of Tropic Biological Resources, Minister of Education, College of Marine Science, Hainan University, Haikou 570228, China [2]School of Life Science, Jilin Normal University, Siping 136000, China

出　　处：《Acta Biochimica et Biophysica Sinica》2015年第5期376-382,共7页生物化学与生物物理学报（英文版）

摘　　要：A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatog- raphy. The trypsin inhibitor, named as AHLTI （A. heterophyllus Lam. trypsin inhibitor）, consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1：2 （AHLTl：trypsin） analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant （Ki） of 3.7 × 10^-8 M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80℃. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis （Meyen）. These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest.A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatog- raphy. The trypsin inhibitor, named as AHLTI （A. heterophyllus Lam. trypsin inhibitor）, consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1：2 （AHLTl：trypsin） analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant （Ki） of 3.7 × 10^-8 M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80℃. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis （Meyen）. These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest.

关 键 词：Artocarpus heterophyllus Lam. Kunitz-type inhibitor PURIFICATION characterization 

分 类 号：S816.34[农业科学—饲料科学] TS214.2[农业科学—畜牧学]