Studies On Low-temperature Lipase Of Cryytococcus Neoformans

Lipase, the abbreviations of TG hydrolase, is an important hydrolase which can hydrolyze triglycerides to fatty acids and glycerol. In recent years, it has been focused on its unquie potential applications in various fields. However, the message of Cryytococcus neoformans which is subject of my research is vacant in the world. Cryytococcus neoformans was come from NO.1 glacier in Xinjiang which character is high elevation and low temputer and separated by Fu Jianhong. It can be realized about the adopting mechanism of psychrophile in Xinjiang and the relation of the structure and fuction in protein .Many factors that affect lipase production were discussed in this paper, it can grow best in the condition that bean oil as carbon sourse, peptone as azote sourse, PEG600 as detergent, adding of MgSO₄,FeSO₄,ZnSO₄, pH8.7 for 48 hours.The optimum temperature of crude lipase is 35℃and the purified is 38℃which is higher than the crude. It is presumed that the foreign protein in the crude extract is the key factor in this variation. The distance of optimum pH between the crude and purified is tiny. The pH is 7.0. The best temperature condition of culture collection is-20℃.We can see that the activity of lipase decline sharply in 4℃, the activity of lipase is stability in -20℃that is adopt for conservation in long period and -70℃that is adopt for conservation in short period.In the experiment , it was realized that the activity of lipase can be sharply improved when the maltose, lactose, glucose, glycerine, sorbitol were added in and mannitol , sodium acetate is also helpful to improve its activity when it reacts long period. Ca²⁺,Fe²⁺,Cu²⁺,Mn²⁺ have positive function to its activity, but Mg²⁺,Zn²⁺ have not any effect. The activity of lipase have not obvious variation when add in 10mmol/L EDTA for about 40 min. It is indicated that the lipase is not metalloenzyme. Lipase extraction of Cryytococcus neoformans by aqueous two-phase system and reverse micelles were studied in this paper. PEG 15%,(NH4)2O₄ 22.5%, pH8.0 without NaCl was the optimized condition of aqueous two-phase system, and the partition coefficient and purification multiple was 6.8 and 7.5 respectively. This can be a precedent on lipase extraction of Cryytococcus neoformans by aqueous two-phase system. In the case of CTAB 150mmol/L,V0/VW,pH8.0,40℃,lipase was extracted with reverse micelles, its specific activity gained the best but also lose to 90% of the former.Chemical modification and the simulation of its dimensional structure were also studied in this paper. In the case of PMSF 30mmol/L, DEPC 20mmol/L, WRK 60mmol/L, the activity of Cryytococcus neoformans lipase was completely lost. It can be inferred that Ser,His,Asp are the components of activity center of this lipase.Compared with traditional extraction, aqueous two-phase systems extraction offer more advantages such as the high water content of both phases, relatively high capacity, ease of control and low material costs and increase space-time yield, allowing process automation and the continuous recycling of chemicals. It has been successfully performed in laboratory scale to separate protein, and will be used for industrial application.According to the work of Fu Jianhong, the gene of Cryytococcus neoformans was found to know the protein sequence, its dimensional structure was formed by the computer. After the step of chemical modification, it can be inferred that Ser,His,Asp are the components of activity center of this lipase. This is also a precedent on lipase research of Cryytococcus neoformans.