Enzymatic Characteristics And Partial Gene Of Trypsin-like Enzymes From Euphausia Pacifica Hansen

In this paper, trypsin-like enzymes (TLEs) were purified from North Pacific krill (Euphausia pacifica Hansen) and characterized. Trypsin genes were cloned, sequenced and analysised. This is aimed to provide primary data and theory support for future commercial utilization of E. pacifica TLEs in science, medicine and industry.At least two TLEs were identified from the crude exact of E. pacifica by 2 dimensional electrophoresis (2-DE) and mass spectrometry (MS). By solid ammonium sulfate fractional precipitation with saturation between 35 % and 65 %, DEAE sepharose Fast Flow ion exchange chromatography, sephadex G100 gel chromatography and Benzamidine Sepharose 4B affinity chromatography, two TLEs, TLE I and TLE II were purified. They had molecular masses of 32.9 and 32.3 kDa respectively. The specific activity and purification fold of TLE I were 91.4 BApNA Unit per mg protein and 177. And those of TLE II were 113 BApNA Unit per mg protein and 218 respectively.The purified TLE I and TLE II were active in extremely wide pH range of 6.0~11.0 and the optimum values were between pH 9.0 and 10.0 at 37°C with BApNA as subtrate. And they were active in temperature range of 20~70°C, extremely wide, with optimum values between 40 and 50°C at pH 9.0, BApNA as subtrate. TLE I and TLE II were stable at 40°C or lower, which were deactivated with temperature over 40°C。With metal ions, Mg2+(≥0.3 mmol L-1)and Ca2+(≥0.2 mmol L-1) had activation on TLE I and TLE II, while Zn2+(≥0.1 mmol L-1), Cu2+(≥0.2 mmol L-1), Hg2+(≥0.1 mmol L-1) and Pb2+(≥0.1 mmol L-1)showed different degree of inhibition on them, especially obvious at the presence of Hg2+ and Pb2+.The activities of TLE I and TLE II were relative stable with ethanol (≤10 %), glycerin (≤5 %) and Dimethyl sulphoxide (DMSO) (≤5 %) and deactivated with concentration over≥15 %,≥10 %,≥10 %10 % (v/v) respectively. Inhibitions of acetone (≥5 %) were significantly.Serine specific inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and soybean trypsin inhibotor (STI), and trypsin specific inhibitors, such as p-amionbenzamidine, chicken egg ovomucoid (CEOM), benzamidine and N-α-tysol-L-lysine chloromethyl ketone (TLCK) had different degree of inhibition on TLE I and TLE II; chymotrypsin specific inhibitor, N-α-tysol-L-phenylalanine chloromethyl ketone had no inhibition on TLE I and TLE II. It confirmed that TLE I and TLE II were of trypsins and belonged to serine proteinase families. The Km, Kcat and Kcat / Km of TLE I was 0.088 mmol L-1, 0.84 S-1 and 9.54 mmol L-1 S-1. And those of TLE II was 0.064 mmol L-1, 0.98 S-1and 15.3 mmol L-1 S-1 respectively。The Kcat / Km of the TLEs were 1.6~6.7 times higher than other organism.The inhibition type of Benzamidine on TLE I is noncompetitive inhibition, and the inhibitor constant of Benzamidine is 0.07 mmol L-1.The functional group study of TLE I showed that, tryptophan residue and histidine residue is essential group to active center. And it was speculated the arginine residue might be an essential group to active center. The freeε-amino group of lysinethe residue had relation with enzyme activity and the free hydroxyl group might have relation with it. According to the result, sulfhydryl group had no relation with enzyme active centre.Study on trypsin gene of 9 kinds of crustaceans revealed the consistence of gene structure in them. There are at least three trypsin gene families, with 13.2~66.1 % identity between introns and 65.9~90 % identity between genes of different families; and 1.9~6.2 % mutation between introns and 2.5~6.7 % mutation between genes of the same families.Analysis on the sequence of deduced protein showed that the essential active center, such as the catalytic triad residues, substrate binding group and disulfide bridge were conservative in crustaceans.The evolution rule revealed by polygenetic analysis on trypsin amino acids sequence might do not consistent with evolution theory nowadays completely.It was speculated that, because of low content of polar amino acid, such as Arg, Lys and Arg/(Arg+Lys) ratio, high Asp, Glu and Ile content, low Met and Pro content in crustacean trypsins, they had lower pH-thermal stability, but higher flexibility, especially higher physiological efficiency with them.