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A putative glutamine synthetase (GS) was detected in a psychrophilic bacterium, Cryobacterium soli GCJ02. For gaining greater insight into its functioning, the gene was cloned and expressed in a heterologous host, Escherichia coli. The monomer enzyme with a molecular weight of 53.03 kDa was expressed primarily in cytosolic compartment. The enzyme activity was detected using glutamate and ATP. The optimum conditions of its biosynthesis were observed to be 60 °C and pH value 7.5. Its thermostability was relatively high with a half-life of 50 min at 40 °C. GS activity was enhanced in the presence of metal ions such as Mg(2+) and Mn(2+), whereas Fe(2+), Cu(2+) and Ca(2+) proved inhibitory. The consensus pattern [EXE]-D-KP-[XGXGXH] in the GS lies between residues 132 and 272. The catalytic active sites consisting of EAE and NGSGMH were verified by site-directed mutagenesis. Based on the analysis of the consensus pattern, the GS/glutamate synthase cycle of C. soli GCJ02 is expected to contribute to the GS synthesic activity.

The green synthesis of the flavor esters, n-propyl acetate, isobutyl acetate and isoamyl acetate had the advantages over the chemical synthesis. The esterase from Candida parapsilosis could transform n-propanol, isobutanol and isoamyl alcohol into n-propyl acetate, isobutyl acetate and isoamyl acetate, respectively. The esterase was expressed in Saccharomyces cerevisiae. At 30 °C for 1 d, the concentration of n-propyl acetate, isobutyl acetate and isoamyl acetate synthesized by the esterase expressed in Saccharomyces cerevisiae was 24.6 mg/100 mL, 8.3 mg/100 mL, 5.6 mg/100 mL, respectively. Expression of the esterase has a practical significance for flavor ester synthesis by green biochemical process.

Cellulase production, lignocellulose saccharification and bioethanol fermentation were integrated to efficiently produce bioethanol. A modified gas lift bioreactor was developed for bioethanol production by the integrated process. Cellulase production was achieved using Aspergillus niger mycelia immobilized within the reactor in wire meshes, and Saccharomyces cerevisiae cells were immobilized in resin beads. During four repeated batches fermentation, cellulase activities were more than 6.28 U/mL and bioethanol production was over 45.9 g/L for 48 h. The factual bioethanol conversion efficiency was 86.8%. By the modification of the modified gas lift bioreactor, immobilization of Aspergillus niger mycelia and Saccharomyces cerevisiae cells, aerobic cellulase production, substrate saccharification and anaerobic bioethanol fermentation were successfully integrated in tandem. The integrated processes is of great significance in bioethanol production.