飼料業界におけるpapainとその用途は何ですか?

Papain酵素主にパパイヤの根、茎、葉、果実に見られ、熟していない果実のラテックスに最も多く含まれています(ye qiteng etal。, 1999;趙元範ほか、1999年。yi yin etal., 2000)。強いタンパク質分解能力とアミド結合とエステル結合を加水分解する能力により、papain酵素は医薬品、食品、繊維、皮革、飼料、染料などの産業で広く使用されている(wu xianrong et al., 1988)。

1 . papain酵素の概要

パパイヤに由来するジュースは純粋な酵素ではありません。これらの粗雑な酵素はpapainだけでなく、リゾチーム、システインプロテアーゼ、セルラーゼ、グルカナーゼ、グルタミン、低分子量チオール化合物を含む。等電点に基づいて、パパイヤラテックスのシステイン含有酵素は、パパイン、パパイン複合体(キモパパイン)、リゾチームの3つの主要なカテゴリーに分類することができる。papainのpiは9.55、chymopapainは10.10、lysozymeはpi >11.0. 中性に近い条件下では、カチオン交換カラムクロマトグラフィーによってこれら3つの酵素を容易に分離することができる。パパイヤラテックスでは、papainが約10%、chymopapainが45%、リゾチームが20%を占める(ling xinghan et al., 1998)。

2代目パチンコ屋

2.1 Papain

パパインは最初の酵素である発見され研究され広く応用されています1937年、ボールズとラインウィーバーは、新鮮なパパイヤジュースから段階的な塩沈殿法を用いて結晶性のパパインを抽出した。1970年にpapainの配列が決定され、1971年にはx線結晶構造解析法を用いてpapainの立体構造が決定された。

papainの相対分子量は21,000で、212からなる1本のポリペプチド鎖から構成されているアミノ酸 residues. In 1969, Husain とTowe reported のamino acid sequence のthis enzyme (see Figure 1).

Papain contains seven cysteine residues, six のwhich form disulfide bonds within three chains, while the remaining free cysteine residue is one of the essential amino acid residues of the active center. Drenth et アルdetermined the tertiary 構造of the papain molecule, which is elliptical in shape and consists of two domains, with a narrow groove at the junction, where the active site of the enzyme is located (Chen Zizhen, 1978).

2.2豆腐Papain

Papain was first discovered, extracted, and named by Jansen and Balls in 1941 (Jansen et al., 1941). のcomposition of papain is relatively complex. In 1967, 国光et al. separated papain using ion exchange columns and classified it into two types based on the order of elution: papain のand B; in 1982, Brocklehurst et al. reported that papain consists of four components. In 1985, they used an extremely slow NaCl gradient elution to obtain five active peaks (Kunimitsu et al., 1985; Brocklehurst et al., 1987).

Silvia et al. (1989) conducted circular dichroism analysis on the four subtypes of papain. Currently, it has been discovered that papain exists in different subtypes, with only one or two amino acid differences between them (Buttleet al., 1984). According to data from databases such as EMBL/GenBank/DDJB, there are at least nine subtypes, each encoded by different genes. Among these, six subtypes consist of polypeptide chains with 218 amino acids, two with 227 amino acids, and one with 226amino acids. のamino acid sequences of the nine subtypes are shown in Figure 2. Comparison results indicate that among these 9 subtypes, only subtype II, subtype III, and subtype V have individual amino acid residues at specific sites that differ from those of other subtypes.

Maes et al. (1996) and Azarkan et al. (1996) determined the three-dimensional structure of papain (see Figure 3). As shown in Figure 3, the polypeptide folding of papain forms two domains of equal size but different shapes—the L domain and the R domain. のL domain is primarily composed of α-helices, while the R domain is primarily composed of anti-parallel β-sheets. のactive site is located at the interface between these two domains. Papain belongs to the α+β class, where the C-terminal domain of papain is a fully α-helical structure, while the N-terminal domain is a fully β-sheet structure (Schulz et al., 1979). In contrast, papain belongs to the α/β class, with its secondary structure containing more α-helices and β-sheets, and the folding patterns of the secondary structures are also different (Ssolis-Mendiola et al., 1992).

Papain contains eight cysteine residues, six of which form intra-molecular disulfide bonds, while the remaining two active free sulfhydryl groups are located at residues 25 and 117. Due to their significant distance and different regions, these two residues cannot form intra-molecular disulfide bonds. The two residues have similar activity. From the three-dimensional model of papain, Cys117 is located on the molecular surface, making it easily accessible to derivatization reagents and susceptible to complete and irreversible oxidation. The catalytic Cys25, along with the conserved Cys22 and Cys56, form the S1 microsite. Due to the relatively large “pocket” of the S1 micro-site, it has almost no effect on the enzyme&#具体的な39;s基板。しかし、67、68、69、133、157、207位のs2マイクロサイトが酵素に影響を与える#39;s基質特異性(maes et al., 1996)。

2.3パパイヤ塩化リゾチーム

In 1955, Smith et al. isolated pure crystalline papain from パパイヤlatex. Later, Haward and Glazer determined the structure and properties of this enzyme. Papain has a molecular weight of 24,000, which is higher than that of animal lysozyme. It consists of 223 amino acids, with the amino acid composition shown in Table 1. Compared to animal lysozyme, papain has significantly higher levels of proline, tyrosine, and phenylalanine.

The arrangement of amino acids at the N-terminal end is: glycine—isoleucine—serine—isoleucine. The arrangement of amino acids at the C-terminal end is: serine—phenylalanine—glycine. The higher structure of papain was determined by circular dichroism spectroscopy, with single helices accounting for approximately 30% of the total. Spectroscopic measurements and chemical reagent reactions indicate that tryptophan residues are entirely embedded within the enzyme molecule and do not participate in the binding of N-acetylglucosamine or active reactions. Papain lysozyme contains 8 cysteine residues, 4 of which form disulfide bonds, and 4 are free SH groups. One SH group is essential for the enzyme&#現場の結39;活躍もらいましょう。papainリゾチームの活性部位構造は、卵白やヒトのリゾチームとは大きく異なる(funatsu et al., 1982)。

3代目パチンコ師

3.1 papainおよびpapainプロテアーゼの性質

The structural similarity of papain proteases determines their significant similarity in properties. Both papain and papain protease exhibit broad substrate specificity, Most peptide bonds can be hydrolyzed to some extent by papain, but the rates of hydrolysis vary greatly among different peptide bonds, with some differing by three orders of magnitude; many amino acid derivatives and peptides can serve as substrates, among which arginine derivatives are particularly sensitive to hydrolysis. However, papain cleaves various bonds much faster than papain.

jansen et al.(1941)は、papainカゼインプロテアーゼがカゼインをpapainの半分の割合で加水分解することを報告した。江原真二郎、Yasunobue papainた1种プロテアーゼを使ってhydrolyzeβチェーン酸化ビタミンインスリン(過マンガン酸カリウムとの酸化)enzyme&を強調し#39;s advantage in hydrolyzing peptide bonds containing acidic amino acid residues and aromatic amino acid residues. When studying the A and B chains of insulin and various peptide segments of different lengths containing glutamic acid, Layle also noted the advantage of papain in hydrolyzing peptide bonds containing glutamic acid. It is worth noting that papain exhibits broader substrate specificity in organic solvent media compared to aqueous media (Jin-Eon So et al., 2000).

Papain and papain chymopapain can also hydrolyze amide bonds and ester bonds (Ling Xinghan et al., 1998). They can catalyze the synthesis of oligopeptides but produce multiple byproducts, which may be due to the broad substrate specificity of the enzyme's s1サイト(jineon so et al., 2000)。

The optimal pH values of papain and papain protease vary depending on the substrate. Papain protease, using casein as a substrate, has an optimal reaction temperature of 80°C (pH 7.0) and an optimal pH range of 3–5 at 37°C. The Michaelis constant (Km) value is 1.25 g/l (at pH 7.0 and a reaction temperature of 37°C) (Zucker et al., 1985). The optimal pH value for papain is 7.0, with pH stability ranging from 4 to 9. Enzyme activity remains stable below 60°C and retains some activity up to 90°C. Due to its high thermal stability and good stability, it is suitable for feed processing.

PCMDchlorobenzyl mercuric acid, iodide acetic acid, iodide acetic acid, hydrogen peroxide, NEM, and heavy metals such as Hg²⁺, Ag⁺, Cu²⁺, and Zn²⁺ can irreversibly inhibit enzyme activity. Papain, containing two free sulfhydryl groups, is more susceptible to oxidation by oxidizing agents or binding with heavy metal ions, leading to inactivation. However, in the presence of various reducing agents (cysteine, thioglycolic acid, グルタチオンの, DTT,etc.) and some metal chelating agents (EDTA), they can reduce the cysteine residues in the active center, thereby activating enzyme activity (Deng Jing et al., 2004).

3.2 papainリゾチームの性質

Lysozyme is an enzyme that can cleave the β-1,4 bond between N-acetylglucosamine and N-acetylglucosamine. Papain lysozyme is an alkaline タンパク質with an isoelectric point of 10.5, exhibiting only one-third of the activity of egg white lysozyme against Listeria monocytogenes, with an optimal pH of 4.5 and an optimal ionic strength of 0.04–0.07. When decomposing cell walls, it behaves similarly to animal-derived lysozyme, it generates N-acetylmuramic acid at the reducing end. Papain exhibits high activity in decomposing chitin. For example, its activity in decomposing gelatinous chitin is 10 times that of egg white lysozyme, and its activity in decomposing (N-acetylglucosamine)₄ is 200 times that of egg white lysozyme. The products of the enzyme' s (N-acetylglucosamine)₅の劣化は(N-acetylglucosamine)₃(N-acetylglucosamine)₂。n-アセチルグルコサミンは遊離状態では存在せず、糖転移反応は稀である(so et al., 2000)。

4酵素活性の決定

4.1教皇活動の決定

The activity of papain is determined by measuring the enzyme'の能力は、酵素活性の単位として機能するタンパク質の加水分解から生成されるアミノ酸の量で、指定された条件(例えば、特定の温度およびph値など)の下で基質タンパク質の加水分解を触媒する。同じ条件で生成されるアミノ酸の量が多いほど、酵素が大きくなります'の触媒反応能力と高い酵素活性。

There are three methods for determining papain activity: the UV spectrophotometric method using casein as the substrate, the indophenol colorimetric method, and the BAEE (benzoyl-L-arginine ethyl ester) method. Each method has its own characteristics. The UV spectrophotometric method using casein as a substrate is simple to operate, low-cost, and suitable for comparing enzyme activity during enzyme purification, as well as for determining the activity of papain products and food enzymes. Although the indophenol colorimetric method is cumbersome to perform, it requires simple equipment, making it usable in areas with limited resources; the BAEE method is stable and reproducible, making it suitable for theoretical studies of enzyme properties and the determination of enzyme activity in reagent enzymes (Wu Xianrong, 2005). The method specified in the United States Pharmacopeia and Chinese pharmaceutical standards for determining papain activity is the ultraviolet spectrophotometric method using casein as the substrate (Luo Yanshou, 2000; Chen Deming et al., 2004).

When using the UV spectrophotometric method to detect enzyme activity, the unit of activity is defined as: under the testing conditions, the amount of enzyme required to release the amount of trichloroacetic acid soluble substance from casein hydrolyzed per minute, when the absorbance at a wavelength of 275 nm is equivalent to the absorbance of 1 μg/ml of tyrosine, is one enzyme activity unit.

そのため、受注生産(受注生産)では、受注生産(受注生産)で使用されるトリクロロ酢酸の濃度が異なることが多い。②ギャバスワッチpapainに厳格な違いは展示活動から輸入されたカゼインは溶解度が高く、酵素活性も高い。③判定最適なpH条件下のみ行われなければならない(趙ホテル・リベラら。1999)。

4.2 papain凝集プロテアーゼ活性の測定

The method for determining the hydrolytic activity of papain coagulating protease uses a UV spectrophotometric method with casein as the substrate. This enzyme also possesses coagulating activity compared to papain, The unit of coagulation activity is defined as the amount of enzyme required to coagulate 1 ml of 10% skim milk (containing 0.01 mol/l CaCl₂) within 40 minutes, which is defined as one enzyme activity unit, i.e., one Soxhlet Unit (SU). The relative activity (RU) is used to indicate the influence of various factors (Arima et al., 1967).

4.3  パパイン・リゾザイム活動決定

リゾチームの活性を決定するには、典型的には3つの方法がある。

受注生産(受注処理)では、受注処理後に受注処理を行い、受注処理後に受注処理を行う。

②として原因になるmonocytogenesの文化媒体を利用する基板、酵素が働きが指示された変更前後の濁りいてばかりはいられない。

Since the above two methods involve solid-liquid phase reactions, it is difficult to accurately measure reaction rates. Therefore, some researchers have prepared a homogeneous substrate using 水溶性多糖類 hexane diol chitin to determine enzyme activity.

③分光试験:450においてnm、一般一定の高野解散細菌粉末や冻Solubulic一定の容积の解決策phosphate-bufferedに細菌休止実现された2004約1.3 absorbance値。この基質溶液と標準酵素溶液を25°cの水浴中で培養する。2.5 mlの基質を水浴中の1 cmのキュベットに入れ、0.5 mlの酵素溶液を加えてタイミングを取り、1分間の読取りe1、2分間の読取りe2を記録し、この式で酵素活性を計算する。考え方酵素愛着変化酵素もっ水準が違う—活動ΔE価値観の违いに対応の酵素が働き使われた標準的な酵素ΔE価値観基づいて計算することができる持てばを検出の精度検証(張勇氏(2004)。

5代目パチンコ屋

Immobilized enzymes are a new technology developed in the 1960s. Immobilization refers to the process of combining free cells or enzymes with a solid insoluble carrier using physical or chemical methods to maintain their activity and enable repeated use. To improve the utilization efficiency of papain and reduce production costs, researchers worldwide have conducted extensive studies on the immobilization and application of papain.

In 1961, J。Cebray successfully immobilized papain on a polyamino acid carrier and used it to hydrolyze fragments of γ-globulin. In 1977, J.W. Finley from the United States employed a glutaraldehyde cross-linking method to immobilize papain on chitin, and used it in the beer production process, achieving good clarification effects. In 1978, P. Monsan et al. in France used aminoalkylated microporous glass as a carrier to cross-link papain on its surface, and also applied it to beer clarification (Ling Xinghan et al., 1998).

Xu Fengcai et al. (1992) used sugarcane bagasse cellulose and nylon as carriers to immobilize papain, determined the enzymatic properties of the immobilized enzyme, and applied it to beer clarification. Li Hong et al. (2001) used chitosan microspheres to immobilize papain, studied the enzymatic properties of the immobilized enzyme, and applied it to casein hydrolysis for tyrosine production. The authors have also conducted a series of studies in this area, including the immobilization of papain using silk fibers from silkworms, investigated the enzymatic properties of the immobilized enzyme and applied it to casein hydrolysis for tyrosine production, as well as silk-immobilized papain in a packed-bed reactor and its application, all of which yielded satisfactory results (Chen Fangyan et al., 2004, 2005a, 2005b), and obtained the corresponding immobilized papain products.

6  飼料産業におけるpapainの応用

During food processing, large amounts of protein by-products are generated, such as animal feathers, animal blood after slaughter, fish trimmings, and fish heads from fish processing. These proteins, when directly ground into feed, are difficult for animals to digest and absorb. Discarding them not only wastes resources but also pollutes the environment. By using proteases to hydrolyze these proteins into soluble small-molecule proteins and amino acids, they become easily digestible and absorbable by animals. This not only enables the development of low-cost, high-quality protein feed but also improves feed utilization efficiency and reduces feed costs (Lü Shimin et al., 2001; Tan Aijuan et al., 1998; Yang Ping et al., 2008). Currently, animal protein enzymes are too expensive for hydrolysis; microbial fermentation methods are prone to contamination by 毒素を含んでいるかもしれませんしかし、papainは高い活性、高い熱安定性、良好な安定性を有しており、飼料加工に適しています。

Additionally, papain, when added as a feed additive to livestock diets, aids in feed digestion, enhances feed efficiency, reduces feed intake, improves growth rates in livestock, and has significant effects on milk production, milk quality, and the prevention of mastitis in dairy cows.

Zhang Qing et al. (1996) added 0.3% papain to shrimp feed and studied the changes in papain activity during production and its effects in aquaculture. They found that under shrimp feed production conditions (85°C, 45 minutes), papain activity loss was severe. However, under poultry feed production conditions (temperature 75°C), protease activity was preserved quite well. Therefore, under pelletizing conditions, the temperature at which most enzyme activity can be preserved is around 75°C. In shrimp farming, it demonstrated a certain growth-promoting effect, increasing yield by 5%.

binshiyuら(1996)は、成長中のブタの飼料に0.1%のpapainを添加し、1日の体重増加と飼料転換率を改善し、最も顕著な効果が10 ~ 20 kgのブタで観察された(p <0.01)。その結果、初期成長期のブタの飼料にpapainを添加することが最も適切であり、中期成長期にも適度に使用できることが示された。

He Ting et al. (1992) fed meat-type chicks a diet supplemented with papain (40,000 units/kg of feed). The results showed that although there was no significant difference in average weight gain between the groups supplemented with papain (P > 0.05), feed consumption was slightly lower in all experimental groups compared to the control group. Additionally, the use of FS protein feed (fermented blood meal) in the diet was more effective than fish meal. The reason may be that FS protein feed contains not only animal blood but also various plant fiber-rich cake and meal carriers, which may allow other enzymes in papain to exert their effects.

7結論

Papain is a pure natural product with strong タンパク質分解hydrolysis and synthesis capabilities, as well as coagulation, lipolysis, and bacteriolytic activity, making it highly versatile. Currently, the global application ratio of papain is as follows: 75% in the brewing and beverage industry, 10% in the meat processing industry, 5% in the fish processing industry, 3% in the pharmaceutical industry, 5% in the feed processing industry, and 2% in other fields. It is evident that the application ratio of papain in the feed industry is relatively low, indicating significant potential for further development. The reasons may include: the relatively high cost of the enzyme during feed processing, significant loss of enzyme activity, which limits its application; or, as a feed additive, the enzyme is not used scientifically during the feeding process. Therefore, how to more effectively and sustainably enhance and maintain the activity of papain; and how to correctly and reasonably use this enzyme in different animal species and at different stages of feeding to maximize its efficacy are key areas worthy of future research and development efforts.

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