Two Escherichia coli expression systems based on T7 RNA polymerase promoter (pET system) and tac promoter (pFLAG system) have been used for the production and secretion of recombinant enzymes in our laboratory. Both E. coli OmpA signal peptide and native Bacillus signal peptide could be used efficiently for the secretion of recombinant enzymes into periplasmic space and culture media. The genes could be induced for overexpression with 0.1–1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) when the OD600 of the culture broth reached 0.6–1.5. The recombinant enzymes could be harvested from whole cell lysate, perimplasmic extract or culture broth after induction for 4–20 hours. Since the enzyme is C-terminally tagged with hexahistidine, the recombinant enzymes could be conveniently purified to apparent homogeneity by onestep immobilized-metal affinity chromatography (IMAC) using Ni-NTA resins. The characteristics of various purified recombinant enzymes have been analyzed. These systems should be applicable for the production and directed evolution of various recombinant bacterial extracellular enzymes.

So far our laboratory has published a number of publications related to molecular enzyme biotechnology field as follows:

1. 1.Pechsrichuang, P., Yoohat, K., Yamabhai, M., 2013. Production of recombinant Bacillus subtilis chitosanase, suitable for biosynthesis of chitosan-oligosaccharides. Bioresour Technol, 127, 407-14. [IF 4.75]
   

2. 2.Sinsuwan, S., Yongsawatdigul, J., Chumseng, S., Yamabhai, M., 2012. Efficient expression and purification of recombinant glutaminase from Bacillus licheniformis (GlsA) in Escherichia coli. Protein Expr Purif, 83, 52-58. [IF 1.429]
   

3. 3.Phrommao, E., Yongsawatdigul, J., Rodtong, S., and Yamabhai, M. (2011). A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillus sp. SK37. BMC Biotechnol 11, 65. [IF 2.165]
   

4. 4.Songsiriritthigul, C., Lapboonrueng, S., Roytrakul, S., Haltrich, D., and Yamabhai, M. (2011). Crystallization and preliminary crystallographic analysis of beta-mannanase from Bacillus licheniformis. Acta Crystallogr Sect F Struct Biol Cryst Commun 67, 217-220. [IF 0.552]
   

5. 5.Juajun, O., Nguyen, T.H., Maischberger, T., Iqbal, S., Haltrich, D., and Yamabhai, M. (2011). Cloning, purification, and characterization of beta-galactosidase from Bacillus licheniformis DSM 13. Appl Microbiol Biotechnol 89, 645-654. [IF 3.689]
   

6. 6.Yamabhai, M., Buranabanyat, B., Jaruseranee, N., and Songsiriritthigul, C. (2011). Efficient E. coli expression systems for the production of recombinant β-mannanases and other bacterial extracellular enzymes. Bioengineered bugs 2, 1-5.
   

7. 7.Songsiriritthigul, C., Lapboonrueng, S., Pechsrichuang, P., Pesatcha, P., Yamabhai, M., (2010) Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste. Bioresour Technol 101, 4096-4103. [IF 4.75]
   

8. 8.Songsiriritthigul, C., Buranabanyat, B., Haltrich, D., Yamabhai, M., (2010) Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli. Microb Cell Fact 9, 20. [IF 3.306]
   

9. 9.Salaheddin, C., Takakura, Y., Tsunashima, M., Stranzinger, B., Spadiut, O., Yamabhai, M., Peterbauer, C.K., Haltrich, D., (2010) Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji). Microb Cell Fact 9, 57. [IF 3.306]
   

10. 10.Maischberger, T., Leitner, E., Nitisinprasert, S., Juajun, O., Yamabhai, M., Nguyen, T.H., and Haltrich, D. Beta-galactosidase from Lactobacillus pentosus: purification, characterization and formation of galacto-oligosaccharides., (2010) Biotechnol J 5, 838-847. [IF 3.446]
    

11. 11.Do, B.C., Dang, T.T., Berrin, J.G., Haltrich, D., To, K.A., Sigoillot, J.C., Yamabhai, M., (2009) Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01. Microb Cell Fact 8, 59. [IF 3.306]
    

12. 12.Spadiut, O., Radakovits, K., Pisanelli, I., Salaheddin, C., Yamabhai, M., Tan, T.C., Divne, C., and Haltrich, D. (2009). A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications. Biotechnol J 4, 525-534. [IF 3.446]
    

13. 13.Yamabhai, M. (2009). Sticky PCR: A PCR-based protocol for targeted protein engineering. Biotechnol J 4, 544-553. [IF 3.446]
    

14. 14.Songsiriritthigul, C., Pesatcha, P., Eijsink, V.G., and Yamabhai, M. (2009). Directed evolution of a Bacillus chitinase. Biotechnol J 4, 501-509. [IF 3.446]
    

15. 15.Yamabhai, M., Emrat, S., Sukasem, S., Pesatcha, P., Jaruseranee, N., and Buranabanyat, B. (2008). Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems. J Biotechnol 133, 50-57. [IF 3.183]
    

16. 16.Na-ngam, N., Kalambaheti, T., Ekpo, P., Pitaksajjakul, P., Jamornthanyawat, N., Chantratita, N., Sirisinha, S., Thamlikitkul, V., Chaicumpa, W., Yamabhai, M., and Ramasoota, P. (2008). Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei. Southeast Asian J Trop Med Public Health 39, 443-451.
    

17. 17.Nguyen, T.H., Splechtna, B., Yamabhai, M., Haltrich, D., and Peterbauer, C. (2007). Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli. J Biotechnol 129, 581-591. [IF 3.183]
    

18. 18.Nakkharat, P., Kulbe, K.D., Yamabhai, M., and Haltrich, D. (2006). Formation of galacto-oligosaccharides during lactose hydrolysis by a novel beta-galactosidase from the moderately thermophilic fungus Talaromyces thermophilus. Biotechnol J 1, 633-638. [IF 3.446]
    

19. 19.Yamabhai, M. (2005). A convenient method for the screening of compounds that inhibit specific molecular interactions using the alkaline phosphatase fusion system. Acta Hort 678, 51-57.