Dissolution and Degradation of Bacillus thuringiensis δ-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori

Abstract

The dissolution and degradation of 8-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.

Lectin‐like binding of Bacillus thuringiensis var, kurstaki lepidopteran‐specific toxin is an initial step in insecticidal action

“…It seems likely that the 54 kDa polypeptide is analogous to the 59 kDa polypeptide produced by treatment of alkali-solubilised S-endotoxin with Bombyx mori gut extract [20]. This 59 kDa polypeptide was shown to be the primary toxic principle responsible for toxicity towards lepidopteran larvae [20].…”

“…It seems likely that the 54 kDa polypeptide is analogous to the 59 kDa polypeptide produced by treatment of alkali-solubilised S-endotoxin with Bombyx mori gut extract [20]. This 59 kDa polypeptide was shown to be the primary toxic principle responsible for toxicity towards lepidopteran larvae [20]. Incubation of soluble toxin with P. brassicae gut extract at 37°C for longer than 30 min resulted in the appearance of a 50 kDa polypeptide concomitant with a decrease in the intensity of the 54 kDa band and a reduction in in vitro toxicity to CFl cells (not shown).…”

EFFECT OF DISSOLUTION AND DEGRADATION OF BAClLUlS THURlNGIENSlS PARASPORAL CRYSTALS ON TOXICITY TO COTTON BOLLWORM*

“…To become insecticidal, the disso-lution and degradation of the crystals were necessary steps. Tojo and Aizawa (1983) reported that crystals swelled in the alkaline environment , and Bombys mori gut juice protease was effective for dissolving only such swollen crystals. Our results showed that the crystal was easier to be digested by H .…”

“…nism and is helpful to use Bt products rationally and effectively. Tojo and Aizawa (1983) reported that crystals of Bt var. kurstaki HD-1 were dissolved in buffers at pH>12 or at pH between 10 and 11 , supplemented with gut juice protease of silkworm Bombyx mm'.…”

Immuno‐analytical method development for detection of transgenic Cry1Ac protein and its validation

“…3 These Cry proteins are synthesized as protoxins of about 130-140 kDa and processed to form 67-70 kDa active toxins. 4 They comprise 50 subgroups with more than 200 members and are closely related based on the sequence. Further, the proteins designated Cry1A (including Cy1Aa, Cry1Ab, and Cry1Ac) are identical (>85%) in the amino acid sequence.…”