| 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. |
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