Recombinant Expression And Properties Of A Thermophilic Lipase From Thermomicrobium Roseum DSM 5159

Lipases are widely used in various fields due to its efficient catalytic activity,substrate specificity and functional versatility.However,there were few reports about the heat-resistant lipases,and the commercial lipases are expensive and scarce.These problems limit the application of lipase.In this study,in order to obtain thermotolerant lipases and reduce the production cost,a predicted lipase gene from the archaebacterium Thermomicrobium roseum DSM 5159 was cloned and expressed in Escherichia coli by genetic engineering.The main research contents and results are as follows:（1）The molecular chaperone thioredoxin from Escherichia coli and lipase co-expression vectors（p TIG-li P and p ET32a-li P）and the fusion expression vectors（p TIG-li P’and p ET32a-li P’）were constructed to enhance the expression of the lipase.The best expression vector p TIG-li P was selected for the subsequent experiments and its specific enzyme activity was 3.23U·mg^-1.After purification by nickel affinity chromatography column,a single protein band was shown in gels by SDS-PAGE,which was consistent with the theoretical molecular mass of 36.7k Da.The results of MALDI-TOF indicated that it was indeed a lipase from Thermomicrobium roseum.By optimizing the induction conditions,the highest enzymatic activity(0.886 U·m L^-1)and specific enzyme activity(0.557 U·mg^-1)of the lipase were obtained after inducing for 24 h by 0.1 mmol·L^-1 IPTG at 20°C.（2）The enzymatic properties of the purified lipase（Lip）were characterized.The optimal temperature and p H for reaction were separately 85°C and 8.5,which suggested that it was suitable for reaction at high temperature and alkaline conditions.The temperature stability of0.1 mg·m L^-1 pure enzyme was investigated after incubating for 12 h from 40°C to 90°C.The results showed that there was almost no loss of enzyme activity below 80℃.The T_m andΔH of the enzyme were 97.53℃and 1637 k J·mol^-1 by Nano-DSC,suggesting a good thermal stability.The remaining enzyme activity was above 80%after incubating for 2 h at p H 5.5-11.0.Moreover,the lipase was verified with effective resistance ability to most of the studied organic solvents.The addition of K⁺and Ca²⁺was verified that have a promotive effect on the enzyme activity.Based on the kinetic study,it has been found that the lipase showed high affinity to p-nitrophenol palmitate.The K_m,k_cat and k_cat/K_m for this substrate were 0.01 mmol·L^-1,3.0 s^-1 and299.0 s^-1·L·mmol^-1,respectively.To improve lipase enzyme activity,two mutant strainsΔhx andΔ（lid+hx）were constructed by resecting C-terminalα-helix and lid from the lipase gene sequence.The specific enzyme activity of the pure enzyme was 2.61 and 1.73 U·mg^-1.Compared with the recombinant lipase,the values of k_cat/K_m ofΔhx andΔ（lid+hx）were separately increased by 9 and 7.4 times when p-nitrophenol acetate was used as substrate.（3）Four kinds of metal ions(Cu²⁺,Ca²⁺,Co²⁺,and Mn²⁺)were selected to prepare hybrid nanoflower immobilized enzyme.The preparation conditions were optimized by comparing the protein encapsulation rate and specific enzyme activity.The results showed that 0.05 mg·m L^-1Lip and 3.6 mmol·L^-1 Cu²⁺,0.1 mg·m L^-1 Lip and 3.6 mmol·L^-1 Ca²⁺,0.05 mg·m L^-1 Lip and 3.6mmol·L^-1 Co²⁺,0.1 mg·m L^-1 Lip and 2.4 mmol·L^-1 Mn²⁺were the optimal preparation conditions.The morphology,functional groups and crystallization of the immobilized enzyme were characterized by scanning electron microscopy（SEM）,Fourier Transform infrared spectroscopy（FTIR）and X-ray diffraction（X-RD）techniques.The results showed that flower-like immobilized enzymes were successfully formed.The enzymatic properties of the immobilized enzyme revealed that Lip-Ca hybrid nanoflower had better catalytic activity and stability than the other three kinds of immobilized enzymes.The relative activity of Lip-Ca hybrid nanoflower was still 50%at 60℃after 7-days storage and around 80%after six consecutively applications.