Prokariotic Expression,Purification And Activity Analysis Of Pyruvate Kinase And Lactate Dehydrogenase From Thermophilic Bacillus

Thermophilic bacteria are one class of microorganisms that can adapt to high temperatures.They,with fast growth rate,efficient metabolism,and a significant survival advantage at high temperatures,have wide applications in environmental protection and energy utilization.The thermophilic enzymes are those isolated from thermophilic bacteria and capable of adapting to high temperature reaction conditions.The coupling method is one of the commonly used methods for measuring enzyme activity in vitro.For example,pyruvate kinase?PK?and lactate dehydrogenase?LDH?are commonly used coupling enzymes for the determination of nucleoside diphosphate?NDP?.The optimum growth temperature of the thermophilic bacteria Aeribacillus pallidus JH-7 was 50?.In order to analyze whether PK and LDH from it can be used as coupling enzymes for 50? reaction conditions,pk and ldh were cloned from A.pallidus JH-7 genome and constructed into expression vector pET24a,which was expressed and purified.apPK and apLDH were obtained with a purity of over 95%.The catalytic efficiency and kinetic constants of apPK and apLDH were investigated by spectrophotometry.We found that the optimal substrate for apPK is ADP,and its catalytic efficiency is 5×10⁴M^-1·s^-1,which is optimal pH is 8.0,and the optimum temperature is 45?;NADH is the optimum substrate for apLDH,the catalytic efficiency is 3.7×10⁵ M^-1·s^-1,the optimum pH is 5.8,and the optimum temperature is 35?.Although 50? is not the optimum temperature for apLDH and apPK,their k_cat at at this temperature is 16.4 s^-1 and 64.5 s^-1,respectively,suggesting that they works efficiently at this temperature.TFSAC is a sulfate-activated enzyme complex of A.thermophilus which lacks the activity of ATP sulfated enzyme but has the activity of adenosine 5'-phosposulfate kinase?APSK?.APSK has an irreplaceable function in the assimilation of sulfur.Using apLDH and apPK as coupling enzymes,the kinetic constants of their substrates were analyzed under the reaction conditions of 50?.We found that K_mAPSAPS was 0.65?M and the k_cat at value was 4.6 s^-1.Under the reaction conditions of 25?,the K_mAPS APS was 0.56?M and the k_cat at was 2.45 s^-1.Therefore,under the reaction conditions of 50?,the k_cat at value of the APSK is 1.9 times of that under the reaction condition of 25?,the K_mAPS APS value is 1.1 times of that under the reaction condition of 25?.Little difference of the APSK activity measured at 50? and 25? was found,which suggested that apLDH and apPK can be used as efficient coupling enzymes for the measurement of NDP.