【作者】 伍善广；

【导师】 廖丹葵；

【作者基本信息】 广西大学 ， 化学工艺， 2013， 博士

【摘要】 海洋生物蛋白是不同结构功能物质的理想、丰富的贮库,尤其是一些海洋低值鱼类是制备生物活性肽理想的初始原料。以海洋低值鱼类为原料制备、分离降血压肽正成为当今一个研究热点。长蛇鲻(Saurida elongata),长蛇鲻属,狗母鱼科,主要分布于西北太平洋,为广西北部湾经济鱼类之一。本实验以长蛇鲻为原料,蛋白组成分析显示长蛇鲻是一种高蛋白原料,湿基粗蛋白15.34%,干基粗蛋白78.35%,含有18种氨基酸,氨基酸总含量达到791.41mg·g-1,其中8种人体必需氨基酸占总量的44.92%,非极性、疏水性氨基酸39.13%,极性氨基酸占总含量的60.87%。本实验选用六种蛋白酶水解长蛇鲻鱼肉蛋白,通过体外ACE抑制活性测定,筛选中性蛋白酶作为制备血管紧张素转化酶抑制肽(ACEI)的蛋白酶,其酶解产物对ACE抑制活性IC50为0.182mg·mL-1。以水解度DH(Y1)和产物对ACE抑制率IP(Y2)双指标为参数,采用响应面设计法考察了酶解温度、pH值、酶/底(E/S)对水解度及抑制率的影响,优化得到二元多次方程：Y1=23.77-0.81X1+3.21X2-1.65X3-0.84X1X2-0.35X1X3-0.95X2X3-1.76X12-0.99X22-0.33X32；Y2=83.32-2.27X1+2.94X2-2.02X3-0.33X1X2-1.76X1X3+1.64X2X3-3.35X12-3.39X22-2.30X32,并确定了最适酶解工艺条件为：E/S为10000U-g-1、温度为48℃、pH为7.0及水解时间120min,在此工艺条件下水解度为24.07%,ACE抑制率为84.00%。利用超滤(UF)和凝胶层析(GFC)对酶解物进行初步分离。酶解物经超滤分离后活性组分得到富集,5kDa渗透液(LFPH-I)的IC50值为0.123mg·mL-1。采用凝胶层析对LFPH-I进一步分离,考察了各因素对凝胶层析的影响,得到最优条件：柱规格(1.6cm×45cm)、蒸馏水洗脱1.0mL·min-1、上样量为20mg。在此条件下,可将LFPH-I分成A. B、C、D、E五个组分,其中C组分(LFPH-I-C)活性最高,IC50为0.110mg·mL-1。考察了LFPH-I的抗胃肠道稳定性能,实验表明LFPH-I经胃蛋白酶消化后活性略有上升；经胰蛋白酶作用稳定性良好,但LFPH-I经胃蛋白酶作用后再用胰蛋白酶处理则活性有所下降。采用离子交换色谱-反相高效液相色谱法结合(IEC/RP-HPLC法)分离纯化LFPH-I-C,得到活性组分G3；同时研究了连续多步反相高效液相色谱法(RP-HPLC/RP-HPLC/RP-HPLC法)分离纯化LFPH-I-C,得到活性组分U73D。经反相高效液相色谱和高效毛细管电泳检测二者均为单一组分。通过飞行时间质谱鉴定了G3和U73D结构,解析其氨基酸序列分别为Arg-Val-Cys-Leu-Pro (RVCLP)和Ser-Pro-Arg-Cys-Arg (SPRCR),ICso值分别为175μM和41μM。固定化金属离子亲和分离技术(IMAC)在重组蛋白和重组多肽的分离纯化中己得到广泛应用。在采用IEC/RP-HPLC法及RP-HPLC/RP-HPLC/RP-HPLC法分离纯化LFPH-I-C得到长蛇鲻降血压肽结构基础上,探索了IMAC分离长蛇鲻降血压肽的方法。以反相悬浮法制备琼脂糖微球载体,确定了最佳制备工艺为琼脂糖浓度2.5%,乳化剂用量1.5%,乳化温度60℃,搅拌速度400rmin-1,此时获得的目标微球载体成球率为70.80%。以环氧氯丙烷为活化剂,确定了最佳活化条件为：NaOH溶液浓度为0.8mol·L-1,环氧氯丙烷体积分数为20%,NaBH4的浓度为3.0g.L-1,活化温度为40℃,活化时间为4h。以活化的琼脂糖微球为载体,亚氨基二乙酸(IDA)为连接臂,Cu2+、Ni2+、 Zn2+为螯合金属离子,分别制备了固定化AS-Cu2+、AS-Zn2+、AS-Ni2+三种亲和介质,其配基密度分别为32.05、33.22、29.17μmol·g-1。考察三种亲和介质在pH3.0-9.0的范围内金属离子的泄露百分率,结果表明酸性环境比碱性环境更容易让金属离子泄露,三种亲和介质中AS-Cu2+最稳定性,AS-Ni2+次之,AS-Zn2+最容易泄露。分别研究了三种亲和介质AS-Cu2+-、AS-Zn2+、AS-Ni2+对LFPH-I的吸附。以LFPH-I为原料,通过对比亲和介质分离ACEI的效果,筛选了AS-Ni2+作为长蛇鲻ACEI分离的亲和介质。其最优层析条件为：0.02mol·L-1磷酸缓冲盐(pH6.8,含1.0mol·L-1NaCl)为平衡液、0.02mol·L-1磷酸缓冲盐(pH4.0,含0.5mol·L-1NaCl)为洗脱液。经AS-Ni2+富集所得组分IC50值为0.116mg·mL-1.通过测定、分析粗蛋白LFPH-I及亲和层析富集组分的氨基酸摩尔百分比的变化,发现在实验条件下,相对其它氨基酸,AS-Ni2+对Met、His、 Tyr、Pro、Ile、Leu具有更高的富集作用。采用反相高效液相色谱对AS-Ni2+富集的组分进一步分离,得到活性组分N5,经反相高效液相色谱和高效毛细管电泳检测为单一组分。经飞行时间质谱测定、解析其氨基酸序列为Arg-Tyr-Arg-Pro (RYRP),其IC50值为52μM,具有较高的活性。更多还原

【Abstract】 Marine proteins are rich reservoirs of structurally diverse biofunctional components. Being rich sources of protein, marine organisms especially some marine low-value fish are ideal starting materials for the preparation of bioactive peptides. The use of marine low-value fish as substrate for preparation and separation of angiotensin-I converting enzyme inhibitory peptides (ACEI) have become a hot topict for research in recent years.The lizard fish(Saurida elongata) is a small marine fish that lives in tropical and subtropical seas. In this paper, component protein of Saurida elongata were determined and18kinds of amino acids in protein of Saurida elongata were determined by RP-HPLC. As the results, protein content of Saurida elongata was15.34%(wet basis) and78.35%(dry basis), and18kinds amino acid of Saurida elongata proteins was about791.41mg·g-1and the total quality of8essential amino acid was about44.92%, aromatic amino acid contained39.13%, polar amino acid contained60.87%.Lizardfish muscle protein was hydrolysed by six proteases. Neutral protease derived hydrolysates showed highest angiotensin-I converting enzyme (ACE) inhibitory activity and have a IC50of0.182mg·mL-1and were used to produce angiotensin-I converting enzyme (ACE) inhibitory peptides. By using degree of hydrolysis (DH, Y1) and ACE-inhibitory activity (IP, Y2) as targets, several factors that influence DH and IP were investigated by response-surface methodology(RSM), including temperature, pH value and E/S. The linear regression models to describe the correlation between the variables and the response were Y1=23.77-0.81X,+3.21X2-1.65X3-0.84X,X2-0.35X1X3-0.95X2X3-1.76X,2-0.99X22-0.33X32and Y2=83.32-2.27X1+2.94X2-2.02X3-0.33X1X2-1.76X1X3+1.64X2X3-3.35X12-3.39X22-2.30X32, respectively. The optimum conditions for producing peptides with the highest angiotensin-I converting enzyme (ACE)-inhibitory activity were the following:E/S of10000U·g-1, temperature of48℃, pH7.0, and hydrolysis time of120min. Under this conditions, the hydrolysis degree of lizardfish was24.07%and ACE-inhibitory activity of the hydrolysates was84.00%.Lizard fish protein hydrolysate (LFPH) was fractionated by ultrafiltration and G15gel filtration chromatography (GFC). The fraction LFPH-I, which was able to pass through the5kDa membrane, have high ACE-inhibitory activity (IC50of0.123mg·mL-1). The fraction LFPH-I was used for further separation by GFC and several factors that affected GFC were investigated. The optimized condition was that loading quantity of LFPH-I was20mg and the column was eluted in a gel filtration column (1.6cm×45cm) with water at a flow rate of1mL·min-1. LFPH-I was fractionated by GFC into five portions:A, B, C, D, and E. Fraction C (LFPH-I-C) was found to possess the strongest activity and its IC50value was0.110mg·mL-1. LFPH-I was used to investigate stability against gastrointestinal proteases in vitro. The ACE-inhibitory activity of LFPH-I showed any change and slight increase after treatment with trypsin and pepsin alone, respectively. However, significant change was observed after combined digestion with pepsin and trypsin.LFPH-I-C was further separated by ion exchange chromatography(IEC) and RP-HPLC (IEC/RP-HPLC method). Fraction G3was obtained and exhibited the strong ACE-inhibitory activity. Furthermore, U73D with the strong ACE-inhibitory activity was isolated from LFPH-I-C by three-step RP-HPLC (RP-HPLC/RP-HPLC/RP-HPLC method). G3and U73D were identified to be single-components by HPLC and HPCE, respectively. The amino acid sequences of G3and U73D were identified by MALDI-TOF-TOF as Arg-Val-Cys-Leu-Pro (RVCLP) and Ser-Pro-Arg-Cys-Arg (SPRCR), and their IC50was175μM and41μM, respectively.Metal ion affinity chromatography(IMAC) has been widely used to fractionate recombinant proteins and polypeptides. After separation of two novel ACE inhibitory peptides by IEC/RP-HPLC and RP-HPLC/RP-HPLC/RP-HPLC methods, IMAC was used to isolate ACE inhibitory peptides from LFPH-I.Agarose microspheres were prepared by means of inverse suspension method. The optimum conditions for producing microspheres were the following:agarose concentration of2.5%, span-80of1.5%, temperature of60℃, stirring rate of400r·min-1. Under this conditions, the rate of target microspheres was70.80%. By using epichlorohydrin as activating agent, agarose microspheres were activated by the following conditions:NaOH concentration of0.8mol·L-1, epichlorohydrin concentration of20%, NaBH4concentration of3.0g·L-1, temperature of40℃, time of4h. IMAC adsorbent (AS-Cu2+、AS-Zn2+、AS-Ni2+) were prepared by using iminodiacetic acid (IDA) as the chelating ligand and Cu2+, Zn2+and Ni2+as metal ion. The concentration of AS-Cu2+、AS-Zn2+、AS-Ni2+was32.05、33.22、29.17μmol·g-1, respectively. Leakage of metal ion at pH3.0-9.0was investigated and the results showed that the IMAC adsorbents were more stable in acidic environment than in alkaline environment. AS-Cu2+was most stable among the three adsorbents and AS-Zn2+was most easily to leach in acidic environment.The adsorption of LFPH-I on AS-Zn2+, AS-Cu2+and AS-Ni2+was investigated. According to ACE-inhibitory activity of fractions separated by different metal ion affinity carrier from LFPH-I, AS-Ni2+was selected for purifying ACE-inhibitory peptides. The optimized conditions of separation of LFPH-I by AS-Ni2+were that the equilibrium solution was0.02mol·L-1phosphate buffer (pH6.8, containing1.0mol·L-1NaCl) and0.02mol·L-1phosphate buffer (pH4.0, containing0.5mol·L-1NaCl) as eluent. A fraction with IC50of0.116mgm·L-1was obtained after the separation of LFPH-I by AS-Ni2+. By comparing amino acid levels (mol%) of LFPH-I and fraction enriched from LFPH-I by AS-Ni2+, it was found that the IMAC fraction was enriched in Met、His、Tyr、Pro、He and Leu.The fraction enriched by AS-Ni2+was further separated by RP-HPLC and the results showed that fraction N5was found to possess the strongest activity. The amino acid sequence of N5was identified by MALDI-TOF-TOF as Arg-Tyr-Arg-Pro (RYRP) and its IC5Owas52μM.更多还原