Mannose-6-phosphate is an important phosphor-sugar, which is involved in many physiological functions and it is used to treat many diseases. Its production is however expensive since it requires costly substrate ATP as phosphorylation agent. This study has focused upon the direct synthesis of M6P by glucomannokinase using inorganic polyphosphate without involvement of ATP. The gene cloned for glucomannokinase has been sequenced from Mycobacterium phlei and it is transformed into Escherichia coli for expression. After purification involving affinity chromatography, a band of 30 kDa corresponding to the enzyme has been isolated from induced crude supernatant. A total amount of 0.69 mg/ml of enzyme has been successively obtained and the purity exceeds 90%. The kinetic assay studies show that this enzyme has more affinity towards polyphosphate and glucose than ATP and mannose respectively. The KM values of the enzyme for glucose, mannose, ATP and hexametaphosphate derived from experiments are 9.5, 203.7, 4.6, 1.7 mu M, respectively. The enzyme has shown a maximum production of mannose-6-phosphate at optimized conditions of pH 8.5, 25 degrees C, poly(P)/mannose ratio 3:1 and in the presence of bivalent ion mg(2+). The results reveal that the glucomannokinase from Mycobacterium phlei suitable for further production of mannose-6-phosphate.