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Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-l-Lys-l-Pro-l-Leu-Gly-l-Leu-[N3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl]-l-Ala-l-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

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Mulberry leaves contain iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin (GAL-DNJ) that inhibit α-glucosidase. In this study, we quantified iminosugars in Morus australis leaves and made the kinetic analysis in the hydrolysis of maltose by α-glucosidase. By LC-MS/MS, the concentrations of 1-DNJ, fagomine, and GAL-DNJ in the powdered leaves were 4.0, 0.46, and 2.5 mg/g, respectively, and those in the roasted ones were 1.0, 0.24, and 0.73 mg/g, respectively, suggesting that the roasting process degraded iminosugars. Steady-state kinetic analysis revealed that the powdered and roasted leaves exhibited competitive inhibition. At pH 6.0 at 37ºC, the IC50 values of the extracts from the boiled powdered or roasted leaves were 0.36 and 1.1 mg/mL, respectively. At the same condition, the IC50 values of 1-DNJ, fagomine, and GAL-DNJ were 0.70 μg/mL, 0.18 mg/mL, and 2.9 mg/mL, respectively. These results suggested that in M. australis, 1-DNJ is a major inhibitor of α-glucosidase. Abbreviations 1-DNJ: 1-deoxynojirimycin; GAL-DNJ: 2-O-α-D-galactopyranosyl-DNJ

The inhibition of α‐amylase and α‐glucosidase are important for the maintenance of blood glucose level. Mammalian α‐glucosidase includes maltase–glucoamylase and sucrase–isomaltase complexes. In this study, we examined the inhibitory effects of Morus australis fruit extract and its components, that is, three iminosugars (1‐deoxynojirimycin [1‐DNJ], fagomine, and 2‐O‐α‐D‐galactopyranosyl deoxynojirimycin), two anthocyanins (cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside), and glucose, against α‐amylase and α‐glucosidase. We also quantified the concentration of each component in M. australis fruit extract. The IC50 values of the fruit extracts of four M. australis subspecies were >10 mg/ml for α‐amylase, 1.1–1.7 mg/ml for maltase, 6.9–8.6 mg/ml for glucoamylase, 0.13–1.0 mg/ml for sucrase, and 0.46–1.4 mg/ml for isomaltase. When the IC50 value of each component and the concentration of each component in the fruit extract were taken into consideration, our results indicated that glucose are involved in the inhibition of α‐amylase, and 1‐DNJ and glucose are involved in the inhibition of α‐glucosidase. This is in contrast to that in M. australis leaf, neither anthocyanin nor glucose are contained, and 1‐DNJ is a main inhibitor. Practical Application It is widely accepted that inhibition of α‐amylase and α‐glucosidase is one of the strategies to treat type‐2 diabetes. Today, acarbose, miglitol, and voglibose are clinically used for this purpose. Our results that 1‐DNJ and anthocyanin are present in Morus australis fruit and are involved in the inhibition of α‐amylase and α‐glucosidase suggest that M. australis fruit is a healthy sweetener.