Expression And Activity Analysis Of A Serine Carboxypeptidase HoSCP From Holotrichia Oblita(Coleoptera: Scarabaeoidea)

The scarab beetle, Holotrichia oblita Faldermann (Coleopera: Scarabaeidae), is apredominant underground pest in the northern parts of China and causes great economicloss. The larvae of scarab are called grubs, which have seriously damage to several majorcrops including peanuts and potatoes, pasture and plants resulting from feeding on theiryoung roots.Insect midgut proteases are essential for insect development and play an importantrole in the digestion of insect food. Serine carboxypeptidases (SCPs) are widely distributedamong fungi, plants and animals and known to play essential roles in a wide range ofbiological processes, including the degradation of regulatory proteins, precursor processing,apoptosis and digestive processes. To date, the SCP proteins that have been biochemicallycharacterized mainly fall into yeast and plant species. In insects, however, there are a fewSCP sequences and their biological roles have been identified.In this study, the hoscp gene of H. oblita was amplified by PCR method and clonedinto the expression vector pPIC9K. The following recombinant vector was transformedinto yeast Pichia pastoris GS115strain. The results showed that HoSCP was stablyexpressed as a50kDa recombinant protein in P. pastoris and enzyme activity wasdetermined to be0.165U/mL based on hydrolysis the synthetic substrateN-(3-[2-furyl]acryloyl)-Ala-Lys(FAAK). The midgut SCPs of3rd-instar larvae exhibitedmaximal activities at pH8.0and their relative activity was0.020U/mg. The midgut SCPsactivities were analyzed in H.oblita larvae of different instar, and the results indicated thatearly2nd-instar and early3rd-instar larvae had higher levels of SCPs activity permilligram of midgut than those of late2nd-instar and late3rd-instar larvae respectively,which had approximately half the level of activity.We report the expression and activity analysis of a serine carboxypeptidase (HoSCP)identified in the midgut of the H.oblita larvae. It is useful for the further study of biologicalfunction of HoSCP in H. oblita.