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Silk fibroin (SF) has potential applications in the biomedical field because of its excellent mechanical properties and biocompatibility. In the current study, chitooligosaccharide (COS) was enzymatically grafted onto SF using laccase. Subsequently, COS-grafted SF (SF-g-COS) was treated enzymatically in the presence of hexokinase and Mg-chelated adenosine triphosphate (ATP), so as to introduce phosphate groups onto the fibroin chains and promote the deposition of hydroxyapatite (HAp) during in situ biomimetic mineralization. The efficacy of phosphorylation and biomimetic mineralization of the SF-g-COS was evaluated by means of HPLC, MALDI-TOF MS, FTIR, XRD and EDS-Mapping. The results indicate that hexokinase has the capability to catalyze the phosphorylation of COS, resulting in an increase in the quantity of phosphorus in the SF-g-COS. Following mineralization of the phosphorylated SF-g-COS, a greater number of mineral phases were detected on its surface, accompanied by a higher content of calcium and phosphorus compared with other specimens. Cell viability tests using NIH/3T3 cells and cellular adhesion potential with MG-63 cells indicated that the fibroin-based biocomposite exhibited acceptable biocompatibility and superior cellular adhesion properties. The present study describes a novel method for preparation of fibroin/HAp biocomposites for bone tissue engineering. [ABSTRACT FROM AUTHOR]