仙人掌桿菌幾丁質酵素的過量表現與催化反應機制的探討

Abstract

從Bacillus cereus NCTU2選殖出來的含訊息月生肽幾丁質酵素基因建構於載體pET-22b(+)上，成功於大腸桿菌BL21 (DE3) 中大量表現，胞內酵素經疏水性管柱層析，得到90%以上均質度之幾丁質酵素，命名為ChiNCTU2，以電灑式質譜儀分析其分子量為36236 Da，此與成熟蛋白之分子量吻合，顯示此訊息月生肽可被大腸桿菌辨識而遭切除。 重組酵素穩定性高，反應最佳活性在pH約5.5左右，在pH 6.0 MES緩衝液條件下，對PNPCB的反應活性之Km為0.27 mM，kcat為66.0 s-1。 由BrØnsted plot顯示ChiNCTU2為兩步驟水解反應機制-中間體的生成與水解，反應中間體的水解步驟是速率決定步驟。由突變株酵素E145G與受質反應過程呈現的雙相動力反應現象 (biphasic kinetic)，顯示有中間體累積，此亦證實中間體水解為速率決定步驟。在Sodium azide濃度為2.5 M時可增強突變株E145G對受質2,4-DNPCB的反應速率近100%，顯示催化反應機制極可能是anchimeric assistance，其反應中間體為oxazoline ring。 經胺基酸比對，E145與Y213和Serratia marcescens ChiA (subfamily A) 之E315與Y390位於相同保留區，經突變後E145G、E145C和E145Q之kcat值較野生株下降3900 ~ 9100倍，kcat /Km下降2900 ~ 5300倍，證實E145為ChiNCTU2催化作用之重要胺基酸。然而Y213F的催化活性kcat與kcat /Km與野生株相近，顯示Y213對subfamily B的ChiNCTU2不是參與水解反應的重要基團，故推論ChiNCTU2與S. marcescens ChiA之結構與催化機制可能不同。

A gene of family 18 chitinase from Bacillus cereus NCTU2 was constructed in pET-22b(+) and over-expressed by E. coli BL21 (DE3) strain. The full gene encodes a signal peptide (27 amino acids) and a mature protein (333 amino acids). The recombinant protein, designated as ChiNCTU2, was purified from the cytosolic crude extract by a hydrophobic interaction column with homogeneity >90%. LC/MS analysis of the purified protein gave a molecular weight of 36236 Da, which is consistent with the calculated value of the mature protein, indicating the signal peptide can be recognized in E. coli. The catalytic activity (kcat) and its Michaelis constant (Km) were characterized to be 66.0 s-1 and 0.27 mM, respectively. The Bronsted plot, log kcat vs the pKa of leaving phenol, was derived from the catalytic activity of ChiNCTU2 towards a series of arylchitobiosides. The slope of the plot is nearly flat, i.e. βlg ~ 0, suggesting that the formation of intermediate is fast step and the breakdown of intermediate is the rate limiting of the reaction. Amino acid multi-alignment revealed that E145 and Y213 are the potential residues mediating the catalytic function of ChiNCTU2. When E145G catalyzed the hydrolysis of 2, 4- dinitrophenyl chitobioside, a biphasic kinetic was observed. The fast step may be attributed to the formation of intermediate and the slow kinetic is simply due to the hydrolysis of intermediate. Sodium azide can serve as the exogenous nucleophile to enhance the breakdown of intermediate. The formation of glycosyl-enzyme intermediate can be ruled out since LC/MS analysis gave the intact molecular weight of E145G. All of these results support the formation of oxazoline intermediate. Mutagenic study showed that the catalytic activities of mutants, E145Q, E145C, and E145G, were reduced 3900-9100-fold, whereas, the activity of Y213F remained unchanged. This finding confirms the essential role of E145 for ChiNCTU2 catalysis. However, the function of Y213 is distinguished from that of conserved Y390 in Serratia marcescens. With the functional inconsistency of Y213 and the discrepancy of amino acid conservation between several family 18 chitinases, we conclude that the protein structure and/or the catalytic mechanism of ChiNCTU2 (subfamily B) is different from other subfamily A chitinase such S. marcescens.