Adamson N J, Reynolds E C
Biochemistry and Molecular Biology Unit, School of Dental Science, University of Melbourne, Vic, Australia.
J Dairy Res. 1997 Nov;64(4):505-14. doi: 10.1017/s0022029997002483.
In an approach to develop a commercial-scale process for the production of casein phosphopeptides containing the cluster sequence-SerP-SerP-SerP-Glu-Glu-, we have studied the relationship between casein hydrolysis and phosphopeptide release. The degrees of hydrolysis (DH) of casein using Novo trypsin PTN 3.0 S and pancreatin 4NF independently, at enzyme to substrate (E:S) ratios of 1:50-1:1600 (by weight), were determined using the pH-stat method. Casein phosphopeptides (CPP) were selectively precipitated using Ca2+ and ethanol from the acid-clarified hydrolysates. The precipitates were analysed by high performance capillary electrophoresis to calculate individual phosphopeptide yields based on extinction coefficients of the purified peptides. Individual peptides were purified by reversed-phase HPLC and anion-exchange FPLC and characterized by MALDI-TOF mass spectrometry and amino acid sequence analysis. For both enzymes, lowering the E:S ratio resulted in reductions in the DH and the release of the CPP, and an increase in peptide chain length. The longer chain length offset the reduction in release such that the gravimetric yields of CPP preparations remained relatively constant. For Novo trypsin the highest yields of the major cluster peptides (beta-casein(CN)f(1-25), alpha s1-CNf(59-79), alpha s2-CNf(1-21), alpha s2-CNf(46-70) and related peptides) in the selective precipitates were obtained at a casein DH of 17%. At lower DH values (9-15%), there was a decrease in yield of the peptides derived from alpha s1-CN and alpha s2-CN while the yield of the beta-CN-derived cluster peptides remained relatively constant. The CPP produced using pancreatin were found to be truncated at all E:S ratios, relative to the tryptic CPP, owing to higher levels of chymotryptic and carboxypeptidase activities in pancreatin. The highest yields of the truncated forms of the major cluster peptides using pancreatin were obtained at a casein DH of 19-23%.
为开发一种用于生产含簇序列-SerP-SerP-SerP-Glu-Glu-的酪蛋白磷酸肽的商业规模工艺，我们研究了酪蛋白水解与磷酸肽释放之间的关系。使用pH计法测定了在酶与底物（E:S）重量比为1:50至1:1600的条件下，分别使用诺维信胰蛋白酶PTN 3.0 S和胰酶4NF对酪蛋白进行水解的水解度（DH）。酪蛋白磷酸肽（CPP）从酸澄清的水解产物中通过Ca2+和乙醇选择性沉淀。通过高效毛细管电泳分析沉淀物，根据纯化肽的消光系数计算各个磷酸肽的产率。通过反相高效液相色谱和阴离子交换快速蛋白质液相色谱纯化各个肽，并通过基质辅助激光解吸电离飞行时间质谱和氨基酸序列分析进行表征。对于这两种酶，降低E:S比会导致DH降低和CPP释放减少，以及肽链长度增加。较长的链长抵消了释放量的减少，使得CPP制剂的重量产率保持相对恒定。对于诺维信胰蛋白酶，在酪蛋白DH为17%时，选择性沉淀物中主要簇肽（β-酪蛋白（CN）f(1-25)、αs1-CNf(59-79)、αs2-CNf(1-21)、αs2-CNf(46-70)及相关肽）的产率最高。在较低的DH值（9-15%）下，源自αs1-CN和αs2-CN的肽的产率降低，而源自β-CN的簇肽的产率保持相对恒定。由于胰酶中胰凝乳蛋白酶和羧肽酶活性水平较高，发现使用胰酶生产的CPP在所有E:S比下相对于胰蛋白酶CPP都被截短。使用胰酶时，主要簇肽截短形式的最高产率在酪蛋白DH为19-23%时获得。
