RNAi (RNA interference) is a tool for silencing of target genes through sequence-specific manner. Spodoptera exigua belongs to Noctuidae family of Lepidoptera and is serious threat to crops of economic importance. One of S. exigua chymotrypsin gene (SeCHY2) was cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3). Oral delivery of bacterially expressed dsRNA gave significant larval mortality. Quantitative real-time PCR results showed that expression level of target SeCHY2 gene in the larval gut tissue was significantly down-regulated. Pretreatment with an ultra-sonication and heating to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria

A blast search using SPODOBASE gave 10 chymotrypsin (CHY) candidate genes with complete open reading frames (ORFs) of Spodoptera exigua. Among these candidates, Se-CHY2 was chosen and further analyzed in expression and RNAi. Se-CHY2 was expressed only in larval stage and especially in midgut. Its RNAi was successfully performed by double injection of specific double-stranded RNA (301 bp). qPCR showed a decrease of Se-CHY2 expression level by ~50% at 48 h and ~83% at 72 h. RNAi resulted in significant larval mortality. All treated larvae died at 96 h after dsRNA injection. In contrast, control dsRNA treatment did not give any mortality. Enzyme activity of gut juice was measured with N-succinyl-alanine-alanine-prolin-phenylalanine-p-nitroanilide substrate and significantly decreased in larvae treated with dsRNA. These results indicate that Se-CHY2 is a chymotrypsin that is secreted into midgut lumen and plays a role in nutrient digestion.

Cheese analogs using lipoxygenase-defected soymilk and α -chymotrypsin modified soy protein isolate (SPI) were prepared. Color, textural properties, sensory attributes and melting spreadability of cheese analogs were evaluated and compared with mozzarella cheese, and relationships between textural properties, sensory attributes and melting spreadability of cheese analogs were analyzed. Off-flavors were not mostly discriminated. Cheese analogs containing 10% SPI untreated and containing 6% and 8% SPI treated by α -chymotrypsin in δE value of color were the most similar to mozzarella cheese. Quality characteristics and melting spreadability of cheese analogs were highly affected and improved by α -chymotrypsin modification. Sensory attributes and melting spreadability of cheese analogs containing 6% SPI treated by α -chymotrypsin were the most similar to mozzarella cheese, while in textural properties, cheese analogs containing 10% SPI were the most similar with mozzarella cheese. Hardness in sensory attributes was highly positively correlated with hardness (r>0.65), adhesiveness (r>0.56), chewiness (r>0.77) and gumminess (r>0.76) in textural properties, while it was highly negatively correlated with melting spreadability (r>-0.68).

Effects of α -chymotrypsin modification on degree of hydrolysis (DH), solubility, emulsifying capacity and thermal aggregation of laboratory-purified soy protein isolate (SPI) using a lipoxygenase-defected soybean (Jinpum-kong) and commercial soy protein isolate (Supro 500E) were compared. SPIs were hydrolyzed by α -chymotrypsin at pH 7.8 and 37~circC for 30 min. DHs of Supro 500E and Jinpum-kong SPI were increased by α -chymotrypsin modification, and DH of Supro 500E was higher than that of Jinpum-kong SPI. DH of α -chymotrypsin treated Jinpum-kong SPI was similar with untreated Supro 500E and DH of treated Supro 500E was the highest. Solubility, emulsifying capacity and thermal aggregation of SPIs were increased by α -chymotrypsin modification, and these changes were highly related to changes in DH. Functional properties of Supro 500E were higher than Jinpum-kong SPI in both of untreated and α -chymotrypsin treated SPIs.