Extracellular amylolytic activity in marine Croceibacter sp. 23-6: Cu 2+ response and optimized wheat starch hydrolysis

Background Marine bacteria are important sources of extracellular hydrolytic enzymes, but the amylolytic activity of many non-model marine bacterial lineages remains insufficiently characterized at the phenotype level. This study evaluated extracellular amylolytic activity in the marine isolate Croceibacter sp. 23-6 and examined its metal-ion response and wheat starch hydrolysis performance under defined laboratory conditions. Methods Strain 23-6 was isolated from mesopelagic seawater of the western Pacific Ocean and identified at the genus level using 16S rRNA gene sequence analysis. Extracellular amylolytic activity was first screened on starch agar. A partially purified extracellular amylolytic protein preparation was then used to determine the effects of temperature, pH, thermal and pH stability, metal ions, apparent kinetic parameters and hydrolysis of different starch substrates. Wheat starch hydrolysis was further evaluated using single-factor experiments and response surface methodology based on a Box–Behnken design. Results Strain 23-6 produced a clear starch-hydrolysis halo and was conservatively assigned to the genus Croceibacter . The partially purified extracellular protein fraction showed maximum amylolytic activity at 50 °C and pH 7.0 and retained comparatively higher activity under near-neutral conditions. Among the tested metal ions, Cu 2+ increased the measured relative activity to 119.0% at 3 mM, whereas K + , Mn 2+ , Zn 2+ and Fe 2+ decreased the measured activity. Wheat starch showed the highest relative reducing-sugar release among the tested insoluble starch substrates. Response surface optimization predicted suitable hydrolysis conditions of approximately 52 min, 50 °C, 4.0 U/mL enzyme and 1.0% wheat starch. Validation under the adjusted conditions yielded 2.43 ± 0.09 mg/mL reducing sugars, expressed as maltose equivalents, consistent with the model prediction. Conclusions These results provide strain-level evidence that Croceibacter sp. 23-6 exhibits extracellular amylolytic activity and indicate that its partially purified extracellular protein fraction displays a measurable Cu 2+ -responsive activity profile and wheat starch hydrolysis capacity. The findings define the functional properties of this marine bacterial extracellular amylolytic fraction while leaving the identity of the responsible enzyme(s), preparation homogeneity, and the mechanism of Cu 2+ response for future work.