1259207 (1) 玖、發明說明 【發明所屬之技術領域】 本發明係關於一種亞硫酸鹽還原酵素之純化方法，詳 而言之’該亞硫酸鹽還原酵素可催化將亞硫酸鹽還原爲硫 化物之反應，將其使用於變性之魚肉中，可有效地使變性 魚肉蛋白質復原，同時亦可提高變性魚肉之可反應硫氫基 及膠強度。本發明亦關於使用該亞硫酸鹽還原酵素之溶液 或粉末以使變性魚肉復原之方法。 【先前技術】 亞硫酸鹽還原酵素廣泛存在於植物及微生物之中，在 硫酸鹽（sulfate)同化作用之最終步驟中將亞硫酸鹽（sulfite) 還原爲硫化物（sulfide)的酵素，爲半胱胺酸生合成之關鍵 步驟。在亞硫酸鹽還原過程中的電子供應者，在大腸桿菌 是以還原菸醯胺腺嘌呤磷二核苷（red uced nicotinamide adenine dinucleotide phosphate，NADPH)作爲電子供應 者，酵母菌則是以 NADPH或還原甲基紫精（reduced methyl viologen，MVH)作爲電子供應者，而於其他酵素中 則係以其他電子予體作爲電子供應者。 肌肉蛋白質大都由L-胺基酸以胜肽鍵、雙硫鍵、氫 鍵、離子鍵、疏水基之相互作用和偶極-偶極間之作用力 等形成各種不同型態和生理功能之蛋白質。當蛋白質分子 之二級、三級和四級立體構造發生改變，但沒有破壞胜肽 -6- 1259207 (2) 鍵時，謂之爲「蛋白質變性」。 變性後之蛋白質構造由於形成隨意螺旋狀、展開形態 或聚集狀態，使胜肽鏈上或胜肽鏈間之官能基相互作用而 失去原有之物理、化學和生物特性。蛋白質變性之原因一 般認爲是因肌肉中之水分被凍結，造成鹽濃度提高，蛋白 質所處的環境pH値改變，蛋白質分子因鹽析而產生沉 澱，分子間或分子內疏水基聚集，同時形成新的氫鍵、雙 硫鍵及離子鍵而使蛋白質凝聚變性。而且蛋白質分子周圍 之水與官能基如-SH、-COOH、-NH和-CO等形成結合水 之狀態，亦因溫度下降，而使結合力較弱之水分子先形成 冰晶，同時因體積膨脹，使蛋白質分子型態改變、官能基 暴露、蛋白質分子間或分子內形成新鍵結而凝聚變性。 凍藏魚肉蛋白之變性所形成之共價結合經証實主要是 由於硫氫基被氧化形成雙硫鍵所造成（Jiang et al.，J. Food Sci. 63 : 777-78 1，1 9 9 8 ； Jiang e t al·，J. Food Sci. 60 : 6 5 2 -65 5, 1 99 8)。經凍藏後之變性魚肉，不但有肉質軟 化、汁液流失、組織成海綿狀等現象發生，且其乳化性、 保水性、成膠能力和黏彈性亦比新鮮者差，此劣變主要是 由於肌纖維蛋白變性所致。 形膠是魚肉蛋白加工製成煉製品時極爲重要的特性之 一，即蛋白質經變性而形成有規則的網狀結構，使肌肉蛋 白加熱固定後具有彈性之特性。蛋白質的凝膠受到許多因 素的影響，如：鹽濃度、pH値、溫度、蛋白質濃度及組 成分、添加物、離子強度、鮮度等。從煉製品的生化特性 -7- 1259207 (3) 得知：形成網狀結構的鍵結包括氫鍵、疏水鍵、離子鍵、 雙硫鍵及其他共價鍵。 另外，膠強度爲魚漿煉製品品質判斷之指標，而影響 煉製品彈性的因素很多，如：鹽濃度、p Η値、溫度、蛋 白質濃度及組成分、添加物、離子強度、鮮度等。因此， 已提出多種改善魚肉煉製品彈性的方法。 魚肉凍藏期間會造成形膠能力降低，主要爲魚肉肌纖 維蛋白質之肌凝蛋白重鏈在凍藏期間逐漸減少，而產生肌 凝蛋白二聚物及多聚物等高分子聚合物，此產物之形成主 要藉由肌凝蛋白之次單元體間之硫氫基形成雙硫鍵所致， 使魚肉肌動凝蛋白凝聚而變性。 如上所述，於凍藏期間會使魚肉之形膠能力下降，且 造成蛋白質不安定，而限制凍藏魚肉在生產蛋白膠體食品 上的應用。 以往均使用鹼洗處理以改善紅色魚肉之形膠能力，然 而並無法改善色澤。增加鹼洗循環次數、抑或使用臭氧脫 色，雖然實質上均可改善鯖魚肉之色澤，但卻會造成形膠 能力劣化。 另外，變性魚肉經添加半胱胺酸、硫酸氫鈉或異抗壞 血酸鹽類等化學還原劑後所製成之冷凍魚漿，其肌動凝蛋 白之總硫氫基、可反應硫氫基及可抽出之肌動凝蛋白量均 較未添加還原劑之冷凍魚漿提高甚多。冷凍雖然造成蛋白 質行膠能力的降低，但添加還原劑後能使其大部分復原， 顯示還原劑能使凝聚變性之肌動凝蛋白復原。還原劑之添 -8- 1259207 (4) 加可使冷凍變性魚肉在擂潰時將雙硫鍵還原並轉換成硫氫 基’在煉製品形膠時重新形成雙硫鍵，使網狀結構更爲穩 固。但是化學還原劑被視爲外來之添加物，較不易被消費 者接受。 本案發明人曾提出以微生物來源之亞硫酸鹽還原粗酵 素’以取代該類之化學還原劑，例如Jiang et al.係探討由 酵母菌生產之亞硫酸鹽還原酵素對臭氧脫色變性及冷凍變 性鯖魚漿之變性肌肉蛋白之復原性，發現亞硫酸鹽還原粗 酵素可使得變性之肌肉蛋白復原，提高煉製品之形膠能力 (Jiang et al.? J. Food Sci. 60 : 6 52-65 5，1 998; Jiang et al·， J· Food Sci· 60: 777-78 1，1 998)。另外，Wu et al.由 Escherichia coli製備亞硫酸鹽還原酵素粗酵素凍乾粉末 應用於冷凍魚肉之加工，亦發現其可提高煉製品之形膠能 力（Wu et al·，J. Food Sci. 65 : 1400-1403，2000)° 但迄今 尙無製備微生物來源之亞硫酸鹽還原酵素，亦無人曾探討 其使變性魚肉復原之功效。 【發明內容】 爲解決上述先前技術中所存在之問題點，本發明之發 明人加以專心探討，硏究微生物來源之純化亞硫酸鹽還原 酵素之純化方法及所得亞硫酸鹽還原酵素使變性魚肉復原 之功效’結果發現，由E s c h e r i c h i a c ο 1 i製造之純化亞硫 酸鹽還原酵素可達成上述目的，藉此完成本發明。 亦即，本發明係提供一種亞硫酸鹽還原酵素，其特徵 - 9- 1259207 (5) 在於具有下述特性： a. 其作用爲催化將亞硫酸鹽還原爲硫化物、復原被 氧化之硫氫基的反應； b. 在該催化反應中，電子供應者爲N ADPH、MVH 或其他電子予體； c. 分子量爲 100,000 至 400,000; d. 作用之適宜溫度範圍爲20°C至30°C ;以及 e. 作用之適宜pH値爲6.5至8.0。 於上述亞硫酸鹽還原酵素中，該電子供應者較佳爲 NADPH。又，該亞硫酸鹽還原酵素較佳係源自 Escherichia coli 〇 另外，本發明亦提供一種製造純化亞硫酸鹽還原酵素 之方法，其特徵爲利用硫酸銨分劃法及層析法，藉以由 Escherichia coli粗酵素液製造純化之亞硫酸鹽還原酵 素。 於上述製造純化亞硫酸鹽還原酵素之方法中，粗酵素 液之製備較佳係包含下述步驟：將Escherichia coli菌體 加入pH値爲6 · 5至8.5之磷酸鹽緩衝液中，於2至1 0 °C 之溫度下’以超音波破碎機破碎達0.1至2小時，於 4,000至1 5,00 0xg之速度下離心〇·!至2小時，收集上澄 液，再加入磷酸鹽緩衝液，再利用相同方式加以破碎、離 心，合倂含有酵素之磷酸鹽緩衝液以得粗酵素液。硫酸銨 分劃法係包含下述步驟：於2至1 下，將固態硫酸銨 緩緩加入以上述方式製備之粗酵素液中，並加以攪拌；於 -10- 1259207 (6) 3,0 0 〇至1 5，0 0 0 x g之速度下離心分離所得物經〇 .丨至2小 時；以及使沉澱物溶液於濃度爲0.01至0.2M之pH値爲 6.5至8.5之磷酸鹽緩衝液中進行達〇.5至2日之透析， 而得透析物。又，係利用選自D E A E S e p h a c e 1管柱層析及 /或Sephacryl S-300 HR管柱層析的方法以進行層析法。 再者’本發明係提供一種使變性魚肉復原之方法，其 特徵在於將本發明之亞硫酸鹽還原酵素以溶液或粉末的形 態使用於變性魚肉中，以使變性魚肉復原，其中相對於每 公克之變性魚肉，係使用〇 · 0 1至0 · 5活性單位之亞硫酸 鹽還原酵素、其粗酵素液或粗酵素粉末，較佳係使用0.03 至〇 · 1活性單位。又，亞硫酸鹽還原酵素於變性魚肉中之 作用時間爲5至40分鐘，較佳爲10至30分鐘。 【實施方式】 本發明係由 Escherichia coli(CCRC 11634)製造亞硫 酸鹽還原酵素、其粗酵素液或粗酵素粉末，並探討其使變 性魚肉復原之功效。 首先詳述所使用之材料及儀器，以及亞硫酸鹽還原酵 素之製造、與相關之試驗或測試方式。 (A)材料及儀器 (1)菌株及菌體1259207 (1) 玖, invention description [Technical field of invention] The present invention relates to a method for purifying sulfite reducing enzyme, in detail, 'the sulfite reducing enzyme can catalyze the reduction of sulfite to sulfide The reaction, which is used in the denatured fish meat, can effectively restore the denatured fish protein, and can also increase the reactable sulfhydryl group and the rubber strength of the denatured fish. The invention also relates to a method of using a solution or powder of the sulfite reducing enzyme to restore denatured fish. [Prior Art] Sulfite reductase is widely present in plants and microorganisms, and is a caspase that reduces sulfite to sulfide in the final step of sulfate assimilation. A key step in the synthesis of amino acids. The electron supplier in the sulfite reduction process uses red uced nicotinamide adenine dinucleotide phosphate (NADPH) as the electron supplier in the E. coli, and the yeast is NADPH or reduced. Reduced methyl viologen (MVH) is used as an electron supplier, while other electron donors are used as electron suppliers in other enzymes. Muscle proteins are mostly composed of L-amino acids with peptides, disulfide bonds, hydrogen bonds, ionic bonds, hydrophobic groups, and dipole-dipole interactions to form proteins of various types and physiological functions. . When the second, third and fourth stereostructures of the protein molecule are changed, but the peptide -6- 1259207 (2) is not destroyed, it is called "protein denaturation". The denatured protein structure loses its original physical, chemical and biological properties by interacting with functional groups on the peptide chain or between the peptide chains due to the formation of random helical, unfolded or aggregated states. The reason for protein denaturation is generally thought to be due to the freezing of water in the muscle, resulting in an increase in salt concentration, a change in the pH of the environment in which the protein is present, precipitation of protein molecules due to salting out, aggregation of molecules in the molecule or in the molecule, and formation New hydrogen bonds, disulfide bonds, and ionic bonds cause denaturation of proteins. Moreover, the water around the protein molecule forms a state of binding water with functional groups such as -SH, -COOH, -NH, and -CO, and the water molecules are weakened by the temperature, and the water molecules with weak binding force first form ice crystals, and at the same time expand due to volume. To change the molecular form of the protein, expose the functional group, form new bonds between the molecules or within the molecule, and coagulate and denature. The covalent bond formed by the denaturation of frozen fish protein has been confirmed mainly due to the oxidation of sulfhydryl groups to form disulfide bonds (Jiang et al., J. Food Sci. 63: 777-78 1,1 9 9 8 Jiang et al., J. Food Sci. 60 : 6 5 2 -65 5, 1 99 8). After being frozen, the denatured fish meat not only has the phenomenon of softening of meat, loss of juice, and spongy structure, but its emulsification, water retention, gel forming ability and viscoelasticity are also worse than those of fresh ones. Degeneration of muscle fibrin. Glue is one of the most important characteristics of fish protein processing into refined products. The protein is denatured to form a regular network structure, which makes the muscle protein elastic and elastic. Protein gels are affected by many factors such as salt concentration, pH, temperature, protein concentration and composition, additives, ionic strength, freshness, and the like. From the biochemical characteristics of refined products -7- 1259207 (3), it is known that the bonds forming the network structure include hydrogen bonds, hydrophobic bonds, ionic bonds, disulfide bonds and other covalent bonds. In addition, the strength of the rubber is an indicator of the quality of the fish paste refined product, and there are many factors affecting the elasticity of the refined product, such as salt concentration, p Η値, temperature, protein concentration and composition, additives, ionic strength, freshness and the like. Therefore, various methods for improving the elasticity of fish meat products have been proposed. During the frozen storage of fish, the gelatinizing ability is reduced, and the myosin heavy chain mainly composed of fish muscle fiber protein is gradually reduced during the freezing period, and a high molecular polymer such as myosin dimer and polymer is produced. The formation is mainly caused by the formation of a disulfide bond by the sulfhydryl group between the subunits of myosin, and the fish muscle actin is coagulated and denatured. As mentioned above, during the freezing period, the gelatinization ability of the fish meat is lowered, and the protein is unstable, and the application of the frozen fish meat to the production of the protein colloid food is restricted. In the past, alkali washing was used to improve the gelling ability of red fish, but it did not improve the color. Increasing the number of alkaline washing cycles or using ozone decoloring can substantially improve the color of the squid meat, but it will cause the gelatinization ability to deteriorate. In addition, the frozen fish paste prepared by adding a chemical reducing agent such as cysteine, sodium hydrogen sulfate or erythorbate to the denatured fish meat, the total sulfur-hydrogen group of the actin, reactive sulfhydryl group and The amount of myosin extracted was much higher than that of frozen fish without added reducing agent. Although freezing causes a decrease in the gelatinizing ability of the protein, the addition of the reducing agent can largely restore it, indicating that the reducing agent can restore the cohesive denaturing actin. Adding Reducing Agent-8- 1259207 (4) Adding can reduce the disulfide bond and convert it into a sulfhydryl group when the frozen denatured fish is in a crucible', re-forms the disulfide bond in the refined rubber to make the network more For stability. However, chemical reducing agents are considered as exotic additives and are less acceptable to consumers. The inventors of the present invention have proposed to reduce the crude enzymes by microbial-derived sulfites to replace such chemical reducing agents. For example, Jiang et al., the sulfite-reducing enzyme produced by yeast to decolorize and degenerate ozone. The resilience of the degenerated muscle protein of fish paste, found that sulfite reduction of crude enzyme can restore the denatured muscle protein and improve the gelatinization ability of refined products (Jiang et al.? J. Food Sci. 60: 6 52-65 5 , 1 998; Jiang et al., J. Food Sci. 60: 777-78 1,1 998). In addition, Wu et al. prepared a sulphite reducing enzyme crude enzyme lyophilized powder from Escherichia coli for processing frozen fish, which was also found to improve the gelling ability of refined products (Wu et al., J. Food Sci. 65). : 1400-1403, 2000) ° But so far no sulfite reductase for the preparation of microbial sources has been prepared, and no one has ever explored its efficacy in restoring denatured fish. SUMMARY OF THE INVENTION In order to solve the problems in the prior art mentioned above, the inventors of the present invention have focused on the purification method of purified sulfite reducing enzyme from microorganisms and the obtained sulfite reducing enzyme to restore the denatured fish. The effect of the present invention found that the purified sulfite reducing enzyme produced by E scherichiac ο 1 i can achieve the above object, thereby completing the present invention. That is, the present invention provides a sulfite reducing enzyme characterized by - 9259 207 (5) having the following characteristics: a. Its function is to catalyze the reduction of sulfite to sulfide and the recovery of oxidized sulphur. The reaction of the base; b. In the catalytic reaction, the electron donor is N ADPH, MVH or other electron donor; c. the molecular weight is 100,000 to 400,000; d. the suitable temperature range of action is 20 ° C to 30 ° C; And e. The appropriate pH for the action is 6.5 to 8.0. In the above sulfite reducing enzyme, the electron supplier is preferably NADPH. Further, the sulfite reducing enzyme is preferably derived from Escherichia coli. In addition, the present invention also provides a method for producing a purified sulfite reducing enzyme, which is characterized by ammonium sulfate partitioning and chromatography, whereby Escherichia is used. Coli crude enzyme solution produces purified sulfite reducing enzyme. In the above method for producing a purified sulfite reducing enzyme, the preparation of the crude enzyme solution preferably comprises the steps of: adding Escherichia coli cells to a phosphate buffer having a pH of 6.5 to 8.5, at 2 to At a temperature of 10 °C, crush with an ultrasonic breaker for 0.1 to 2 hours, centrifuge at 4,000 to 1,500 rpm for 2 hours, collect the supernatant, and add phosphate buffer. Then, it is crushed and centrifuged in the same manner, and the phosphate buffer containing the enzyme is combined to obtain a crude enzyme solution. The ammonium sulfate partitioning method comprises the steps of: slowly adding solid ammonium sulfate to the crude enzyme solution prepared in the above manner at 2 to 1, and stirring; -10- 1259207 (6) 3,0 0 The mixture was centrifuged at a speed of 1 5,0 0 0 xg for 2 hours; and the precipitate solution was subjected to a phosphate buffer having a concentration of 0.01 to 0.2 M and a pH of 6.5 to 8.5. Daxie. 5 to 2 days of dialysis, and get dialyzate. Further, the chromatography is carried out by a method selected from the group consisting of D E A E S e p h a c e 1 column chromatography and/or Sephacryl S-300 HR column chromatography. Further, the present invention provides a method for restoring denatured fish meat, characterized in that the sulfite reducing enzyme of the present invention is used in a form of a solution or a powder in a denatured fish meat to restore the denatured fish meat, wherein each gm is recovered. The modified fish meat is a sulfite reducing enzyme, a crude enzyme solution or a crude enzyme powder of 活性·0 1 to 0·5 active units, preferably 0.03 to 〇·1 active unit. Further, the sulfite reducing enzyme is allowed to act in the denatured fish for 5 to 40 minutes, preferably 10 to 30 minutes. [Embodiment] In the present invention, eutectic acid reducing enzyme, a crude enzyme solution or a crude enzyme powder is produced by Escherichia coli (CCRC 11634), and its effect of restoring the variable fish meat is examined. First, detail the materials and instruments used, as well as the manufacture of sulfite reducing enzymes, and the associated tests or test methods. (A) Materials and instruments (1) Strains and bacteria
Escherichia coli(CCRC 11634):購自食品工業發展硏 究所菌種硏究中心。將經活化之E s c h e r i c h i a c ο 1 i接種1 % 於5 00毫升三角瓶，內裝200毫升之培養液，培養液組成 -11 - 1259207 (7) 爲 4.0%蔗糖、0.4%酵母萃取物及 0.4%胰蛋白腺 (tryptone)，調整 pH 値至 7.5，於 37t、1〇〇 rpm 下振盪 培養1 8小時後，以5，〇 〇 0 X g離心3 0分鐘，收集菌體。 (2) 樂品 DEAE Sephacel、Sephacryl S-300 HR、電泳分子量標 準品和凝膠過濾分子量標準品：Pharmacia製造。 酵母萃取物、胰蛋白腺：Difco製造。 硫酸銨：Merck製造。 牛血淸蛋白（BSA)、黃素核苷酸（FMN)、還原菸醯胺 腺嘌呤磷二核苷（NAD PH)、膠胺基硫、二硫蘇糖醇、光胱 胺酸、5,5'-二硫代雙-(2-硝基苯甲酸）（DTNB)、β-酼基乙 醇（β-Me)及十二烷基硫酸鈉（SDS) : Sigma化學公司製 造。 碘醋酸（IAA)、N-乙基馬來醯亞胺（NEM)、對氯汞苯 甲酸酯（PCMB)、苯基甲基磺醯基氟化物（PmsF)、 Ν，Ν，Ν'，Ν、四甲基-乙二胺（TEMED): Bio-Rad 製造。 其他化學藥品均爲生化藥品級。 (3) 儀器 1·低溫振盪培養箱：台灣台北Hotech儀器公司製造 之 Hotech 718 〇 2. 低溫高速離心機：日本日立公司製造之SCR20B。 3. 超音波破碎機：日本超音波公司製造之W-200型。 4 ·物性測定儀：日本東京太陽化學公司製造之CR-200 型 〇 1259207 (8) 5. 分光光度計：日本日立公司製造之 Hitachi U-200 1 〇 6. 迷你電泳：採用 Mini-PROTEAN Π電池，其電源 供應器爲美國Bio-Rad製造之Bio-Rad 200/2.0型。 7·ρΗ測定儀：日本TOA電子公司製造之HM-30S。 8.水浴槽：台灣MARATON公司製造之Β204型。 9 .擂潰機：台灣僑法有限公司製造之CF 5公斤。 10.管柱：瑞士烏普撒拉Pharmacia LKB生技公司製 造之 C 26/40 及 C 26/ 1 00。 1 1 ·部分收集器（Fraction collector):瑞士烏普撒拉 Pharmacia LKB生技公司製造之FRAC_2〇〇。 12·快速蛋白液相層析系統（FPL C):瑞士烏普撒拉 Pharmacia LKB生技公司製造之控制器LCC-500+，P-500 泵。 13. -20°C冷凍櫃：美國Forma科學公司製造之生化冷 凍器，8 4 4 2型。 14. Amicon超過濾濃縮裝置：購自美國 Amicon公 司，主要構造爲濃縮槽（攪拌電池8 0 5 0型）及過濾膜（Y Μ 10，43毫米之薄膜）。 15·止泡均質機：Waring掺合器（附加擋板）。 (B)亞硫酸鹽還原酵素之製造及其生化特性試驗 (1)粗酵素液之製備Escherichia coli (CCRC 11634): purchased from the Center for Research and Development of Food Industry Development Institute. Inoculate 1% of the activated E scherichiac ο 1 i into a 500 ml flask and hold 200 ml of the culture medium. The medium composition is -11 - 1259207 (7) 4.0% sucrose, 0.4% yeast extract and 0.4% The tryptone was adjusted to pH 7.5, shaken at 37 t, 1 rpm for 18 hours, and then centrifuged at 5, 〇〇 0 X g for 30 minutes to collect the cells. (2) Music products DEAE Sephacel, Sephacryl S-300 HR, electrophoresis molecular weight standards, and gel filtration molecular weight standards: manufactured by Pharmacia. Yeast extract, trypsin gland: manufactured by Difco. Ammonium sulfate: manufactured by Merck. Bovine blood scorpion protein (BSA), flavin nucleotide (FMN), reduced nicotinamide adenine dinucleoside (NAD PH), amide sulfide, dithiothreitol, photocysteine, 5, 5'-Dithiobis-(2-nitrobenzoic acid) (DTNB), β-mercaptoethanol (β-Me) and sodium dodecyl sulfate (SDS): Sigma Chemical Co., Ltd. Iodoacetic acid (IAA), N-ethyl maleimide (NEM), p-chloromercaptobenzoate (PCMB), phenylmethylsulfonyl fluoride (PmsF), hydrazine, hydrazine, hydrazine, Bismuth, tetramethyl-ethylenediamine (TEMED): Manufactured by Bio-Rad. Other chemicals are biochemical grades. (3) Instruments 1. Low-temperature oscillation incubator: Hotech 718 manufactured by Hotech Instruments, Taipei, Taiwan 2. Low-temperature high-speed centrifuge: SCR20B manufactured by Hitachi, Japan. 3. Ultrasonic Wave Crusher: W-200 manufactured by Japan Ultrasonic Corporation. 4 · Physical property tester: CR-200 model manufactured by Tokyo Sun Chemical Co., Ltd., Japan 〇 1259207 (8) 5. Spectrophotometer: Hitachi U-200 manufactured by Hitachi, Ltd. Japan 〇 6. Mini electrophoresis: using Mini-PROTEAN Π battery The power supply is Bio-Rad 200/2.0 manufactured by Bio-Rad, USA. 7·ρΗ analyzer: HM-30S manufactured by TOA Electronics Co., Japan. 8. Water bath: Model 204 manufactured by MARATON Corporation of Taiwan. 9. Crushing machine: CF 5 kg manufactured by Taiwan Overseas Chinese Law Co., Ltd. 10. Pipe column: C 26/40 and C 26/ 1 00 manufactured by Pharmacia LKB Biotech, Uppsala, Switzerland. 1 1 · Fraction collector: FRAC_2〇〇 manufactured by Pharmacia LKB Biotech, Inc., Uppsala, Switzerland. 12. Fast Protein Liquid Chromatography System (FPL C): UCP-500+, P-500 pump manufactured by Pharmacia LKB Biotech, Switzerland. 13. -20 °C freezer: Biochemical chiller manufactured by Forma Scientific, USA, 8 4 4 2 type. 14. Amicon Ultrafiltration Concentrator: purchased from Amicon, USA, mainly constructed as a concentration tank (stirring battery type 850) and a filter membrane (Y Μ 10, 43 mm film). 15. Stop bubble homogenizer: Waring blender (additional baffle). (B) Preparation of sulfite reducing enzyme and its biochemical characteristics test (1) Preparation of crude enzyme solution
於菌體中加入兩倍量之磷酸鹽緩衝液（含〇 . 5 mM ~ 13- 1259207 (9) E D T A之〇 · 1 Μ磷酸鉀或磷酸鈉，p H値爲7 · 7 )，於4 °C 下’利用超苜波破碎機破碎3 0分鐘（輸出功率3 0 %)，於 5，〇〇〇xg下離心30分鐘，取上澄液，再加入等量之磷酸鹽 緩衝液’再破碎、離心’合倂含有酵素之磷酸鹽緩衝液以 得粗酵素液。 (2) 硫酸銨分劃 將一定體積之粗酵素液於4 t下攪拌，同時緩慢添加 定量之硫酸銨粉末’收集3 0至6 0 %飽和硫酸銨濃度之沉 激部分後，將之溶解於磷酸鹽緩衝液中，再以磷酸鹽緩衝 液透析24小時，去除硫酸銨後，製得粗亞硫酸鹽還原酵 素溶液。 (3) DEAE Sephacel 管柱層析 將硫酸錢分劃所得到之粗酵素液，以 DEae S e p h a c e 1 (2 · 6 X 2 0公分）管柱層析，收集含有活性的溶離 液，再以Amicon超過濾濃縮器加以濃縮；最後再以 Sephacryl S-3 00 HR 及 / 或 DEAE Sephadex A-50 管柱層 析純化之。 (4) 蛋白質濃度測定 蛋白質濃度乃依據蛋白質染料結合方法測定之，標準 檢量曲線以牛血淸蛋白做爲標準蛋白質。 (5) 蛋白質分子量測定 酵素係以Sephacryl S-3 00 HR膠過濾層析測定其分子 量。所使用的標準蛋白質爲甲狀腺球蛋白（669 kD a)、鐵 蛋白（440 kDa)、接觸酵素（23 2 kDa)、丁醛醇酵素（158 -14- (10) 1259207 kDa)及 BSA(67 kDa)。 (6) 酵素活性測定 依據Siegel et al.( 1 9 73 )測定活性的方法，將酵素液 加入1 · 0毫升反應試劑，包含〇 ·丨M磷酸鹽緩衝液（pH値 7.7)、0.5 mM NaHS03、1 μΜ FMN 及 0.2 mM NADPH，於 2 5 °C下反應5分鐘，同時以分光光度計做時間掃描，以 3 40奈米測定吸光値。一個酵素活性單位定義爲每分鐘催 化1微莫耳NADPH氧化所須之酵素量。 (7) 最適酸鹼度 將酵素分別在不同酸鹼度之緩衝溶液（0.2 Μ檸檬酸鹽 緩衝液：pH値爲4.0至6.0 ; 0·2 Μ磷酸鹽緩衝液：pH値 爲6.0至8.0;及0·2 Μ重碳酸鹽緩衝液：pH値爲8.0至 1〇·〇)中，測定其作用之最適酸鹼度。 (8) 酸鹼度安定性 將酵素分別溶於不同酸鹼度之緩衝溶液中（〇.2 Μ檸檬 酸鹽緩衝液：pH値爲4.0至6.0 ; 0.2 Μ磷酸鹽緩衝液： pH値爲6.0至8.0 ;及0.2 Μ重碳酸鹽緩衝液：pH値爲 8.0至10.0)，並置於25°C反應30分鐘後，測定其酸鹼度 安定性。 (9) 最適溫度 將酵素加入反應試劑，分別在5至6 0 °C下測定其活 性，以測試其最適溫度。 (1〇)熱安定性 將酵素液置於5至6 0 °C下反應3 0分鐘後’於冰水中 •15- 1259207 (11) 冷卻5分鐘，測定其殘存活性，以測試其熱安定性。 (11)還原劑的影響 將酵素置於含1 0 mM還原劑之反應試劑，還原劑分 別爲膠胺基硫、二硫蘇糖醇、β-锍基乙醇及光胱胺酸，在 2 5 °C反應3 0分鐘後。 (1 2 )抑制劑的影響 將酵素置於含1 mM抑制劑之反應試劑中，抑制劑分 別爲：苯基甲基磺醯基氟化物（PMSF)、碘醋酸（IAA)、對 氯汞苯甲酸酯（PCMB)、N-乙基馬來醯亞胺（NEM)及氰化鉀 (KCN)，在25 t：反應30分鐘後，以測試抑制劑的影響。 (13) 金屬離子的影響 將酵素分別加入最終濃度爲0.5、1.0、5.0及10 mM 的鈉、鉀、鎂、鈣、錳、鐵、鋇、鈷、鎳、銅、鉛、鋅和 汞之氯化鹽中，於2 5 °C下靜置3 0分鐘後，以測試金屬離 子對酵素活性之影響。 (14) 低溫儲存之安定性 將酵素液分別在4及-20 °C溫度下儲存，定時取出測 定其殘存酵素活性，以測試低溫儲存之安定性。 (C)亞硫酸鹽還原酵素、其粗酵素液或粉末對冷凍變性 鯖、金線鰱及白帶魚肉之復原測試 (1)冷凍鯖、金線鰱及白帶魚漿之製備 以鯖魚、金線鰱及白帶魚爲原料，置於-2 5 °C冷凍櫃 貯藏六個月，使其冷凍變性。後採其魚肉經細碎除筋後， -16- 1259207 (12) 以冰水水漂三次後以離心式脫水機脫水至水分含量爲7 8 % 左右。將所得之魚漿擂潰1〇分鐘並混合0.2%之重合磷酸 鹽及5%之糖或醣醇類，置於-40 °C之送風式凍結設備中使 中心溫度快速降至-1 8 °C實施急速凍結，再於-25 °C之凍藏 庫中貯藏備用。 (2) 煉製品之製造 將上述之冷凍魚漿置於5至1 0 °C之冷房中使中心溫 度上升至-2至-5 °C止實施解凍，再加入上述製得之亞硫酸 鹽還原酵素、其粗酵素液或粗酵素粉末，同時利用擂潰機 擂潰20分鐘，加入2.5%鹽後再擂潰20分鐘，最後加入 3 %馬鈴薯澱粉混合均勻，灌入腸衣，在25°C下靜置30 分鐘，再以95 °C加熱20分鐘，經流水冷卻後，置於4°C 貯藏並於次日測定其品質。 (3) 魚漿性質之測定 魚漿肌動凝蛋白之萃取乃依據 Noguchi and Mat sumoto( 1 97 8)方法製備；而肌動凝蛋白之可反應基則 依據Itoh et al.( 1 979)之改良方法測定。至於形膠能力之 測定，係將魚漿製成之煉製品切成3 · 0公分高，以物性測 定儀測定膠強度，柱塞直徑爲5毫米。 (D)實施例 以下茲例舉製備例及使用例以更具體地說明本發明， 惟本發明並不限定於此等例示中。 -17- 1259207 (斗 製備例1 :源自Escherichia c〇l i之亞硫酸鹽還原酵素之 製造以及生化特性 將Escherichia coli菌體（CCRC 11634)加入兩倍量之 磷酸鹽緩衝液（含0.5 mM EDTA之0.1 Μ磷酸鉀或磷酸 鈉，pH値爲7.7)，於下利用超音波破碎機破碎30分 鐘（輸出功率30%)，將菌體染色，以顯微鏡觀察其細胞是 否完全破碎，於5,000xg下離心30分鐘，取上澄液，再 加入等量之磷酸鹽緩衝液，再以相同方式加以破碎、離 心，合倂含有酵素之磷酸鹽緩衝液以製得粗酵素液。 源自 Escherichia coli之亞硫酸鹽還原酵素經超音波 破碎細胞，在5，0 0 0 X g下離心3 0分鐘，所得上澄液即爲 粗酵素液，其經硫酸銨分劃，可發現在 3 0至60%飽和 硫酸銨沉澱部分之活性最高，沉澱部分用兩倍量 0.1 Μ 之磷酸鹽緩衝液（pH値爲7.7)溶解，並進行透析處理。再 經DEAE Sephacel離子交換管柱層析（梯度條件：〇.〇至 1·〇 M NaCl ;溶離速度：1.0毫升/分鐘；5.0毫升/管），被 吸附之酵素蛋白在NaCl 0·35 Μ濃度下被溶離，收集具活 性部分，利用Amicon濃縮成4毫升。使用Sephacryl S-3 00 HR膠過濾管柱層析，收集具有活性之部分進行電泳 分析，確定已被純化。 經測定得知E s c h e r i c h i a c ο 1 i之回收率及比活性分別 爲3 1 · 7 %及1 〇 · 0 3單位/毫克，且其純化倍率爲5 7 9 · 5倍 (如表1所示）。 -18- (14) 1259207 表1 源自Escherichia coli之亞硫酸鹽還原酵素之製造 步驟 蛋白質 總量 (毫克） 總活性 (單位） 比活性 (單位/毫克） 回收率 (%) 純化 (倍率） 粗酵素液 3660.1 63.27 0.017 100.0 1.0 30 至 60%(NH4)2S〇4 1401.6 44.23 0.032 69.9 1.8 DEAE-Sephacel 599.5 35.25 0.059 55.7 3.4 1st Sephacryl S-300 HR 34.2 30.00 0.877 47.4 50.7 2nd Sephacryl S-300 HR 15.8 25.98 1.644 41.1 95.1 3rd Sephacryl S-300 HR 2.0 20.05 10.025 31.7 579.5 源自 Escherichia coli之亞硫酸鹽還原酵素之生化特 性如下： (1 )分子量 利用Sephacryl S-3 00 HR膠過濾管柱層析，配合標準 蛋白質所作得之檢量曲線圖，估計源自E s c h e r i c h i a c ο 1 i 之亞硫酸鹽還原酵素還原酵素之分子量爲1 1 9,00 0(如圖1 所示）。 (2)最適酸鹼度及酸鹼度安定性 源自Escherichia coli之亞硫酸鹽還原酵素之最適pH 値爲7.7，於pH値爲6.5至8.0下非常穩定（如圖2所 示）。在25t：、pH値爲4.0至10.0下靜置30分鐘後測定 其活性，結果發現，在pH値爲6.5至7.5時仍保有95% 以上之活性，pH値爲6,5時酵素殘存活性仍爲73.7%， -19- 1259207 (t5) p H値爲8.0時酵素殘存活性仍爲7 9.0 %，由此可知酵素在 中性環境下之安定性較佳。 (3) 最適溫度及熱安定性 源自 Escherichia coli之亞硫酸鹽還原酵素之最適作 用溫度爲2 5 °C。於熱安定性方面，在5至2 5 °C範圍內該 酵素極爲安定，酵素殘存活性仍爲98.0%以上，溫度超過 2 5 °C時酵素逐漸失活，至5 時則完全沒有活性（如圖3 所示）。 (4) 抑制劑對酵素活性之影響 PCMB及KCN可完全抑制源自Escherichia coli之亞 硫酸鹽還原酵素之催化活性，而NEM、PMSF和IAA可部 分抑制還原酵素之催化活性。 (5) 金屬離子對酵素活性之影響 各種金屬離子對源自Escherichia coli之亞硫酸鹽還 原酵素活性之影響如下·· Hg2+、Fe2+、Fe3+、Ca2+、Co2 + 和 Cu2 +具強烈抑制作用，Cd2+、Zn2+、Mn2 +和Ba2 +具部 分抑制作用，而Na+、K +和Mg2 +對酵素活性則無影響。 其中Hg2+、Co2+、Zn2 +及Fe2 +會與酵素上的硫氫基結合而 抑制其活性，本項結果再次證實該酵素之活性部位含有光 胱胺酸殘基。 (6) 還原劑對酵素活性之影響 膠胺基硫、二硫蘇糖醇、β-毓基乙醇及光胱胺酸等還 原劑均可促進源自 E s c h e r i c h i a c ο 1 i之亞硫酸鹽還原酵素 之活性。 -20- (16) 1259207 (7)低溫儲存之安定性 將源自Escherichia c〇ii之亞硫酸鹽還原酵素之酵素 液分別在4°C及-20°C溫度下儲存，定時取出測定其殘存 酵素活性，測試結果發現，在4 °C冷藏2 8天後，其酵素 活性仍爲77.8%，而在-2(TC貯藏兩個月，其活性幾乎不 變 0 使用例1 :源自 Escherichia coli之亞硫酸鹽還原酵素對 冷凍變性鯖魚漿之影響 源自 Escherichia coli之亞硫酸鹽還原酵素之添加量 對冷凍變性鯖魚漿之影響如圖4所示。添加不同酵素活性 單位之源自Saccharomyces cerevisiae之亞硫酸鹽還原酵 素於冷凍變性鯖魚漿中，隨酵素添加量增加，鯖魚漿中可 反應硫氫基明顯增加，當亞硫酸鹽還原酵素添加量爲〇.〇3 活性單位/克時，可反應硫氫基由控制組之4.19x 1(Τ5莫耳 /克增至7.23 xl 0_5莫耳/克，若超過0.03單位，則其可反 應硫氫基並無明顯變化。而膠強度亦有相同之結果，源自 Escherichia coli之亞硫酸鹽還原酵素之添加量爲〇.〇3活 性單位/克時，膠強度會由1 15.0克X公分增加至2 3 5.7克X 公分，超過0.03單位後，膠強度並無明顯變化。 作用時間對添加源自Escherichia coli之亞硫酸鹽還 原酵素之冷凍變性鯖魚漿品質之影響如圖5所示。添加 0.03活性單位/克之源自Escherichia coli之亞硫酸鹽還原 酵素，作用時間越長，可反應硫氫基明顯增加，作用至 -21 - (17) 1259207 2 5分鐘時，可反應硫氫基由控制組之4.2 〇 χ 1 〇·5莫耳/克增 至7·65χ10_5莫耳/克。膠強度結果亦相似，於作用25分 鐘後，膠強度由109.4克X公分增加至211.2克X公分，之 後趨於平緩。 (Ε)產業上可利用性 由本發明之亞硫酸鹽還原酵素之最適pH値及pH値 安定性可知，本發明之亞硫酸鹽還原酵素極適合用於水產 製品之加工領域中。 另外，由於大部分之魚漿加工均於冷卻溫度（通常係 低於5°C )下進行，故由本發明之亞硫酸鹽還原酵素之熱 安定溫度範圍可知，本發明之亞硫酸鹽還原酵素極適合用 於魚漿加工上。 【圖式簡單說明】 於圖1中，係利用Sephacryl S-300 HR膠過濾管柱層 析’以檢量曲線圖估計源自E s c h e r i c h i a c ο 1 i之亞硫酸鹽 還原酵素之分子量；圖中之 A點爲甲狀腺球蛋白（669 kDa)、B爲點鐵蛋白（440 kDa)、C點爲接觸酵素（232 k〇a)、D點爲丁醛醇酵素（158 kDa)、E點爲 BSA(67 kDa)。 圖2係圖示pH値對源自Escherichia coli之亞硫酸鹽 還原酵素之影響；圖中之「〇」表示與pH値相關之活 性、「·」表示pH値安定性。 -22- (18) 1259207 圖3係圖示溫度對源自E s c h e r i c h i a c ο 1 i之亞硫酸鹽 還原酵素之影響；圖中之「〇」表示與溫度相關之活性、 1 ·」表不熱安定性。 圖4係圖不源自Escherichia coli之亞硫酸鹽還原酵 素之添加量對冷凍變性鯖魚漿中可反應硫氫基及膠強度之 影響；圖中之「·」表示可反應硫氫基、「」表示膠強 度。 圖5係圖示源自Escherichia coli之亞硫酸鹽還原酵 素之作用時間對冷凍變性鯖魚漿中可反應硫氫基及膠強度 之影響；圖中之「·」表示可反應硫氫基、「」表示膠 強度。 圖6係圖示源自Add twice the amount of phosphate buffer (containing 〇. 5 mM ~ 13- 1259207 (9) EDTA · 1 Μ potassium phosphate or sodium phosphate, p H値 is 7 · 7 ) at 4 ° C under 'crushing with ultra-chopper crusher for 30 minutes (output power of 30%), centrifuge at 5, 〇〇〇xg for 30 minutes, take the liquid, then add the same amount of phosphate buffer 're-broken , centrifugation 'synthesis of phosphate buffer containing enzyme to obtain a crude enzyme solution. (2) Ammonium sulphate partitioning A certain volume of the crude enzyme solution is stirred at 4 t while slowly adding a quantitative amount of ammonium sulphate powder to collect the sensitized portion of the concentration of 30 to 60% saturated ammonium sulfate, and then dissolve it in In the phosphate buffer solution, the phosphate buffer solution was dialyzed for 24 hours to remove the ammonium sulfate, and a crude sulfite reducing enzyme solution was obtained. (3) DEAE Sephacel column chromatography The crude enzyme solution obtained by dividing the sulfuric acid money into a column of DEae S ephace 1 (2 · 6 X 2 0 cm) to collect the active solution and then Amicon The ultrafiltration concentrator is concentrated; finally purified by Sephacryl S-3 00 HR and / or DEAE Sephadex A-50 column chromatography. (4) Determination of protein concentration The protein concentration is determined by the protein dye binding method, and the standard calibration curve uses bovine blood scorpion protein as a standard protein. (5) Determination of protein molecular weight The enzyme was assayed for molecular weight by Sephacryl S-3 00 HR gel filtration chromatography. The standard proteins used were thyroglobulin (669 kD a), ferritin (440 kDa), contact enzyme (23 2 kDa), butyraldehyde (158 -14-(10) 1259207 kDa) and BSA (67 kDa). ). (6) Determination of enzyme activity According to the method of measuring activity by Siegel et al. (1 9 73), the enzyme solution was added to 1.0 ml of reagent, including 〇·丨M phosphate buffer (pH 値7.7), 0.5 mM NaHS03. 1 μΜ FMN and 0.2 mM NADPH were reacted at 25 ° C for 5 minutes while scanning with a spectrophotometer to measure the absorbance at 3 40 nm. An enzyme activity unit is defined as the amount of enzyme required to catalyze the oxidation of 1 micromolar NADPH per minute. (7) Optimum pH and alkalinity enzymes in different pH buffer solutions (0.2 Μ citrate buffer: pH 4.0 4.0 to 6.0; 0·2 Μ phosphate buffer: pH 値 6.0 to 8.0; and 0·2 ΜHypercarbonate buffer: pH 値 8.0 to 1 〇·〇), the optimum pH of the action was determined. (8) pH stability The enzyme is dissolved in a buffer solution of different pH (〇.2 Μ citrate buffer: pH 4.0 4.0 to 6.0; 0.2 Μ phosphate buffer: pH 6.0 6.0 to 8.0; 0.2 Μ bicarbonate buffer: pH 8.0 8.0 to 10.0), and after reacting at 25 ° C for 30 minutes, the pH stability was measured. (9) Optimum temperature The enzyme is added to the reagent and its activity is measured at 5 to 60 °C to test its optimum temperature. (1〇) Thermal stability The enzyme solution was reacted at 5 to 60 ° C for 30 minutes, then cooled in ice water • 15 - 1259207 (11) for 5 minutes, and its residual viability was measured to test its thermal stability. . (11) Effect of reducing agent The enzyme is placed in a reaction reagent containing 10 mM reducing agent, and the reducing agents are respectively amide sulfide, dithiothreitol, β-mercaptoethanol and photocytosine, at 25 After °C reaction for 30 minutes. (1 2) Effect of inhibitors The enzymes were placed in a reaction reagent containing 1 mM inhibitors: phenylmethylsulfonyl fluoride (PMSF), iodine acetate (IAA), p-chloromercurybenzene Formate (PCMB), N-ethylmaleimide (NEM) and potassium cyanide (KCN) were tested for inhibitors after 25 t: 30 min reaction. (13) Effects of metal ions The enzymes were added to the final concentrations of 0.5, 1.0, 5.0 and 10 mM of sodium, potassium, magnesium, calcium, manganese, iron, strontium, cobalt, nickel, copper, lead, zinc and mercury. In the salt, after standing at 25 ° C for 30 minutes, the effect of metal ions on the activity of the enzyme was tested. (14) Stability of low-temperature storage The enzyme solution was stored at 4 and -20 °C, and the residual enzyme activity was measured periodically to test the stability of low-temperature storage. (C) Restoration test of sulfite reducing enzyme, its crude enzyme solution or powder on frozen denatured mites, golden mites and leucorrhea fish (1) preparation of frozen carp, golden linden and leucorrhea fish carp, gold thread The oysters and leucorrhea were used as raw materials and stored in a freezer at -2 °C for six months to freeze-denature them. After the fish is finely crushed and removed, -16- 1259207 (12) is rinsed three times with ice water and then dehydrated by a centrifugal dehydrator to a moisture content of about 78%. The resulting fish slurry is smashed for 1 minute and mixed with 0.2% of the phosphate and 5% sugar or sugar alcohol. The temperature is rapidly lowered to -1 8 ° in a supply-type freezing device at -40 °C. C is rapidly frozen and stored in a frozen reservoir at -25 °C for later use. (2) Manufacture of refined products The above-mentioned frozen fish paste is placed in a cold room at 5 to 10 °C to raise the center temperature to -2 to -5 °C, and then thawed, and then the sulfite reduction obtained above is added. Enzyme, its crude enzyme solution or crude enzyme powder, while using a smashing machine to smash for 20 minutes, add 2.5% salt and then smash for 20 minutes, and finally add 3% potato starch and mix evenly, into the casing, at 25 ° C After standing for 30 minutes, it was heated at 95 ° C for 20 minutes, cooled by running water, stored at 4 ° C and measured for quality the next day. (3) Determination of the properties of fish paste The extraction of fish muscle actin is prepared according to the method of Noguchi and Mat sumoto (1 97 8); the reactive group of actin is based on Itoh et al. (1 979) Improved method of determination. As for the determination of the gel-forming ability, the refined product made of the fish paste was cut into a height of 3 · 0 cm, and the strength of the glue was measured by a physical property measuring instrument, and the diameter of the plunger was 5 mm. (D) EXAMPLES Hereinafter, the present invention will be more specifically described by way of Preparation Examples and Examples, but the present invention is not limited thereto. -17- 1259207 (Bucket Preparation Example 1: Manufacture of sulfite reductase derived from Escherichia c〇li and biochemical properties Escherichia coli cells (CCRC 11634) were added to twice the amount of phosphate buffer (containing 0.5 mM EDTA) 0.1 Μ potassium phosphate or sodium phosphate, pH 7.7 7.7), crushed for 30 minutes (output power 30%) with an ultrasonic breaker, stained the cells, and observed whether the cells were completely broken under a microscope at 5,000 xg Centrifuge for 30 minutes, take the clear solution, add the same amount of phosphate buffer, crush and centrifuge in the same way, and combine the phosphate buffer containing the enzyme to prepare the crude enzyme solution. From Escherichia coli The sulfate reducing enzyme is ultrasonically disrupted and centrifuged at 5,0 0 X g for 30 minutes. The obtained supernatant is a crude enzyme solution, which can be found to be saturated at 30 to 60% by ammonium sulfate partitioning. The ammonium sulfate precipitated fraction had the highest activity, and the precipitated fraction was dissolved with twice the amount of 0.1 磷酸盐 phosphate buffer (pH 7.7), and subjected to dialysis treatment, and then subjected to DEAE Sephacel ion exchange column chromatography (gradient conditions: 〇. 〇 to 1· 〇M NaCl; dissolution rate: 1.0 ml/min; 5.0 ml/tube), the adsorbed enzyme protein was dissolved at a concentration of NaCl 0·35 ,, and the active fraction was collected and concentrated to 4 ml by Amicon. Sephacryl S- was used. 3 00 HR gel filtration column chromatography, collecting the active part for electrophoresis analysis and confirming that it has been purified. It is determined that the recovery rate and specific activity of E scherichiac ο 1 i are 3 1 · 7 % and 1 分别, respectively. 0 3 units/mg, and its purification ratio is 5 7 9 · 5 times (as shown in Table 1) -18- (14) 1259207 Table 1 Production steps of sulfite reducing enzyme derived from Escherichia coli (mg) Total activity (unit) Specific activity (units/mg) Recovery (%) Purification (rate) Crude enzyme solution 3660.1 63.27 0.017 100.0 1.0 30 to 60% (NH4)2S〇4 1401.6 44.23 0.032 69.9 1.8 DEAE-Sephacel 599.5 35.25 0.059 55.7 3.4 1st Sephacryl S-300 HR 34.2 30.00 0.877 47.4 50.7 2nd Sephacryl S-300 HR 15.8 25.98 1.644 41.1 95.1 3rd Sephacryl S-300 HR 2.0 20.05 10.025 31.7 579.5 From the sulfur of Escherichia coli The biochemical properties of the salt-reducing enzyme are as follows: (1) The molecular weight is determined by Sephacryl S-3 00 HR gel filtration column chromatography, and the calibration curve obtained with the standard protein is estimated to be derived from the sulfite reduction of E scherichiac ο 1 i . The molecular weight of the enzyme reducing enzyme is 1 19,00 0 (as shown in Figure 1). (2) Optimum pH and pH stability The optimum pH of sulfite reductase from Escherichia coli is 7.7, which is very stable at pH 6.5 6.5 to 8.0 (as shown in Figure 2). The activity was measured after standing at 25t:, pH 4.0 4.0 to 10.0 for 30 minutes, and it was found that 95% or more of the activity was maintained at pH 6.5 6.5 to 7.5, and the residual activity of the enzyme remained at pH 66,5 When 73.7%, -19-1259207 (t5) p H値 was 8.0, the enzyme residual activity was still 79.0%, which indicates that the enzyme has better stability in a neutral environment. (3) Optimum temperature and thermal stability The optimum temperature for sulfite reductase from Escherichia coli is 25 °C. In terms of thermal stability, the enzyme is extremely stable in the range of 5 to 25 °C, and the enzyme residual activity is still above 98.0%. When the temperature exceeds 25 °C, the enzyme is gradually inactivated, and when it is 5, it is completely inactive (such as Figure 3)). (4) Effect of inhibitors on enzyme activity PCMB and KCN completely inhibit the catalytic activity of sulfite reductase derived from Escherichia coli, while NEM, PMSF and IAA can partially inhibit the catalytic activity of reductase. (5) Effect of metal ions on enzyme activity The effects of various metal ions on the activity of sulfite reductase derived from Escherichia coli are as follows: · Hg2+, Fe2+, Fe3+, Ca2+, Co2+ and Cu2+ have strong inhibitory effects, Cd2+, Zn2+, Mn2+, and Ba2+ have partial inhibition, while Na+, K+, and Mg2+ have no effect on enzyme activity. Among them, Hg2+, Co2+, Zn2+ and Fe2+ bind to the sulfhydryl group on the enzyme to inhibit its activity. This result again confirms that the active site of the enzyme contains a cysteine residue. (6) Effect of reducing agent on enzyme activity Reducing agents such as amide sulfide, dithiothreitol, β-mercaptoethanol and photocysteine can promote sulfite reductase derived from E scherichiac ο 1 i Activity. -20- (16) 1259207 (7) Stability of low-temperature storage The enzyme solution of sulfite-reducing enzyme derived from Escherichia c〇ii was stored at 4 ° C and -20 ° C, respectively, and was taken out at regular intervals to determine its residual value. Enzyme activity, the test results showed that after 24 hours of storage at 4 °C, the enzyme activity was still 77.8%, while at -2 (TC stored for two months, its activity is almost unchanged 0 use case 1: from Escherichia coli The effect of sulphite reducing enzyme on frozen denatured squid pulp is derived from the effect of the addition of sulfite reductase from Escherichia coli on frozen denatured squid pulp as shown in Figure 4. Adding different enzyme activity units from Saccharomyces Cerevisiae sulfite reducing enzyme in frozen denatured squid pulp, with the increase of enzyme addition, the reaction sulphur-hydrogen group in squid pulp increased significantly, when the sulfite reducing enzyme added amount is 〇.〇3 active unit / gram The reaction sulfhydryl group is 4.19x 1 (Τ5 mol/g to 7.23 xl 0_5 mol/g of the control group, and if it exceeds 0.03 unit, the reactable sulfhydryl group does not change significantly. The same result, from Esc When the addition amount of sulfite reducing enzyme of herichia coli is 〇.〇3 activity unit/g, the rubber strength will increase from 1 15.0 g X cm to 2 3 5.7 g X cm. After more than 0.03 units, the rubber strength is not obvious. The effect of the action time on the quality of the frozen denatured squid pulp added with sulfite reductase from Escherichia coli is shown in Figure 5. Add 0.03 activity units/gram of sulfite reductase from Escherichia coli, time of action The longer the reaction, the more obvious the reaction of sulfhydryl groups, the effect of the reaction to the sulphur-hydrogen group from 4.2 〇χ 1 〇·5 m /g to 7.65 χ10_5 Mohr/g. The rubber strength results were similar. After 25 minutes of action, the rubber strength increased from 109.4 g X cm to 211.2 g X cm, and then tended to be gentle. (Ε) Industrial availability by the sulfite of the present invention The optimum pH and pH stability of the reducing enzymes show that the sulfite reducing enzyme of the present invention is very suitable for use in the processing of aquatic products. In addition, since most of the processing of the fish paste is at a cooling temperature (usually lower than 5 ° C According to the thermal stability temperature range of the sulfite reducing enzyme of the present invention, the sulfite reducing enzyme of the present invention is extremely suitable for use in fish paste processing. [Simplified Schematic] In Fig. 1, the system utilizes Sephacryl S-300 HR Glue Filter Column Chromatography' uses a calibration curve to estimate the molecular weight of the sulfite reductase derived from E scherichiac ο 1 i; the point A in the figure is thyroglobulin (669 kDa), B is Point ferritin (440 kDa), point C is contact enzyme (232 k〇a), point D is butyryl alcohol (158 kDa), point E is BSA (67 kDa). Fig. 2 is a graph showing the effect of pH 値 on the sulfite reducing enzyme derived from Escherichia coli; "〇" in the figure indicates the activity related to pH 、, and "·" indicates pH 値 stability. -22- (18) 1259207 Figure 3 shows the effect of temperature on the sulfite reductase derived from E scherichiac ο 1 i; the “〇” in the figure indicates the temperature-related activity, and the expression is not heat stable. Sex. Figure 4 is a graph showing the effect of the amount of sulfite reducing enzyme added from Escherichia coli on the reactivity of sulfhydryl groups and the strength of the gel in freeze-denatured squid pulp; "·" in the figure indicates the reaction of sulfhydryl groups, "Expresss the strength of the glue. Figure 5 is a graph showing the effect of the action time of sulfite reductase derived from Escherichia coli on the reactivity of sulfhydryl groups and the strength of the gel in freeze-denatured squid pulp; "·" in the figure indicates that the sulfhydryl group can be reacted, "Expresss the strength of the glue. Figure 6 is a diagram derived from
Escherichia coli之亞硫酸鹽還原酵素之添加量對臭氧脫 色鯖魚漿中可反應硫氫基及膠強度之影響；圖中之「〇」 表不可反應硫氣基、「❿」表示膠強度。 -23-The effect of the amount of sulfite reducing enzyme added by Escherichia coli on the reactive sulfur-hydrogen group and the strength of the rubber in the ozone decolorized salmon slurry; the "〇" table in the figure does not reflect the sulfur gas base, and the "❿" indicates the strength of the rubber. -twenty three-