Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues

Enzyme pre‐milling treatments improved milling performance of chickpeas by targeting mechanisms of seed coat and cotyledon adhesion with various effects on dhal quality

BACKGROUND: Dehulling and splitting are important elements of the milling process to produce dhal from pulses. However, grain that is difficult-to-mill because of tightly adhered seed coats or cotyledons that resist separation makes it difficult to achieve high quality dhal. Milling yields are reduced, energy inputs into the milling process are increased, and the resulting dhal can be of poorer quality, chipped or abraded. RESULTS: Eight enzyme pre-treatments were chosen based on the hypothesised mechanisms of seed coat and cotyledon adhe-sion established previously. Using a difficult-to-mill chickpea (Cicer arietinum L.) genotype, we examined the effects of these pre-treatments, over time, on laboratory-scale milling performance and dhal quality. We pioneered a texture analyser method to measure the flex of the cotyledons and the force required to cleave the cotyledons. The enzyme-induced changes ranged from negative (tough seed coat, weight loss, deleterious colour and texture, increased visual damage to cotyledons and increased kibble loss, concave cotyledons, increased flex, and changes in taste) to positive (brittle seed coat, increased seed vol ume, improved dehulling efficiency and splitting yield, reduced cotyledon cleavage force, and acceptable dhal quality and taste). CONCLUSION: All pre-treatments improved milling performance compared to milling the raw seed, although there was consid-erable variation between them. Two pre-treatments showed no improvement in milling yields compared to the water control, and several pre-treatments resulted in unacceptable qualities. Three pre-treatments, endo-polygalacturonanase, α-galactosidase and cellulase, show potential for commercial milling applications and could assist pulse millers globally to achieve high quality dhal at the same time as minimising milling effort