Petry Inga, Ganesan Ashok, Pitt Andrew, Moore Barry D, Halling Peter J
Westchem, Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, G1 1XL Glasgow, UK.
Biotechnol Bioeng. 2006 Dec 5;95(5):984-91. doi: 10.1002/bit.21074.
Methods adapted from proteomics can directly characterize proteins present in immobilized biocatalysts. Complete hydrolysis followed by HPLC analysis of Tyr and Phe estimates total protein bound, and is preferable to conventional difference methods, as tested with subtilisin Carlsberg on silica. This new method shows that various treatments give quantitative desorption of proteins immobilized by adsorption. Intact desorbed proteins may be analyzed by electrospray mass spectrometry. The Candida antarctica lipase B from Novozyme 435 was shown to be heavily glycosylated, while the lipase from Lipozyme RM IM was a mixture of four N-terminally truncated forms. Peptides from selective cleavage were analyzed by tandem mass spectrometry, leading to automatic identification of proteins present. A second major protein present in Lipozyme RM IM was thus found to be alpha-amylase from Aspergillus oryzae. These methods should be valuable complements to activity measurements in understanding immobilized enzyme activity and stability.
源自蛋白质组学的方法可直接表征固定化生物催化剂中存在的蛋白质。用HPLC分析酪氨酸和苯丙氨酸的完全水解产物可估算结合的总蛋白量，与传统的差值法相比，这种方法更可取，如在硅胶上对嗜热栖热菌蛋白酶进行的测试所示。这种新方法表明，各种处理可使通过吸附固定的蛋白质定量解吸。完整的解吸蛋白质可用电喷雾质谱法进行分析。结果表明，诺维信435中的南极假丝酵母脂肪酶B高度糖基化，而Lipozyme RM IM中的脂肪酶是四种N端截短形式的混合物。通过串联质谱法分析选择性裂解产生的肽段，从而自动鉴定出存在的蛋白质。因此发现Lipozyme RM IM中存在的第二种主要蛋白质是米曲霉的α-淀粉酶。在理解固定化酶的活性和稳定性方面，这些方法应是对活性测量的有价值补充。
