仿刺参(Apostichopus japonicus)和海地瓜(Acaudina leucoprocta)体壁多肽的响应面法酶解和N末端测序

ENZYMATIC HYDROLYSIS TECHNOLOGY OF APOSTICHOPUS JAPONICUS AND ACAUDINA LEUCOPROCTA BASED ON RESPONSE SURFACE METHODOLOGY AND N-TERMINAL AMINO ACID SEQUENCING

为充分开发利用海参资源,优化海参体壁酶解工艺,鉴定海参多肽,本文以水解度为指标,设计了3因素(时间、温度和加酶量)3水平的响应面实验,得到海参水解的最优条件是:仿刺参(Apostichopus japonicus)体壁在加酶量1.97%、温度55.7°C、水解136.8min的条件下,水解度为83.39%;海地瓜(Acaudina leucoprocta)体壁在加酶量1.70%、温度55.4°C、水解115.6min的条件下,水解度为63.68%。之后通过使用超滤膜、反相高效液相色谱(RP-HPLC)、基质辅助激光解析电离飞行时间质谱(MALDI-TOF)技术和蛋白测序仪等分析手段从水解产物中分离鉴定出4种多肽。其中,经N端序列鉴定出仿刺参多肽S1、S2氨基酸残基序列分别为Gly-Pro-Val-Gly-Ala-Ser-Gly-Pro-Gln-Gly-ProGln-Gly-Pro-Gln-Gly-Leu-Ser-Ala-Leu和Trp-Pro-Pro-Gly-Asn-Ser-Gly-Ile-Gln-Gly。海地瓜多肽A1和A2氨基酸残基序列分别为Gly-Ala-Asn-Gly-Asn和Trp-Leu-Pro-Gly-Asp-Thr-Gly-Pro-Gln-GlyVal-Thr-Gly-Pro-Val-Gly-Pro-Ala-Gly。

We optimized the hydrolytic conditions of two sea cucumber species with protamex in respond surface methodology(RSM) to take full advantages of this valuable marine resources. Using hydrolysis degree(HD) as an indicator, we designed an RSM experiment with three factors of temperature, enzyme amount, and the treatment time. The best hydrolysis conditions for Apostichopus japonicus were at 55.7°C in enzyme amount of 1.97% for 136.8 min treatment, at which the HD reached 83.39%; and for Acaudina leucoprocta, there were 55.4°C, 1.70%, and 115.6 min, respectively in HD of 63.68%. In addition, after the enzyme solutions were separated and purified by centrifugation, ultra-filtrating, RP-HPLC(reverse phase high-performance liquid chromatography), MALDI-TOF(matrix-assisted laser desorption/ ionization time of flight mass spectrometry) and N-terminal sequencing, the main constituents of the solutions were analyzed, from which we harvested peptide S1(Gly-Pro-Val-Gly-Ala-Ser-Gly-Pro-Gln-Gly-Pro-Gln-Gly-Pro-Gln-GlyLeu-Ser-Ala-Leu), S2(Trp-Pro-Pro-Gly-Asn-Ser-Gly-Ile-Gln-Gly), Acaudina polypeptide A1(Gly-Ala-Asn-Gly-Asn), and A2(Trp-Leu-Pro-Gly-Asp-Thr-Gly-Pro-Gln-Gly-Val-Thr-Gly-Pro-Val-Gly-Pro-Ala-Gly).