Functional expression of a <italic>Bombyx mori</italic> cocoonase: potential application for silk degumming

Abstract

Cocoon, a shelter for larva development to silk moth, contains the fibrous protein fibroin, which is coated by the globular protein sericin. Emergence of the silk moth requires the action of cocoonase, a protease secreted by the pupa. The full-length prococoonase cDNA, with 780 bp open reading frame encoding 260 amino acids, was cloned by reverse transcription from total RNA of the head of 6-dayold Thai-silk Bombyx mori pupa. Only the gene fragment lacking the propeptide encoding sequence was successfully expressed in Pichia pastoris, yielding an extracellularly active cocoonase. The recombinant cocoonase was purified to homogeneity by 80% ammonium-sulfate fractionation and CM-Sepharose chromatography, and its internal peptide sequences were analyzed by nano liquid chromatographymass spectrometry/mass spectrometry. This monomeric protein has native molecular weight of 26 kDa by gel exclusion analysis and 25 kDa subunit size by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme hydrolyses sericin but does not hydrolyse fibroin, as shown by radial diffusion on thin-layer enzyme assay (RD-TEA). Scanning electron microscopy showed that purified recombinant cocoonase could remove sericin from natural silk completely in 24 h, without damaging fibroin, using only 1 immobilized sericin unit (ISU) of enzyme as determined by RD-TEA. Natural cocoonase isolated from B. mori pupa could also digest sericin effectively, but required more enzymes (2 ISU) and longer time (48 h). In comparison, a commercial enzyme, alcalase, with the same activity not only showed less complete digestion of sericin but also caused damage of fibroin. These results suggest that recombinant B. mori cocoonase is potentially useful for silk degumming.

An active recombinant cocoonase from the silkwormBombyx mori: bleaching, degumming and sericin degrading activities

“…The fibroin damage analysis, weight loss, SEM analysis and OPA assay experiments supported the same result: cocoonase had no effect on fibroin fiber. The data coincided with other degumming proteolytic enzymes such as the bromelain from pineapple fruit or protease from Bacillus subtilis C4 isolated from degumming wastewater (data not shown) or recombinant cocoonase from Rodbumrer et al 21 This current study clearly demonstrated that the cocoonase had no effect on fibroin fiber. The catalytic function of cocoonase in the silkworm during developmental stages needs to be further characterized.…”

“…More than 90% of purified recombinant BmCoc was recovered from yeast culture medium and retained activity for as long as 1 h of incubation at pH 8 and 40 ∘ C. Obtaining recombinant BmCoc expression was enhanced by removing the signal peptide at the N-terminus of the protein. The data obtained in this study are closely related to the report of Rodbumrer et al, 21 which expressed B. mori cocoonase using pPICz-A expression vector with minor adjustments in vector construction, i.e. size of expressed mature cocoonase with the removal of a signal protein and addition of histidine tag for protein purification.…”

“…Our data clearly demonstrated that the recombinant BmCoc was suitable for the silk degumming process, i.e. using a short degumming time (1-2 h) was superior to the recombinant enzyme from Rodbumrer et al, 21 which used 24 h for complete degumming.…”

“…It could therefore be suitable for preparing low-molecular-weight sericin for cosmetic applications. Even though this study and Rodbumrer et al 21 expressed the recombinant cocoonase from Thai B. mori using the same Pichia expression vector backbone, the minor adjustment made by deleting the signal peptide finally resulted in three characters of recombinant BmCoc, i.e. sericin degumming, sericin degrading and color cocoon bleaching properties.…”

“…Both the natural function and industrial applications of cocoonase are emerging topics. Owing to the difficulties in the preparation of natural cocoonase, the production of recombinant Bombyx mori cocoonase (BmCoc) has been investigated in various kinds of expression systems, namely Escherichia coli, insect cells and yeast cells . This present study explores production of the active recombinant B. mori cocoonase in a yeast expression system and explored its successful applications in Thai silk degumming processing, which include degumming, color bleaching and sericin‐degrading properties.…”