Mechanism Research On Extraction Protein And Amino Acids From The Excess Sludge By Enzymatic Hydrolysis

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 sludgeThe 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 hydrolysisES 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 ESThe 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 hydrolysateEighteen 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-compositesGraphene 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 proteinAlkaline 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 carpThe 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 ofAHs 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.