Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes. Two of the biggest challenges for its commercialization are: effective release of fermentable sugars from pretreated biomass, and preparations of thermostable enzymes with low-cost. The hydrolysis performance of 21 reconstituted bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase, family 9 Clostridium phytofermentans processive endoglucanase, and family 48 Clostridium phytofermentans cellobiohydrolase was investigated on microcrystalline cellulose (Avicel) and regenerated amorphous cellulose (RAC). The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was most important for high cellulose digestibility regardless of substrate type. These results suggested that the hydrolysis performance of reconstituted cellulase cocktail strongly depended on experimental conditions. Thermobifida fusca YX was hypothesized to have a thermophilic polyphosphate glucokinase. T. fusca YX ORF Tfu_1811 encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was over expressed in Escherichia coli. By a simple one-step immobilization, the half-life time increased to 2 h, at 50 °C. These results suggest that this enzyme was the most thermostable PPGK reported. My studies would provide important information for the on-going project: high-yield hydrogen production from cellulose by cell-free synthetic enzymatic pathway. / Master of Science