Studies On Screening,Fermentation Conditions Optimization And Enzymatic Properties Of Cutin Degrading Bacteria

The cuticle covered on plant surface is airtight, water impermeable, acid resistant, alkali resistant and oxidation resistant. It is the physical and chemical barrier between plant cell walls and the surrounding environment, and could inpair the digestion of plant fiber to a certain extent. The main component of the cuticle is cutin. Cutinase can efficiently degrade the cutin in mild conditions with no pollution. The present study was designed to screen the cutin-degrading strains, and their fermentation conditions and enzymatic properties will be explored to provide guidance for cutin degrading efficiently.Screening bacteria with high cutinolytic esterase capacity: In this work, 8 cutin-degrading bacteria were screened from mixed-plant compost and animal feces by use of cutin-indicator discolor circle method and PCL-indicator discolor circle method, which was named as L3-2, L14, L16 L, X3 PB, X3 PH, X4, X8 P, X16. Furthermore, Comparing the ability of extracellular esterase activity induced by cutin, we selected L3-2 and X8 P successfully, identified as Klebsiella pneumoniae L3-2 and Acetobacter orientalis X8 P by colony morphological characteristic observing and 16 SrDNA sequeneing.Optimization of the fermentation conditions of Klebsiella pneumoniae L3-2 and Acetobacter orientalis X8P: The results showed that the optimal fermentation conditions of Klebsiella pneumoniae L3-2 were temperature 40℃ and fermentation time for 3d, Adding carbon and nitrogen sources, we found that 1% corn cob and 0.5% beef extract supplemented in the LB medium could significantly promote microbial enzymes production（P<0.05）. Morever,The results showed that the optimal fermentation conditions of Acetobacter orientalis X8 P were temperature 37℃and fermentation time for 4d, and the LB medium supplemented with 1% olive oil was enough to promote enzyme production significantly. Medium supplemented with 1% soluble starch inhibited the enzyme production of two strains, while 1% glucose added promoted microbial enzyme production.The properties of Klebsiella pneumoniae L3-2 and Acetobacter orientalis X8 P crude enzymes: The optimum temperature and pH of Klebsiella pneumoniae L3-2 extracellular crude enzymes were 55℃ and pH 6.5. The pH stability of the crude enzymes is not very well. Most of tested organic solvents significantly inhibited the cutinolytic esterase activity. However, cutinolytic esterase activity had a 1-fold and 1.85-fold in presence of Methanol（50%） and DMSO（50%）, respectively. Glycerol（50% and 80%） had a 3.4-fold and 6-fold on cutinolytic esterase activity. Tween-20, Triton-100 and Metal ions such as Na⁺,Mg²⁺,Mn²⁺,Ca²⁺,Ba²⁺（1 mM and 10 mM）promoted extracellular enzymes activity by 2%-31%. The optimum temperature and pH of Acetobacter orientalis X8 P extracellular crude enzymes with good pH stability were 45℃ and pH 6.5. In tested organic solvents, the enzymes exhibited part of cutinolytic esterase activity only in DMSO（50%）. Tween-20（1mM）, Tween-80（10mM） and Triton-100（1mM and 10mM） slightly promote the extracellular enzyme activity by 3%-35%, and Metal ions such as K⁺, Mn²⁺（1mM and 10mM）could improve extracellular enzyme activities by 2%-20%（P <0.05）.