The Protein Engineering of Hydrogenase and Its Application

本研究旨在通过蛋白质工程改善氢酶的酶学性质。该酶控制厌氧细菌的氢代谢，因此，该酶的改进将提高微生物的产氢效率。首先，我们研究了HID A和HID B的卷曲和产物。根据基因产物的分析，我们认为1)HyA产物似乎控制了体内氢酶的表达水平，2)HyB产物似乎是构成氢酶的亚基之一，然后我们尝试通过DNA合成器(Biosearch；Cyclone)合成的DNA对HyA进行定点突变，以提高体内氢酶的活性。半胱氨酸转化为丝氨酸，氢酶活性提高(野生菌株的133%)。我们推测HydA中氨基酸序列的改变可能会影响和改变氢酶的表达水平，我们还发现hvdB突变体通过在含有镍离子的培养基中培养来恢复其氢酶活性。我们还发现，在含有铁或钒离子的培养基中培养的hyB突变株只恢复了氢酶的吸氢活性，而不恢复了氢酶的放氢活性。在这些金属的存在下培养，可能会导致这些金属取代镍而进入氢酶活性中心，从而导致氢酶性质的改变。这些结果将有助于微生物产氢的研究。

This research aimed the improvement of the enzyme character of hydrogenase by protein engineering. This enzyme dominates the hydrogen metabolism in anaerobic bacteria, therefore, the improvement of this enzyme will enhance the microbial hydrogen production efficiency.First, we investigated the roll and the products of both hydA and hydB. According to the gene product analyses, we assumed, 1)hydA product seems to control the expression level of hydrogenase in vivo, and 2)hydB product seems to be one of the subunit constructing hydrogenase.We then attempted the improvement of hydrogenase activity in vivo, by modifing hydA by site directed mutagenesis using synthetic DNA, synthesized by DNA synthesizer (Biosearch; Cyclone). The alternation of one amino acid, cysteine to serine, resulted in the improvement of hydrogenase activity (133% of wild strain). We assumed that the alternation of amino acid sequence in hydA might affect and change the expression level of hydrogenase.We have also found that hvdB mutant restored its hydrogenase activity by cultivating in a medium containing nickel ion. We also found that hydB mutant cultivated in media containing iron or vanadium ion restored only hydrogen uptake activity but not hydrogen evolution activity of hydrogenase. The cultivation in the presence such metals might result in incorporation of these metals in active center of hydrogenase instead of nickel, hence the alternation in its character might occur.In conclusion, we have been suceeded in the modification of hydrogenase by means of proteinengineering. These results will contribute to the research of microbial hydrogen production.