剩余污泥酶法水解制备蛋白质、氨基酸及其机理研究
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摘要
剩余污泥(excess sludge, ES)有机成分中含量最高的是微生物单细胞蛋白质,而微生物单细胞蛋白质是品质好、氨基酸种类齐全的高价值饲料,但尚未得到充分利用。如果应用大量剩余污泥为原料,运用现代食品、生物加工技术,对污泥蛋白资源进行精细加工,生产出动物饲料添加剂,既可综合利用大宗蛋白质资源,避免和减少资源的浪费,提高剩余污泥的附加值；另一方面将ES蛋白应用于动物饲料中,又扩大了优质氨基酸的来源问题。
从污泥中提取蛋白质和氨基酸的研究方法为酸水解、碱水解法及其超声波辅助法,但还存在固定投资大,资金回收周期长,反应条件剧烈,对生产设备要求较高,对环境造成严重污染,且水解产物极易发生构型改变从而造成水解产物成分极为复杂和蛋白特性变化,导致水解蛋白不能被生物体吸收利用。另外碱性水解易引起几种氨基酸结构变化或外消旋化。水解产生的废水处理需要大量的酸碱,处理费用高。
本课题针对上述问题,选用蛋白酶水解剩余污泥提取蛋白质,并将其有效转化为氨基酸,产品纯度较高且质量高于国家有关动物饲料标准。此外,利用氧化石墨烯固定化酶反应条件温和、温度低、对蛋白质的破坏作用小等特点,发明一套可循环利用并提高蛋白质水解率的固定酶酶解方法,利用异硫氰酸苯酯(PITC)高效液相色谱、聚丙烯酰胺凝胶电泳对水解蛋白质、氨基酸进行分离纯化和鉴定,利用化学反应动力学原理探讨水解参数与固定酶深度酶解速度之间的关系,在此基础上探索深度酶解的反应动力学和机理,并对蛋白质氨基酸提取物进行重金属含量及多环芳烃(PAHs)含量分析、动物急慢性毒理学研究。
论文的主要研究内容及结果如下：
(1)剩余污泥主要成分及利用价值分析
对选取的ES样品进行了主要有机组分成分分析,发现干物质ES中有机质占73.85%,其中脂肪含量约为0.6%,总糖含量为10.2%,蛋白质含量58.1%,此剩余污泥样品应用于提取蛋白质具有可行性。
(2)蛋白酶优选及剩余污泥的水解
考察了木瓜、碱性、中性、酸性、胰以及胃蛋白酶六种不同蛋白酶对ES蛋白质的提取效果。结果发现,碱性蛋白酶提取ES中蛋白质提取率最高。其次为中性蛋白酶和木瓜蛋白酶,胃蛋白酶、酸性蛋白酶和胰蛋白酶的提取效果较差。对碱性蛋白酶水解剩余污泥提取蛋白质的条件进行了优化,考察了不同影响因素,得到碱性蛋白酶最适工作条件为：pH值为8.0,温度55℃,加酶量为2%,反应时间4小时,液固比4：1。在此工作条件下,52.5%的蛋白质被提取出来,占干污泥重量的30.5%。
(3)酶水解剩余污泥机理分析
利用SDS-PAGE考察了污泥中蛋白质分布情况,结果表明,不同提取方法的SDS-PAGE蛋白质条带不同,酶法水解ES蛋白质分子量范围大约在14-100kDa；通过Michaelis-Menten方程,采用双倒数作图法绘图,得到碱性蛋白酶催化水解污泥蛋白反应的米氏常数和最大反应速率分别为Km=34.9g/L, Vmax=32.2g/(L·min)。以碱性蛋白酶水解污泥蛋白水解度为参数,推导出反映酶法水解污泥蛋白动力学模型,所建立的模型在一定程度上能模拟水解体系的酶解过程,能较准确地计算出碱性蛋白酶酶解ES蛋白的水解度,理论DH值与实际DH值基本吻合,对酶解过程的实际应用具有很高的利用价值。
(4)酶法水解制取氨基酸的分析
PITC柱前衍生反相高效液相色谱法可以在35min内同时分析18种氨基酸,18种氨基酸得到完全分离,相邻氨基酸峰之间均达到基线分离。说明该方法应用在分离氨基酸溶液具有较好的专属性。利用异硫氰酸苯酯(PITC)柱前衍生反相高效液相色谱法检测酶法水解污泥氨基酸溶液含有17种氨基酸,分别为必需氨基酸7种和非必需氨基酸10种。人体所必需氨基酸含量高达40%,说明酶法水解剩余活性污泥制备的氨基酸作为家禽家畜饲料应用价值高。
(5)蛋白酶固定化及酶学性质考察
以氧化石墨烯为基底材料,对碱性蛋白酶进行固定化,得到固定化的最优组合,即当温度为25℃,pH值为8,酶用量为600μL,戊二醛浓度2%时,碱性蛋白酶酶活力回收率可达90%左右；同时对固定化蛋白酶最适工作条件进行了考察,固定酶的最适工作条件是,体系pH为8,温度60℃C；考察了固定化酶的热稳定性、pH稳定性、储存稳定性以及重复使用性。同时确定了以酪蛋白为底物时的动力学参数,动力学分析得到游离酶和固定酶的Km常数分别是4.85和8.57mg/mL,最大速率常数max分别是5.7和6.10μg/min。固定酶的米氏常数是游离酶的1.8倍。
(6)固定酶水解制备氨基酸的研究
考察了氧化石墨烯固载酶体系水解ES蛋白影响因素,在温度为65℃,pH值为10,酶用量8mg/mL,时间为6h条件下,氧化石墨烯固载酶水解剩余污泥氨基酸结果达到8.23g/100g DS。固定酶与游离酶对剩余污泥粗蛋白质的水解效果几乎一样,水解得到的总氨基酸分别是8.23和10.154g/100g DS。从XRD、XPS、SEM、AFM以及TGA等表征手段分析得出：碱性蛋白酶通过戊二醛交联法已经成功地固载在氧化石墨烯载体上,并且可以了解氧化石墨烯-碱性蛋白酶复合材料的稳定性能。
(7)污泥水解产品中重金属和持续性有机污染物的分析及对锦鲤的急性毒理学
考察了污泥及蛋白质制品中重金属微量元素(Cu, Ni, Zn, Fe, Mn)和有害重金属(Pb, Cr, Cd, Hg, As)等金属的含量,发现污泥中铁的含量非常高,达到10247.59mg/kg DS。另外,污泥中除锌含量超标外,基本都在1984年颁布的《农用污泥中污染物控制标准》(GB4284-84)的限值之内。酶法污泥蛋白提取液中重金属微量元素析出量和有害重金属浸出量远远小于碱法、酸法提取污泥蛋白。酶法提取污泥蛋白质中有害重金属Pb、Cr、Cd、Hg、As的含量均低于饲料卫生标准(GB13078-2001),其中Pb浸出率最高。
经微波消解、固相微萃取后,采用GC/MS分析方法,检测了污泥、三种不同方法提取的污泥蛋白及酶提污泥蛋白饲喂锦鲤鱼体内的18种多环芳烃含量,结果发现,碱法提取的污泥蛋白中,ΣPAHs含量最高,酸法次之,酶法提取的污泥蛋白ΣPAHs最少,最安全。同时考察了酶法提取粗蛋白作为饲料的安全性,锦鲤体内ΣPAHs和致癌性多环芳烃(ΣAHscare)含量普遍比较低。
总之,本文选用ES为研究对象,利用碱性蛋白酶以及固定化碱性蛋白酶酶法水解为研究方法,系统研究了游离酶与固定酶酶法水解ES中蛋白质、氨基酸,对其水解特性及水解动力学进行了深入探讨。此外,重点考察了酶法提取ES粗蛋白中有毒有害金属及重金属、PAHs含量分析,以及ES粗蛋白应用在锦鲤鱼饲料上的初步研究。本研究为蛋白酶在ES处理中的应用提供了理论依据和实验基础,通过固定化酶技术实现了酶的回收利用、降低了酶解ES成本,并为ES中蛋白质资源化利用奠定了理论和实验基础。酶法提取出的ES蛋白可以作为动物饲料添加剂、氨基酸水溶肥料、氨基酸绿色缓蚀剂以及其他工业应用中的原材料。
Protein is thought to be the major organic compounds in excess sludge (ES), those microbial single cell protein have not been took full advantage although they are of good quality, with all kinds and reasonable ratio of amino acids. Reutilization of ES as crude material is attracting more interest, which products a series of animal feed additive through food biological and processing technology by deep processing and utilization of ES protein resource. Therefore, protein recovery from excess sludge can also provide other important benefits. For example, recovers useful biomaterials directly from excess sludge could avoid and reduce the waste of resources, improve the added value of surplus sludge, and also expanded alternative sources of protein and good quality amino acids.
Methods of protein and amino acids recovery from excess sludge have been investigated for years. Many chemical, physical or biological treatments, such as alkali treatment and ultra-sonication have been reportedly used to disrupt the sludge floc structure and release the intracellular contents into the aqueous phase. But there have been lots of problems, such as huge fixed assets investment, a long recovery period, intense reaction conditions, the higher requirement in producing equipment is needed, cause serious pollution to the environment. More than that, the protein product cannot be absorption and utilization by organism because hydrolysis product make configuration change and protein characteristic change easily which also resulting in hydrolysis product composition is very complicated. In addition, alkkaline hydrolysis lead to several amino acid structure change or racemic change easily. Last but not least, expenses of subsequent processing are costly because of wastewater generated by base or acid hydrolysis need a lot of acid or alkali.
For the problems above-mentioned, this paper study enzymatic hydrolysis of ES to extract protein and and translating them into amino acids effectively. The product was owned high purification and superior to the national animal feed standard. In addition, an improved rate and can be recycled method was invented by using graphene oxide immobilized enzyme have mild reaction conditions, low temperature and small destructibility on protein structure. The molecular weight ranges of protein product were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The contens of amino acids were detected by Reverse phase-high performance liquid chromatography (RP-HPLC) with phenyl isocyanate (PITC) as reagent. Furthermore, based on chemical reaction kinetics, the relationship between hydrolysis parameters and the rate of extensive hydrolysis of immobilized enzyme was dicussed. The compositions of the recovered protein, heavy metals and polycyclic aromatic hydrocarbons (PAHs) were determined. Finally, toxicity of using the protein recovered from ES was assessed by feeding Brocarded carp. The main contents and results of this study are as follows.
(l)Analysis of main components and indudtrial value of the excess sludge
The research indicated that the ES (dried sludge, DS) consisted of73.85%organic matter, which include58.1%protein,10.39%carbohydrate and less than1%lipid. This ES samples used in the extraction of proteins with good feasibility.
(2)The optimal selection of protease and ES hydrolysis
ES was hydrolyzed by six different commercial proteolytic enzymes (Papain, Alcalase, Trypsin, Pepsin, Acid proleslytic enzyme, Neutral protease). The results showed that alkaline protease has the highest extraction rate of protein from ES, followed by neutral protease and papain, the other three protease has poor effect. The protein content in the hydrolysate was determined by the Kjeldahl method, and alkaline protease was chosen for further analysis. The optimal hydrolysis conditions are pH8.0, temperature550C, enzyme to substrate (E:S)2%, reaction time4h and ratio of liquid to solid4:1. Experimental results have been shown that52.5%of protein was extracted from ES (accounting for30.5%of initial DS weight) under optimum hydrolysis conditions.
(3)Mechanism of enzymatic hydrolysis of ES
The subproteomes generated from ES were analyzed by SDS-PAGE. The results showed that each extraction method led to unique SDS-PAGE protein profiles. Several distinct protein bands were present in ES enzymatic hydrolysate, predominantly in the molecular weight region between14to100kDa. The kinetics of ES enzymatic hydrolysis by alkaline protease was discussed in this article. Lineweaver-Burke plot was used to assess the values of Michaelis constant (Km) and maximum effective velocity (Vmax). The kinetic analysis showed that Km and Vmax values were34.9g/L and32.2g/(L·min).
The degree of hydrolysis (DH) of protein from ES was determined. The hydrolysis kinetic model was expressed and the theoretical DH from the hydrolysis kinetic model is consistent well with the practical DH. It shows that the kinetic model has significant application value.
(4)Analysis of free amino acids in ES protease hydrolysate
Eighteen kinds of free amino acids were separated completely in35min by reversed-phase high performance liquid chromatography with pre-column PITC derivatization. The method has good specificity in the separation of amino acid solution. An RP-HPLC method based on modified pre-column derivatization with PITC was applied to quantify free amino acids in a protein hydrolysate extracted from ES. As shown in results, there were17kinds of natural amino acids emerging in the ES hydrolysate, which consisted of seven kinds of essential amino acids and ten kinds of non-essential amino acids. ES enzymatic hydrolysate has great potential of application in swine and poultry diets.
(5)Alkaline preotease was immobilized and the properties of graphene oxide-alkaline protease bio-composites
Graphene oxide (GO) was used as substrate for immobilizing alkaline protease. The immobilizing conditions were optimized though single-factor test and orthogonal design. The test indicated that the optimum technics were as follows:the enzyme amount to1mg carriers was500μL, the concentration glutaraldehyde is2%, pH was8.0, temperature was25℃, crosslinking and absorption time were4hours and1hour. Then the activity recovery was raised to90.0%in the optimum conditions of immobilization. The optimal hydrolysis conditions were60℃and pH8.0. The results showed that the thermostability and reusability of immobilized protease have been obviously improved compared to the free enzyme. However, there was no significant change in optimum pH value between the free and immobilized protease. The immobilized protease exhibited good operational stability. Various concentrations of casein solution were used as substrate. Lineweaver-Burke plot was used to assess the values of Michaelis constant (Km) and maximum effective velocity (Vmax). The kinetic analysis showed that Km values of free and immobilized protease were4.85and8.57mg/mL, and Vmax were5.7and6.10μg/min, respectively. The proteases immobilized GO exhibited apparent Km value which was about1.77-fold higher than that of free protease.
(6) Highly efficient catalysts of immobilized enzyme for the proteolysis of ES crude protein
Alkaline protease immobilized on GO showed significant activity towards ES protein hydrolysates, attractive for practical applications. The optimal hydrolysis conditions for immobilized protease were65℃, pH10.0, dosage of enzyme8mg/mL and reaction time6h. The free amino acids formed by free protease and enzyme immobilized on GO were generally similar. The total contents of amino acids yield was10.154and8.23g/100g DS for free enzyme and immobilized enzyme.
(7) Content of heavy metals and PAHs in the ES crude protein and toxicity on Brocarded carp
The contents of trace metals (Cu, Ni, Zn, Fe, Mn) and harmful heavy metals (Pb, Cr, Cd, Hg, As) in theES sample and ES crude protein samples were determined. The results showed that iron is the highest which reaching to10247.59mg/kg DS. The content of heavy metals-with the exception of zinc-in ES sample was basiclly within the limit "agricultural sludge the pollutant control standards"(GB4284-84), which promulgated in1984by China. Contents of metals in the ES crude protein which extracted by enzymatic were far lower than that made by alkali and acid extraction. Through the recovery processes of protein, significant amounts of the heavy metals in the raw sludge were removed.
Microwave-assisted extraction and a solid-phase microextraction (SPME) as adsorbent coupled with gas chromatography-mass spectrometry (GC-MS) method was developed for the determination of18polycyclic aromatic hydrocarbons (PAHs) in ES, proteins by three different extraction methods and enzymatic proteins feeding Brocarded carp samples. It was found that,AHs concentrations was highest in protein by alkaline hydrolysis, and acid hydrolyzation take second place. It was least in proteins extraction by enzymatic hydrolysis, and more safer. In addition, the toxicity of proteins by enzymatic hydrolysis was investigated, the contents ofAHs and carcinogenic PAHs (ΣPAHscare) in Brocarded carp samples were generally in a low level.
Overall, we choose the ES as the research object, the protein and amino acid content in the enzymatic hydrolysate by alkaline protease were investigated systematically. The hydrolysis characteristics and kinetics of hydrolysis were further investigated. Furthermore, contents of poisonous and danger metal, heavy metal and PAHs in the crude protein by enzymatic hydrolysis ES were studied. The research include a pilot study of crude protein applied to the feeding Brocarded carp. This study not only provided theoretical basis for alcalase hydrolysates of ES containing high protein, but also estabilised foundation and experimental basis for resource utilization of sludge. The immobilized enzymatic technology makes the recycle of protease is realized and reduces the cost of enzymatic hydrolysis of ES. The curde protein extraction from ES by enzymatic could be used as animal feed additive, water-soluble fertilizers containing amino acids, environment green amino acid inhibitor and raw and processed materials in other industrial applications.
从污泥中提取蛋白质和氨基酸的研究方法为酸水解、碱水解法及其超声波辅助法,但还存在固定投资大,资金回收周期长,反应条件剧烈,对生产设备要求较高,对环境造成严重污染,且水解产物极易发生构型改变从而造成水解产物成分极为复杂和蛋白特性变化,导致水解蛋白不能被生物体吸收利用。另外碱性水解易引起几种氨基酸结构变化或外消旋化。水解产生的废水处理需要大量的酸碱,处理费用高。
本课题针对上述问题,选用蛋白酶水解剩余污泥提取蛋白质,并将其有效转化为氨基酸,产品纯度较高且质量高于国家有关动物饲料标准。此外,利用氧化石墨烯固定化酶反应条件温和、温度低、对蛋白质的破坏作用小等特点,发明一套可循环利用并提高蛋白质水解率的固定酶酶解方法,利用异硫氰酸苯酯(PITC)高效液相色谱、聚丙烯酰胺凝胶电泳对水解蛋白质、氨基酸进行分离纯化和鉴定,利用化学反应动力学原理探讨水解参数与固定酶深度酶解速度之间的关系,在此基础上探索深度酶解的反应动力学和机理,并对蛋白质氨基酸提取物进行重金属含量及多环芳烃(PAHs)含量分析、动物急慢性毒理学研究。
论文的主要研究内容及结果如下：
(1)剩余污泥主要成分及利用价值分析
对选取的ES样品进行了主要有机组分成分分析,发现干物质ES中有机质占73.85%,其中脂肪含量约为0.6%,总糖含量为10.2%,蛋白质含量58.1%,此剩余污泥样品应用于提取蛋白质具有可行性。
(2)蛋白酶优选及剩余污泥的水解
考察了木瓜、碱性、中性、酸性、胰以及胃蛋白酶六种不同蛋白酶对ES蛋白质的提取效果。结果发现,碱性蛋白酶提取ES中蛋白质提取率最高。其次为中性蛋白酶和木瓜蛋白酶,胃蛋白酶、酸性蛋白酶和胰蛋白酶的提取效果较差。对碱性蛋白酶水解剩余污泥提取蛋白质的条件进行了优化,考察了不同影响因素,得到碱性蛋白酶最适工作条件为：pH值为8.0,温度55℃,加酶量为2%,反应时间4小时,液固比4：1。在此工作条件下,52.5%的蛋白质被提取出来,占干污泥重量的30.5%。
(3)酶水解剩余污泥机理分析
利用SDS-PAGE考察了污泥中蛋白质分布情况,结果表明,不同提取方法的SDS-PAGE蛋白质条带不同,酶法水解ES蛋白质分子量范围大约在14-100kDa；通过Michaelis-Menten方程,采用双倒数作图法绘图,得到碱性蛋白酶催化水解污泥蛋白反应的米氏常数和最大反应速率分别为Km=34.9g/L, Vmax=32.2g/(L·min)。以碱性蛋白酶水解污泥蛋白水解度为参数,推导出反映酶法水解污泥蛋白动力学模型,所建立的模型在一定程度上能模拟水解体系的酶解过程,能较准确地计算出碱性蛋白酶酶解ES蛋白的水解度,理论DH值与实际DH值基本吻合,对酶解过程的实际应用具有很高的利用价值。
(4)酶法水解制取氨基酸的分析
PITC柱前衍生反相高效液相色谱法可以在35min内同时分析18种氨基酸,18种氨基酸得到完全分离,相邻氨基酸峰之间均达到基线分离。说明该方法应用在分离氨基酸溶液具有较好的专属性。利用异硫氰酸苯酯(PITC)柱前衍生反相高效液相色谱法检测酶法水解污泥氨基酸溶液含有17种氨基酸,分别为必需氨基酸7种和非必需氨基酸10种。人体所必需氨基酸含量高达40%,说明酶法水解剩余活性污泥制备的氨基酸作为家禽家畜饲料应用价值高。
(5)蛋白酶固定化及酶学性质考察
以氧化石墨烯为基底材料,对碱性蛋白酶进行固定化,得到固定化的最优组合,即当温度为25℃,pH值为8,酶用量为600μL,戊二醛浓度2%时,碱性蛋白酶酶活力回收率可达90%左右；同时对固定化蛋白酶最适工作条件进行了考察,固定酶的最适工作条件是,体系pH为8,温度60℃C；考察了固定化酶的热稳定性、pH稳定性、储存稳定性以及重复使用性。同时确定了以酪蛋白为底物时的动力学参数,动力学分析得到游离酶和固定酶的Km常数分别是4.85和8.57mg/mL,最大速率常数max分别是5.7和6.10μg/min。固定酶的米氏常数是游离酶的1.8倍。
(6)固定酶水解制备氨基酸的研究
考察了氧化石墨烯固载酶体系水解ES蛋白影响因素,在温度为65℃,pH值为10,酶用量8mg/mL,时间为6h条件下,氧化石墨烯固载酶水解剩余污泥氨基酸结果达到8.23g/100g DS。固定酶与游离酶对剩余污泥粗蛋白质的水解效果几乎一样,水解得到的总氨基酸分别是8.23和10.154g/100g DS。从XRD、XPS、SEM、AFM以及TGA等表征手段分析得出：碱性蛋白酶通过戊二醛交联法已经成功地固载在氧化石墨烯载体上,并且可以了解氧化石墨烯-碱性蛋白酶复合材料的稳定性能。
(7)污泥水解产品中重金属和持续性有机污染物的分析及对锦鲤的急性毒理学
考察了污泥及蛋白质制品中重金属微量元素(Cu, Ni, Zn, Fe, Mn)和有害重金属(Pb, Cr, Cd, Hg, As)等金属的含量,发现污泥中铁的含量非常高,达到10247.59mg/kg DS。另外,污泥中除锌含量超标外,基本都在1984年颁布的《农用污泥中污染物控制标准》(GB4284-84)的限值之内。酶法污泥蛋白提取液中重金属微量元素析出量和有害重金属浸出量远远小于碱法、酸法提取污泥蛋白。酶法提取污泥蛋白质中有害重金属Pb、Cr、Cd、Hg、As的含量均低于饲料卫生标准(GB13078-2001),其中Pb浸出率最高。
经微波消解、固相微萃取后,采用GC/MS分析方法,检测了污泥、三种不同方法提取的污泥蛋白及酶提污泥蛋白饲喂锦鲤鱼体内的18种多环芳烃含量,结果发现,碱法提取的污泥蛋白中,ΣPAHs含量最高,酸法次之,酶法提取的污泥蛋白ΣPAHs最少,最安全。同时考察了酶法提取粗蛋白作为饲料的安全性,锦鲤体内ΣPAHs和致癌性多环芳烃(ΣAHscare)含量普遍比较低。
总之,本文选用ES为研究对象,利用碱性蛋白酶以及固定化碱性蛋白酶酶法水解为研究方法,系统研究了游离酶与固定酶酶法水解ES中蛋白质、氨基酸,对其水解特性及水解动力学进行了深入探讨。此外,重点考察了酶法提取ES粗蛋白中有毒有害金属及重金属、PAHs含量分析,以及ES粗蛋白应用在锦鲤鱼饲料上的初步研究。本研究为蛋白酶在ES处理中的应用提供了理论依据和实验基础,通过固定化酶技术实现了酶的回收利用、降低了酶解ES成本,并为ES中蛋白质资源化利用奠定了理论和实验基础。酶法提取出的ES蛋白可以作为动物饲料添加剂、氨基酸水溶肥料、氨基酸绿色缓蚀剂以及其他工业应用中的原材料。
Protein is thought to be the major organic compounds in excess sludge (ES), those microbial single cell protein have not been took full advantage although they are of good quality, with all kinds and reasonable ratio of amino acids. Reutilization of ES as crude material is attracting more interest, which products a series of animal feed additive through food biological and processing technology by deep processing and utilization of ES protein resource. Therefore, protein recovery from excess sludge can also provide other important benefits. For example, recovers useful biomaterials directly from excess sludge could avoid and reduce the waste of resources, improve the added value of surplus sludge, and also expanded alternative sources of protein and good quality amino acids.
Methods of protein and amino acids recovery from excess sludge have been investigated for years. Many chemical, physical or biological treatments, such as alkali treatment and ultra-sonication have been reportedly used to disrupt the sludge floc structure and release the intracellular contents into the aqueous phase. But there have been lots of problems, such as huge fixed assets investment, a long recovery period, intense reaction conditions, the higher requirement in producing equipment is needed, cause serious pollution to the environment. More than that, the protein product cannot be absorption and utilization by organism because hydrolysis product make configuration change and protein characteristic change easily which also resulting in hydrolysis product composition is very complicated. In addition, alkkaline hydrolysis lead to several amino acid structure change or racemic change easily. Last but not least, expenses of subsequent processing are costly because of wastewater generated by base or acid hydrolysis need a lot of acid or alkali.
For the problems above-mentioned, this paper study enzymatic hydrolysis of ES to extract protein and and translating them into amino acids effectively. The product was owned high purification and superior to the national animal feed standard. In addition, an improved rate and can be recycled method was invented by using graphene oxide immobilized enzyme have mild reaction conditions, low temperature and small destructibility on protein structure. The molecular weight ranges of protein product were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The contens of amino acids were detected by Reverse phase-high performance liquid chromatography (RP-HPLC) with phenyl isocyanate (PITC) as reagent. Furthermore, based on chemical reaction kinetics, the relationship between hydrolysis parameters and the rate of extensive hydrolysis of immobilized enzyme was dicussed. The compositions of the recovered protein, heavy metals and polycyclic aromatic hydrocarbons (PAHs) were determined. Finally, toxicity of using the protein recovered from ES was assessed by feeding Brocarded carp. The main contents and results of this study are as follows.
(l)Analysis of main components and indudtrial value of the excess sludge
The research indicated that the ES (dried sludge, DS) consisted of73.85%organic matter, which include58.1%protein,10.39%carbohydrate and less than1%lipid. This ES samples used in the extraction of proteins with good feasibility.
(2)The optimal selection of protease and ES hydrolysis
ES was hydrolyzed by six different commercial proteolytic enzymes (Papain, Alcalase, Trypsin, Pepsin, Acid proleslytic enzyme, Neutral protease). The results showed that alkaline protease has the highest extraction rate of protein from ES, followed by neutral protease and papain, the other three protease has poor effect. The protein content in the hydrolysate was determined by the Kjeldahl method, and alkaline protease was chosen for further analysis. The optimal hydrolysis conditions are pH8.0, temperature550C, enzyme to substrate (E:S)2%, reaction time4h and ratio of liquid to solid4:1. Experimental results have been shown that52.5%of protein was extracted from ES (accounting for30.5%of initial DS weight) under optimum hydrolysis conditions.
(3)Mechanism of enzymatic hydrolysis of ES
The subproteomes generated from ES were analyzed by SDS-PAGE. The results showed that each extraction method led to unique SDS-PAGE protein profiles. Several distinct protein bands were present in ES enzymatic hydrolysate, predominantly in the molecular weight region between14to100kDa. The kinetics of ES enzymatic hydrolysis by alkaline protease was discussed in this article. Lineweaver-Burke plot was used to assess the values of Michaelis constant (Km) and maximum effective velocity (Vmax). The kinetic analysis showed that Km and Vmax values were34.9g/L and32.2g/(L·min).
The degree of hydrolysis (DH) of protein from ES was determined. The hydrolysis kinetic model was expressed and the theoretical DH from the hydrolysis kinetic model is consistent well with the practical DH. It shows that the kinetic model has significant application value.
(4)Analysis of free amino acids in ES protease hydrolysate
Eighteen kinds of free amino acids were separated completely in35min by reversed-phase high performance liquid chromatography with pre-column PITC derivatization. The method has good specificity in the separation of amino acid solution. An RP-HPLC method based on modified pre-column derivatization with PITC was applied to quantify free amino acids in a protein hydrolysate extracted from ES. As shown in results, there were17kinds of natural amino acids emerging in the ES hydrolysate, which consisted of seven kinds of essential amino acids and ten kinds of non-essential amino acids. ES enzymatic hydrolysate has great potential of application in swine and poultry diets.
(5)Alkaline preotease was immobilized and the properties of graphene oxide-alkaline protease bio-composites
Graphene oxide (GO) was used as substrate for immobilizing alkaline protease. The immobilizing conditions were optimized though single-factor test and orthogonal design. The test indicated that the optimum technics were as follows:the enzyme amount to1mg carriers was500μL, the concentration glutaraldehyde is2%, pH was8.0, temperature was25℃, crosslinking and absorption time were4hours and1hour. Then the activity recovery was raised to90.0%in the optimum conditions of immobilization. The optimal hydrolysis conditions were60℃and pH8.0. The results showed that the thermostability and reusability of immobilized protease have been obviously improved compared to the free enzyme. However, there was no significant change in optimum pH value between the free and immobilized protease. The immobilized protease exhibited good operational stability. Various concentrations of casein solution were used as substrate. Lineweaver-Burke plot was used to assess the values of Michaelis constant (Km) and maximum effective velocity (Vmax). The kinetic analysis showed that Km values of free and immobilized protease were4.85and8.57mg/mL, and Vmax were5.7and6.10μg/min, respectively. The proteases immobilized GO exhibited apparent Km value which was about1.77-fold higher than that of free protease.
(6) Highly efficient catalysts of immobilized enzyme for the proteolysis of ES crude protein
Alkaline protease immobilized on GO showed significant activity towards ES protein hydrolysates, attractive for practical applications. The optimal hydrolysis conditions for immobilized protease were65℃, pH10.0, dosage of enzyme8mg/mL and reaction time6h. The free amino acids formed by free protease and enzyme immobilized on GO were generally similar. The total contents of amino acids yield was10.154and8.23g/100g DS for free enzyme and immobilized enzyme.
(7) Content of heavy metals and PAHs in the ES crude protein and toxicity on Brocarded carp
The contents of trace metals (Cu, Ni, Zn, Fe, Mn) and harmful heavy metals (Pb, Cr, Cd, Hg, As) in theES sample and ES crude protein samples were determined. The results showed that iron is the highest which reaching to10247.59mg/kg DS. The content of heavy metals-with the exception of zinc-in ES sample was basiclly within the limit "agricultural sludge the pollutant control standards"(GB4284-84), which promulgated in1984by China. Contents of metals in the ES crude protein which extracted by enzymatic were far lower than that made by alkali and acid extraction. Through the recovery processes of protein, significant amounts of the heavy metals in the raw sludge were removed.
Microwave-assisted extraction and a solid-phase microextraction (SPME) as adsorbent coupled with gas chromatography-mass spectrometry (GC-MS) method was developed for the determination of18polycyclic aromatic hydrocarbons (PAHs) in ES, proteins by three different extraction methods and enzymatic proteins feeding Brocarded carp samples. It was found that,AHs concentrations was highest in protein by alkaline hydrolysis, and acid hydrolyzation take second place. It was least in proteins extraction by enzymatic hydrolysis, and more safer. In addition, the toxicity of proteins by enzymatic hydrolysis was investigated, the contents ofAHs and carcinogenic PAHs (ΣPAHscare) in Brocarded carp samples were generally in a low level.
Overall, we choose the ES as the research object, the protein and amino acid content in the enzymatic hydrolysate by alkaline protease were investigated systematically. The hydrolysis characteristics and kinetics of hydrolysis were further investigated. Furthermore, contents of poisonous and danger metal, heavy metal and PAHs in the crude protein by enzymatic hydrolysis ES were studied. The research include a pilot study of crude protein applied to the feeding Brocarded carp. This study not only provided theoretical basis for alcalase hydrolysates of ES containing high protein, but also estabilised foundation and experimental basis for resource utilization of sludge. The immobilized enzymatic technology makes the recycle of protease is realized and reduces the cost of enzymatic hydrolysis of ES. The curde protein extraction from ES by enzymatic could be used as animal feed additive, water-soluble fertilizers containing amino acids, environment green amino acid inhibitor and raw and processed materials in other industrial applications.
引文
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