Amornrat Promboon

Cloning and expression analysis of the Bombyx mori α-amylase gene (Amy) from the indigenous Thai silkworm strain, Nanglai

Abstract &agr;-Amylase is a common enzyme for hydrolyzing starch. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), &agr;-amylase is found in both digestive fluid and hemolymph. Here, the complete genomic sequence of the Amy gene encoding &agr;-amylase from a local Thai silkworm, the Nanglai strain, was obtained. This gene was 7981 bp long with 9 exons. The full length Amy cDNA sequence was 1749 bp containing a 1503 bp open reading frame. The ORF encoded 500 amino acid residues. The deduced protein showed 81–54% identity to other insect &agr;-amylases and more than 50% identity to mammalian enzymes. Southern blot analysis revealed that in the Nanglai strain Amy is a single-copy gene. RT- PCR showed that Amy was transcribed only in the foregut. Transgenic B. mori also showed that the Amy promoter activates expression of the transgene only in the foregut.

An active recombinant cocoonase from the silkworm Bombyx mori: bleaching, degumming and sericin degrading activities.

BACKGROUND Cocoonase is a serine protease produced by silk moths and used for softening the cocoons so that they can escape. Degumming is one of the important steps in silk processing. This research aimed to produce an active recombinant Bombyx mori cocoonase (BmCoc) for the silk degumming process. RESULTS A recombinant BmCoc was successfully expressed in a Pichia pastoris system. The purified enzyme showed specific activity of 227 U mg(-1) protein, 2.4-fold purification, 95% yield and a molecular weight of 26 kDa. The enzyme exhibited optimal temperature at 40 °C and optimal pH at 8, and showed thermal stability at 25-45 °C and pH stability at 5-9. The recombinant enzyme exhibited sericin degumming ability and color bleaching characteristics, and did not affect the fibroin fiber. The enzyme also degraded sericin substrate with a product size about 30-70 kDa. CONCLUSION In this study, we successfully produced the active recombinant BmCoc in P. pastoris with promising functions for the Thai silk degumming process, which includes degumming, sericin degrading and color bleaching activities. Our data clearly indicated that the recombinant enzyme had proteolytic activity on sericin but not on fibroin proteins. The recombinant BmCoc has proven to be suitable for numerous applications in the silk industry.

Physico-chemical properties and in vitro response of silk fibroin from various domestic races.

In this study, the characteristics of silk fibroin films obtained from regenerated aqueous silk fibroin solution of various races were evaluated. Three races based on original sources of silkworms were selected: Nangnoi-Sisaket 1 (NN), Nakhon Ratchasima 1 (K1), and Nakhon Ratchasima 2 (K8). The Tg and Td of silk fibroin films were around 147-156°C and 277-279°C, respectively. FTIR result revealed that all silk fibroin films had beta sheet conformation. K1 and K8 films were found to be more hydrophilic than NN film. From in vitro tests using L929 mouse fibroblast and rat bone marrow-derived stem cells (rMSCs), the cell attachment was slightly greater on K1 film than the other two films. The enhanced cell growth on K1 film might be related to its slightly high hydrophilicity and the higher content of serine. The result of osteogenic differentiation test showed that alkaline phosphatase and deposited mineral of rMSCs were higher on all silk fibroin films than on tissue culture plate. K1 film tended to promote osteogenic differentiation of rMSCs to a higher extent than NN and K8 films. K1 silk fibroin film exhibited a higher potential to support cell attachment, proliferation, and differentiation than other two films.