JP2004350586A - Sulfite reductase, method for producing the same, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sulfite reductase, a method for producing the sulfite reductase, and use of the sulfite reductase. <P>SOLUTION: The purified sulfite reductase has the following characteristics: (a) acting so as to catalyze the reduction of the sulfite to a sulfide, or so as to recover a sulfhidryl group from a disulfide group; (b) acting so that a reduced nicotinamide adenine dinucleotide phosphate (NADPH), a methyl viologen (MVH) or another donor group may act as an electron donating group in the catalytic reaction of the reduction; (c) having 100,000-400,000 molecular weight; (d) having the optimum temperature of the activity at 20-30°C; and (e) having the optimum pH at 6.5-8.0. The method for producing the purified sulfite reductase, and the method for recovering a denatured fish protein by using the sulfite reductase in a solution or powder shape are also provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

大腸菌由来の亜硫酸レダクターゼの生化学的特性は以下の通りであった。
【００５４】
（１）分子量
セファクリル Ｓ−３００ＨＲゲル濾過カラム・クロマトグラフィーによって標準タンパク質から得られた検量線（図１に表示する）により、大腸菌由来の亜硫酸レダクターゼの分子量は１１９，０００と推定された。
【００５５】
（２）至適ｐＨ値およびｐＨ安定性
大腸菌由来の亜硫酸レダクターゼは、７．７に至適ｐＨを有し、ｐＨ６．５から８．０で非常に安定していた（図２に示す）。２５℃、ｐＨ４．０から１０．０で静置した後、亜硫酸レダクターゼの活性を測定した。ｐＨ６．５から７．５では９５％以上の活性が残留していたことがわかった。ｐＨ６．５では７３．７％以上の活性が残り、ｐＨ８．０では７９．０％以上の活性が残っていた。したがって、酵素の安定性は若干酸性またはアルカリ性よりも中性状態の方が良好であることがわかった。
【００５６】
（３）至適温度および熱安定性
大腸菌由来の亜硫酸レダクターゼの至適温度は２５℃であった。熱安定性としては、酵素は５から２５℃の温度範囲で非常に安定であり、９８％以上の活性が残っていた。２５℃より上の温度では、酵素は徐々に不活性化され、５０℃に達すると完全に不活性化された（図３に示す）。
【００５７】
（４）阻害剤の酵素活性に対する影響
大腸菌由来の亜硫酸レダクターゼの触媒活性は、ＰＣＭＢおよびＫＣＮで完全に阻害され、ＮＥＭ、ＰＭＳＦおよびＩＡＡで部分的に阻害された。
【００５８】
（５）金属イオンの酵素活性に対する影響
大腸菌由来の亜硫酸レダクターゼの触媒活性は、Ｈｇ^２＋、Ｆｅ^２＋、Ｆｅ^３＋、Ｃａ^２＋、Ｃｏ^２＋、およびＣｕ^２＋によって強く阻害され、Ｃｄ^２＋、Ｚｎ^２＋、Ｍｎ^２＋、およびＢａ^２＋によって中等度の阻害を受けた。しかし、Ｎａ^＋、Ｋ^＋およびＭｇ^２＋は酵素活性に影響を与えなかった。金属イオンの内、Ｈｇ^２＋、Ｆｅ^２＋、Ｃｕ^２＋、およびＺｎ^２＋は酵素のスルフヒドリル基と相互作用し、その活性を阻害する。この項の結果から、酵素の活性部位にはシステイン残基が含まれていることが証明された。
【００５９】
（６）還元剤の酵素活性に対する影響
グルタチオン、ジチオスレイトール、β−メルカプトエタノール、およびシステイン等の還元剤はすべて大腸菌由来の亜硫酸レダクターゼの活性を改善した。
【００６０】
（７）低温貯蔵安定性
大腸菌由来の亜硫酸レダクターゼの酵素溶液を４℃および−２０℃の温度でそれぞれ貯蔵した。残留酵素活性を一定の時間間隔で測定した。テストの結果、４℃で２８日間貯蔵した後、酵素活性はまだ７７．８％であり、−２０℃で２カ月間貯蔵した後には、活性はほとんど変化していないことがわかった。
【００６１】
調製実施例２：サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの生産、精製および生化学的特性
サッカロミセス・セレビシアエ（ＣＣＲＣ２２２２３）の細胞の１体積部に、０．１ｍＭのＥＤＴＡ（ｐＨ７．３）を含む０．３Ｍリン酸緩衝液２体積部を加えた。細胞を４℃で３０分間３０％出力の超音波処理器を使用して破砕し、染色した。顕微鏡を使用して細胞が完全に破砕されたかどうかを観察した。細胞緩衝液混合物を次に１３，０００×ｇで６０分間遠心分離した。その上澄み液を採取した。同体積部の同リン酸緩衝液を残りの破砕細胞中に添加した。破砕細胞は同様な方法で再び細かくつぶされ遠心分離し、上澄み液を採集した。
【００６２】
採集した複数の粗酵素溶液上澄み液を混合した。固体硫酸アンモニウムを攪拌しながら静かに粗酵素液に添加した。３０から６０％飽和率での硫酸アンモニウム沈殿物が最も高い活性を有していたことがわかった。３０から６０％飽和率での沈殿物を０．１Ｍリン酸緩衝液（ｐＨ７．７）に溶解し、同緩衝液に対して２４時間透析した。全ての工程は４℃で行った。
【００６３】
透析された粗酵素は、０．５ｍＭ ＥＤＴＡ（ｐＨ７．７）を含む０．１Ｍリン酸緩衝液で予め平衡化されたＤＥＡＥ セファセル（２．６×２０ｃｍ）上で色層分析（クロマトグラフ）した。１０体積量のリン酸緩衝液で洗浄した後、吸着されたタンパク質を０．１Ｍリン酸緩衝液中０．１Ｍの塩化ナトリウムの直線濃度勾配によって、流速１ｍＬ／分で溶出した。５ｍＬの画分を複数、フラクション・コレクターを使用して採取した。粗レダクターゼを０．３２から０．４０Ｍの塩化ナトリウム濃度で溶出した。
【００６４】
ＤＥＡＥセファセル・クロマトグラフィーでレダクターゼ活性を有した画分を濃縮し、０．５ｍＭ ＥＤＴＡ（ｐＨ７．７）を含む０．１Ｍリン酸緩衝液でアミコン限外濾過を使用して平衡化した。得られたサンプルを、０．５ｍＭ ＥＤＴＡ（ｐＨ７．７）を含む０．１Ｍリン酸緩衝液で予め平衡化されたセファクリル Ｓ−３００ ＨＲカラム（２．６×９０ｃｍ）に加えた。流速は１ｍＬ／分であり、１画分あたり５または２．５ｍＬを採取した。このセファクリル Ｓ−３００ ＨＲクロマトグラフィーを３回行った。精製した酵素は使用するまで−７０℃で使用時まで貯蔵した。
【００６５】
分析後、サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの収量および比活性はそれぞれ１４．２％および１．３４単位／ｍｇであった。更に、４３２．３倍の精製を行った（表２に表示）。
【００６６】
【表２】
表２−サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの生産および精製のまとめ

【００６７】
サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの生化学的特性は以下の通りである。
（１）分子量
セファクリル Ｓ−３００ＨＲゲル濾過カラム・クロマトグラフィーによって、標準タンパク質から得られた検量線により、サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの分子量は３５８，０００と推定された。
【００６８】
（２）至適ｐＨおよびｐＨ安定性
サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼは、７．３に至適ｐＨを有し、ｐＨ６．５から７．５で非常に安定していた。
【００６９】
（３）至適温度および熱安定性
サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの至適温度は２５℃であった。５から３０℃の温度範囲で非常に安定であった。
【００７０】
（４）阻害剤の酵素活性に対する影響
サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの触媒活性は、ＰＣＭＰＳ、ＰＣＭＢおよびＫＣＮで完全に阻害され、ＮＥＭ、ＰＭＳＦおよびＩＡＡで部分的に阻害された。
【００７１】
（５）金属イオンの酵素活性に対する影響
サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの触媒活性は、Ｈｇ^２＋、Ｚｎ^２＋、Ｐｂ^２＋、およびＣｕ^２＋によって強く阻害され、Ｃｏ^２＋、Ｎｉ^２＋、Ｆｅ^２＋、Ｍｎ^２＋およびＢａ^２＋によって部分的に阻害を受けた。しかしながらＮａ^＋、Ｋ^＋、Ｃａ^２＋およびＭｇ^２＋は酵素活性に影響を与えなかった。
【００７２】
（６）還元剤の酵素活性に対する影響
グルタチオン、ジチオスレイトール、β−メルカプトエタノール、およびシステイン等の還元剤はすべてサッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの活性を改善した。
【００７３】
（７）低温貯蔵安定性
サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの酵素溶液を４℃および−２０℃でそれぞれ貯蔵した。残留酵素活性を定時間隔をおいて測定した。テストの結果、４℃で２８日間貯蔵した後、酵素活性はまだ７７．８％であり、−２０℃で２カ月間貯蔵した後、活性はほとんど変化していないことがわかった。
【００７４】
使用実施例１：大腸菌由来の亜硫酸レダクターゼの凍結変性されたサバのすり身に対する影響
凍結変性されたサバのすり身における反応性スルフヒドリル基の数およびゲル強度に対する添加された大腸菌由来の亜硫酸レダクターゼ量の影響を図４に示した。様々な活性単位の大腸菌由来の亜硫酸レダクターゼを凍結変性したサバのすり身に添加した。反応性スルフヒドリル基は、添加レダクターゼの量の増加に伴い顕著に増加した。ミンチ肉１ｇあたり０．０３活性単位の亜硫酸レダクターゼを添加した場合、サバのすり身の反応性スルフヒドリル基の数は４．１９×１０^−５から７．２３×１０^−５モル／ｇミンチへと増加した。レダクターゼを０．０３単位／ｇミンチより多く添加しても、反応性スルフヒドリル基に明らかな変化はみられなかった。同様の傾向はゲル強度の変化においても観察された。ゲル強度は、大腸菌由来の亜硫酸レダクターゼを０．０３単位／ｇミンチを添加したとき、１１５．０ｇ×ｃｍから２３５．７ｇ×ｃｍに増加した。レダクターゼを０．０３単位より多く添加してもゲル強度に明らかな変化はみられなかった。
【００７５】
凍結変性されたサバのすり身に対する大腸菌由来の亜硫酸レダクターゼの反応時間の影響を図５に示した。０．０３活性単位／ｇミンチの大腸菌由来の亜硫酸レダクターゼを添加した。反応時間が長くなるほど、反応性スルフヒドリル基の数が顕著に増加した。反応性スルフヒドリル基の数は、対照の４．２０ｘ１０^−５モル／ｇからインキュベーション２５分後には７．６５×１０^−５モル／ｇミンチに増加した。同様の傾向はゲル強度の変化においても観察された。ゲルの強度は１０９．４ｇ×ｃｍから２１１．２ｇ×ｃｍに増加し、その後緩やかに変化する傾向にあった。
【００７６】
使用実施例２：サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼの凍結変性されたサバのすり身に対する影響
様々な活性単位の大腸菌由来の亜硫酸レダクターゼを凍結変性されたサバのすり身中に添加した。反応性スルフヒドリル基の数は、添加レダクターゼの量の増加に伴い顕著に増加した。サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼを０．５単位の／ｇミンチを添加した場合、反応性スルフヒドリル基の数は対照の２．１９×１０^−５モル／ｇから３．４８×１０^−５モル／ｇミンチへと増加した。同様の傾向はゲル強度の変化においても観察された。図６に示したように、ゲル強度は、サッカロミセス・セレビシアエ由来の亜硫酸レダクターゼを０．５単位／ｇミンチを添加した時、１１５．０ｇ×ｃｍから３６９．９ｇ×ｃｍに増加した。
【００７７】
上記記載の例は、説明としてあげられたもので本発明を制限するものではなく、本発明の意図および範囲を逸脱することなしに様々な変化および変更がなされ得る。
【図面の簡単な説明】
【図１】大腸菌由来の亜硫酸レダクターゼの分子量をセファクリルＳ−３００ＨＲゲル透過カラム・クロマトグラフィーを使用して決定するための検量線を示す。図において、ポイントＡはチログロブリン（６６９ｋＤａ）、ポイントＢはフェリチン（４４０ｋＤａ）、ポイントＣはカタラーゼ（２３２ｋＤａ）、ポイントＤはアルドラーゼ（１５８ｋＤａ）およびポイントＥはウシ血清アルブミン（６７ｋＤａ）を示す。
【図２】大腸菌由来の亜硫酸レダクターゼの活性に対するｐＨ値の影響を示す。図において、「〇」はｐＨ依存活性を、「●」は表示ｐＨにおける安定性を示す。
【図３】大腸菌由来の亜硫酸レダクターゼの安定性に対する温度の影響を示す。図において、「〇」は温度依存活性を、「●」は表示温度における安定性を示す。
【図４】凍結変性したサバのすり身の反応性スルフヒドリル基の数およびゲルの強度に対する、添加した大腸菌由来の亜硫酸レダクターゼ量の影響を示す。図において、「●」は反応性スルフヒドリル基の数、「■」はゲル強度を示す。
【図５】凍結変性したサバのすり身の反応性スルフヒドリル基の数およびゲルの強度に対する、大腸菌由来の亜硫酸レダクターゼが凍結変性したサバのすり身の肉に作用する時間の影響を示す。図において「●」は反応性スルフヒドリル基の数、「■」はゲル強度を示す。
【図６】オゾンで脱色したサバのすり身の反応性スルフヒドリル基の数およびゲルの強度に対する、添加したサッカロミセス・セレビシアエ由来の亜硫酸レダクターゼ量の影響を示す。図において「〇」は反応性スルフヒドリル基の数、「●」はゲル強度を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to purified sulfite reductase. More specifically, the sulfite reductase of the present invention catalyzes the reduction of sulfite to sulfide, and when used in denatured fish, effectively regenerates denatured fish meat protein and reduces the denatured fish protein. Increases the number of reactive sulfhydryl groups and gel strength. The present invention also relates to a method for producing purified sulfite reductase and a method for regenerating denatured fish protein using said solution or powdered form of sulfite reductase.
[0002]
[Prior art]
Sulfite reductase, which is widely present in plants and microorganisms, is an enzyme that catalyzes the reduction of sulfite to sulfide in the final step of assimilated sulfate reduction, which is an important step in cysteine biosynthesis. Six electrons are provided to the reaction as follows: SO₃ ^2-+ 6e⁻+ 6H⁺→ S^2-+ 3H₂During the reduction reaction of O and sulfite to sulfide, reduced nicotinamide adenine dinucleotide phosphate (NADPH) acts as an electron donor in Escherichia coli, and NADPH or reduced methyl viologen (MVH) in yeast. Acts as an electron donating group, and in other organisms or tissues, other donating groups act.
[0003]
Muscle proteins are a large variety of proteins, mostly with various physiological effects, by the interaction of L-amino acids via peptide bonds, disulfide bonds, hydrogen bonds, ionic bonds and hydrophobic bonds, and by dipole moments Formed by action. "Protein denaturation" refers to a secondary, tertiary, or quaternary three-dimensional structural change of a protein without breaking peptide bonds.
[0004]
The structure of the denatured protein forms a random helix in an unfolded or aggregated state, where the functional groups on or between the peptide chains interact, thereby causing the Loss of physiological, chemical and biological properties. The reason why proteins are denatured is generally considered as follows. The water in the muscle freezes, which raises the salt concentration and changes the pH around the protein, which may cause protein molecules to precipitate, due to salting out or aggregation of intermolecular or intramolecular hydrophobic groups. At the same time, new hydrogen bonds, disulfide bonds, and ionic bonds are formed to aggregate and denature proteins. In addition, the water around the protein molecule forms bound water with functional groups such as -SH, -COOH, -NH and -CO of the protein molecule. Further, due to the temperature drop, the water molecules whose bonding strength is reduced first form ice crystals. At the same time, volume expansion changes the conformation of the protein molecule, exposing functional groups, and forming new bonds between or within the molecules, which aggregate and denature the protein molecules.
[0005]
It has been found that the reason for the formation of covalent bonds when cryopreserved fish proteins are denatured is that sulfhydryl groups are oxidized to disulfide groups (Jiang et al., J. Food Sci. 63: 777-781, 1998). Jiang et al., J. Food Sci. 60: 652-655, 1998). Fish meat denatured by frozen storage not only causes fish meat softening, tissue fluid outflow, spongy tissue formation, but also has worse emulsifying properties, water retention ability, gel forming ability, and viscoelasticity than fresh fish . The reason for these degradations is mainly due to denaturation of myofibrillar proteins.
[0006]
After denaturation, the protein forms a normal network, which after curing by heating, imparts elastic properties to the muscle protein. Gelation of fish proteins imparts elasticity to fish proteins and is one of the most important properties during processing of fish proteins into purified products. Gelation of proteins is affected by many factors such as salt concentration, pH value, temperature, protein concentration, constituents, additives, ionic strength, freshness and the like. The biochemical properties of the purified product indicate that the bonds that form the network include hydrogen bonds, hydrophobic bonds, ionic bonds, disulfide bonds, and other covalent bonds.
[0007]
In addition, the gel strength is an index that determines the quality of the refined surimi product. The elasticity of a purified product is affected by many factors such as salt concentration, pH value, temperature, protein concentration, constituents, additives, ionic strength, freshness and the like. Therefore, many methods have been proposed to increase the elasticity of refined fish meat products.
[0008]
The gel-forming ability of fish meat decreases during frozen storage. The reason for this is mainly due to the gradual decrease in the solubility of fish muscle proteins, which form macromolecules such as dimers and multimers of myosin due to the formation of disulfide bonds and other covalent bonds. It is believed to result. The formation of disulfide bonds between muscle proteins results in aggregation and denaturation of fish muscle actomyosin.
[0009]
As mentioned above, the gel forming ability of fish meat is reduced during frozen storage, which makes the fish meat unsuitable for processing into colloidal protein foods. Thus, the application of frozen stored fish meat to the production of colloidal protein foods is limited.
[0010]
Alkaline washing treatments have long been used to improve the gel-forming ability of mackerel surimi, but do not help improve color. Although the color of mackerel can be substantially improved by increasing the alkaline washing cycle or using ozone bleaching, the gel-forming ability of mackerel muscle proteins is degraded.
[0011]
In addition, for frozen surimi obtained by adding a chemical reducing agent such as cysteine, sodium disulfite, and ascorbic acid to denatured fish muscle, all sulfhydryl groups and reactive sulfhydryl groups of actomyosin and extractable acthydrin groups were obtained. The amount of all myosin is much higher than frozen surimi without added reducing agent. Although the gel-forming ability of a protein is reduced by freezing, most of the protein can be recovered (regenerated) by adding a reducing agent. Therefore, it is understood that the reducing agent can recover the aggregated and denatured actomyosin. As a result of the addition of the reducing agent, the disulfide bond of the frozen denatured fish meat is reduced to the sulfhydryl bond in the minced meat. If the disulfide bonds reform during the gelation of the purified product, this makes the network more rigid and stable. However, chemical reducing agents are considered external additives and are not easily accepted by consumers.
[0012]
The inventor of the present invention has proposed to use a microorganism-derived sulfite reductase instead of a chemical reducing agent. For example, Jiang et al. Studied the effect of yeast-derived sulfite reductase on the recovery of denatured fish muscle protein from decolorized and freeze-denatured mackerel surimi, which was decolorized with ozone, and crude sulfite reductase recovered denatured muscle protein, (Jiang et al., J. Food Sci. 60: 652-655, 1998; Jiang et al., J. Food Sci. 60: 777-781, 1998). Further, Wu et al. Also found that the crude lyophilized powder of sulfite reductase prepared from Saccharomyces cerevisiae was applied to the processing of frozen fish to significantly enhance the gel-forming ability of the purified product (Wu et al., J. Food Sci. 65: 1400-1403, 2000). However, microorganism-derived sulfite reductase has never been prepared and its effect on the recovery of denatured fish muscle has never been studied.
[0013]
[Problems to be solved by the invention]
In order to overcome the above-mentioned problems in the prior art, the present inventors studied a method for producing a microorganism-derived sulfite reductase and the effect of the obtained sulfite reductase on the recovery of native muscle protein from denatured fish meat, It has been discovered that the object can be achieved by using purified sulfite reductase from E. coli or one of the genus Saccharomyces. The present invention has been completed.
[0014]
[Means for Solving the Problems]
Therefore, the present invention has the following characteristics:
a. Has the effect of catalyzing the reduction to sulfite sulfide or recovering (regenerating) sulfhydryl groups from disulfide groups;
b. In the reduction catalytic reaction, the reduced nicotinamide adenine dinucleotide phosphoric acid (NADPH), methyl viologen (MVH), or another donor group acts as an electron donor group;
c. Have a molecular weight of 100,000 to 400,000;
d. The optimal temperature of activity is between 20 ° C and 30 ° C; and
e. Optimum pH of activity is 6.5 to 8.0
And a purified sulfite reductase characterized by the following:
[0015]
Preferably, the electron donor for sulfite reductase is NADPH. In addition, it is preferred that the sulfite reductase is derived from Escherichia coli or one species of the genus Saccharomyces. One species of the genus Saccharomyces is Saccharomyces cerevisiae, Saccharomyces bayanas, Saccharomyces ellipsoidus, and Saccharomyces acechary. Among the above, Saccharomyces cerevisiae is preferred.
[0016]
In addition, the present invention also provides a method for producing purified sulfite reductase, comprising purifying sulfite reductase from a crude enzyme solution of Escherichia coli or one of the genus Saccharomyces using ammonium sulfate fractionation or chromatography. .
[0017]
In a method for producing purified sulfite reductase, the crude enzyme solution comprises the following steps:
adding a phosphate buffer having a pH of 6.5 to 8.5 to a cell of Escherichia coli or one of the genus Saccharomyces, using an ultrasonicator at a temperature of 2 to 10 ° C. for 0.1 to 2 hours. Crushing step, centrifuging at 5000 × g for 30 minutes, collecting the supernatant, suspending the remaining fragmented cells in the same buffer, grinding the fragmented cells, and centrifuging as described above. After separation, it is produced by a step of collecting a supernatant and a step of mixing a plurality of supernatants.
[0018]
Further, the ammonium sulfate fractionation comprises the following steps: a step of gently adding ammonium sulfate to the crude enzyme solution produced as described above (in claim 6); a step of 0.1 to 35,000 × g at a rate of 3000 to 15,000 × g. After centrifugation for 2 hours from, the precipitate at 30 to 60% saturation is collected and the solution of the precipitate is washed against 0.1 M to 0.2 M phosphate buffer (pH 6.5 to 8.5). It involves dialysis to obtain a dialysate, all steps being performed at 2 to 10 ° C.
[0019]
Still further, said chromatography is selected from the group consisting of DEAE Sephacel column chromatography and / or Sephacryl S-300HR column chromatography.
[0020]
The invention also provides a method of regenerating a denatured fish protein comprising applying to the denatured fish protein. The sulfite reductase of the present invention may be used in solution or powder form in an amount of 0.01 to 0.5 activity units per gram of denatured fish protein. The time for the sulfite reductase in solution or powder form to act on the denatured fish protein is 5 to 40 minutes.
[0021]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the sulfite reductase, that is, the starting enzyme solution and the starting enzyme powder were obtained from Escherichia coli or Saccharomyces cerevisiae. In addition, the effect of the enzyme on the regeneration of denatured fish meat protein was also studied.
[0022]
The materials and equipment used, sulfite reductase production and related experimental or test methods are first described.
(A) Materials and equipment
(1) Bacteria and yeast cells
1. Culture media containing 4.0% sucrose, 0.4% yeast extract, 0.4% tryptone (pH 7.5) in Erlenmeyer flasks, purchased from Taiwan Culture Collection and Research Center (Hisnchu, Taiwan) 1% of the activated E. coli (CCRC11634) thus obtained was inoculated in a loop and cultured at 37 ° C. for 18 hours while shaking at 100 rpm. E. coli cells were collected by centrifugation at 5000 xg for 30 minutes.
[0023]
2. In an Erlenmeyer flask, YM broth culture medium containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1.0% dextrose (pH 7.0) was added to Taiwan Culture Collection and Research Center. Saccharomyces cerevisiae (CCRC22223) purchased from (Hisnchu Taiwan) was inoculated in a loop and cultured at 24 ° C. for 60 hours with shaking at 100 rpm. Saccharomyces cerevisiae cells were collected by centrifugation at 4000 × g for 30 minutes.
[0024]
(2) Reagent
DEAE Sephacel, Sephacryl S-300HR, electrophoresis molecular weight standards and gel permeation filtration molecular weight standards were purchased from Pharmacia Biotech (Uppsala, Sweden).
Yeast extract, tryptone, was purchased from Difco.
Ammonium sulfate was purchased from Merck (Darmstadt Germany).
[0025]
Bovine serum albumin (BSA), flavin mononucleotide (FMN), reduced nicotinamide adenine dinucleotide phosphate (NADPH), glutathione, dithiothreitol, cysteine, 5,5'-dithiobis- (2-nitrobenzoic acid) (DTNB) ), Β-mercaptoethanol (β-Me) and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Co. (St. Louis, Mo., USA).
[0026]
Iodoacetic acid (IAA), N-ethylmaleimide (NEM), P-chloromercuryphenylsulfonic acid (PCMPS), P-chloromercurybenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), and N, N. N ', N'-tetramethyl-ethylenediamine (TEMED) was purchased from Bio-Rad (Richmond, CA, USA).
All other chemicals were biochemical reagent grade.
[0027]
(2) Equipment
1. Low-temperature oscillation culture box: Hotek 718, manufactured by Hotech Equipment Co., Ltd. (Taipei, Taiwan)
2. Low temperature and high speed centrifugation: SCR20B, Hitachi, Japan
3. Ultrasonicator; Model W-200, Ultrasonic, Japan
4. Physical property terminator: Model CR-200, Sun Chemical, Tokyo, Japan
5. Spectrophotometer: Hitachi U-2001, Hitachi, Ltd., Japan
6. Mini-electrophoresis: Mini PROTEAN II cell, power supply Bio-Rad model 200 / 2.0, Bio-Rad USA
7. pH meter: HM-30S, TOA Electric Co., Ltd., Japan
8. Bath tank: Model B204, Marathon (MARATON, Taiwan),
9. Grinder: CF5kg, Cheu Far Co., Taiwan,
10. Columns: C26 / 40, C26 / 100, Pharmacia LKB Biotechnology, Uppsala, Sweden
11. Fraction collector: FRAC-200, Pharmacia LKB Biotechnology, Uppsala, Sweden
12. High Performance Protein Liquid Chromatography (FPLC) System: Controller LCC-500 Plus and Pump P-500, Pharmacia LKB, Biotechnology, Uppsala, Sweden
13. -20 ° C freezer, biofreezer, model 8442, Forma Scientific, USA
14． Amicon Ultrafiltration Concentrator: Purchased from Amicon, USA, main structures are concentration tank (stirred cell, model 8050) and filtration film (YM1043mm membrane)
15. Defoaming homogenizer: Waring blender with baffle
[0028]
(B) Production and purification of sulfite reductase
Extraction: Escherichia coli was cultured in a medium containing 4.0% sucrose, 0.4% yeast extract, and 0.4% tryptone (pH 7.5) at 37 ° C. for 18 hours, and then centrifuged at 5000 × g for 30 minutes. Separately collected and mixed with 2 volumes (based on cell volume) of 0.1 M potassium phosphate buffer containing 0.5 mM EDTA (pH 7.7). The harvested cells were then disrupted using a sonicator at 4 ° C. for 30 minutes and harvested by centrifugation at 5000 × g for 30 minutes. The resulting sample was suspended in 0.1 M phosphate buffer (pH 7.7), crushed and centrifuged again as described above. On the other hand, Saccharomyces cerevisiae cells were cultured in a medium containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1.0% dextrose (pH 7.0) for 60 hours. The cells were collected by centrifugation at 4000 × g for 30 minutes. The cells are mixed with 2 volumes (based on cell volume) of 0.3 M potassium phosphate buffer containing 1 mM EDTA (pH 7.3) for 30 minutes on an ultrasonic model W-200 sonicator. Crushed at 4 ° C. Cells and debris were collected by centrifugation at 13000 xg for 30 minutes. The residue was suspended in a 0.3 M phosphate buffer (pH 7.0), crushed again, and centrifuged.
[0029]
Ammonium sulfate fractionation: Solid ammonium sulfate was gently added to the crude enzyme with stirring. The precipitate at 30-60% saturation was collected by centrifugation at 5000 xg for 30 minutes and then dialyzed against 0.1 M phosphate buffer (pH 7.7) for 24 hours. All steps were performed at 4 ° C.
[0030]
DEAE-Sephacel column chromatography: The dialyzed crude enzyme is colored on DEAE Sephacel (2.6 x 20 cm) pre-equilibrated with 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7). Layer analysis (chromatography) was performed. After washing with 10 volumes of phosphate buffer, the adsorbed protein was eluted at a flow rate of 1 mL / min with a linear gradient of 0 to 1.0 M sodium chloride in 0.1 M phosphate buffer. Multiple 5 mL fractions were collected using a fraction collector. Crude reductase was eluted at a concentration of 0.32 to 0.40 M sodium chloride.
[0031]
Sephacryl S-300 HR column chromatography: The fraction having reductase activity is concentrated by DEAE Sephacel chromatography and contains 0.5 mM EDTA (pH 7.7) using Amicon ultrafiltration. Equilibrated with 0.1 M phosphate buffer. The resulting sample was applied to a Sephacryl S-300 HR column (2.6 x 90 cm) that had been pre-equilibrated with 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7). The flow rate was 1 mL / min and 5 or 2.5 mL was collected per fraction. This Sephacryl S-300HR chromatography was performed three times. The purified enzyme was stored at -70 ° C until use.
[0032]
(C) Testing the biochemical properties of sulfite reductase
Measurement of enzyme activity
Sulfite reductase activity was measured by spectrophotometry under aerobic conditions at 340 nm, following the oxidation of NADPH with sulfurous acid by the enzyme activity determination method of Siegel et al. (Siegel, 1973). 0.9 mL reaction mixture [0.5 mM EDTA, 0.5 mM NaHSO₃0.1 M phosphate buffer (pH 7.7) containing 1 μM FMN and 0.2 mM NADPH], and incubated at 25 ° C. Activity was determined according to the initial rate after addition of the reductase and corrected by the slow oxidation of NADPH in the absence of sulfite. One activity unit was defined as the amount of enzyme that catalyzes the oxidation of 1 micromole of NADPH within 1 minute at 25 ° C under the above conditions.
[0033]
Measurement of protein concentration
Protein concentration was measured by the dye binding method. Bovine serum albumin was used as a marker.
[0034]
Molecular weight
The molecular weight of the sulfite reductase was measured using Sephacryl S-300HR column chromatography. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa) and bovine serum albumin (67 kDa) were used as standard proteins.
[0035]
Temperature and pH effects
Optimal temperature: 0.9 mL reaction mixture [0.5 mM EDTA, 0.5 mM NaHSO₃0.1 M phosphate buffer (pH 7.7) containing 1 μM FMN and 0.2 mM NADPH], and 0.1 mL reductase was added thereto at various temperatures (5, 10, 15, 20, 25, 30, 35, (40, 45, 50, 55, and 60 ° C.) for 5 minutes. Activity was determined from the initial rate and corrected by oxidation of NADPH in the absence of sulfite according to the method of Siegel et al. (Siegel et al., 1973).
[0036]
Thermostability: Purified sulfite reductase in 0.1 M phosphate buffer (pH 7.7) was prepared at various temperatures (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, And 60 ° C) for 30 minutes. Residual activity was measured according to the method of Segel et al. (Seigel (1973)).
[0037]
Optimal pH values: various pH (0.2 M citrate buffer: pH 4.0 to 6.0; 0.2 M phosphate buffer: pH 6.0 to 8.0; 0.2 M bicarbonate buffer: pH 8) 0.0 to 10.0; all buffers were 0.5 mM EDTA, 0.5 mM NaHSO₃0.1 mL of reductase was added to a 0.9 mL reaction mixture (containing 1 μM FMN and 0.2 mM NADPH) and incubated at 25 ° C. for 5 minutes. Activity was determined from the initial rate and corrected for the oxidation of NADPH in the absence of sulfite according to the method of Siegel et al. (Seigel (1973)).
[0038]
pH stability: various pH (0.2 M citrate buffer: pH 4.0 to 6.0; 0.2 M phosphate buffer: pH 6.0 to 8.0; 0.2 M bicarbonate buffer: pH 8. Purified sulfite reductase in a buffer solution (0 to 10.0) was incubated at 25 ° C. for 30 minutes. Residual activity was measured according to the method of Segel et al. (Seigel (1973)).
[0039]
Effects of metal ions or inhibitors
Purified sulfite reductase in 0.1 M phosphate buffer (pH 7.7) was incubated at 25 ° C. with various metals or inhibitors. The final metal concentrations were 0.5, 1.0, 5.0, and 10.0 mM, and the inhibitor concentration was 1.0 mM. After 30 minutes of incubation, the residual activity was measured according to the method of Segel et al. (Seigel (1973)).
[0040]
Cold storage stability
The enzyme solution was stored at 4 ° C and -20 ° C, respectively. Residual enzyme activity was measured at regular time intervals to test the stability of cold storage.
[0041]
(D) Frozen denatured fish muscle recovery test of mackerel, scorpionfish and swordfish
Preparation of Frozen Mackerel, Tigerfish and Swordfish
As raw materials, the fish muscles of mackerel, scorpionfish and swordfish were stored in a freezer at -25 ° C for 6 months and freeze-denatured. The frozen fish muscle was decapitated, the intestine was removed, the bone was removed, washed three times with ice water, and centrifuged with a centrifugal dehydrator until the water content was about 78%. The resulting surimi is ground for 10 minutes, mixed with 0.2% polyphosphoric acid and 5% sugar or glycitol, put in a -40 ° C blast freezer to lower the central temperature below -18 ° C, and quickly freeze And stored in frozen storage for later use.
[0042]
Production of refined products
The above-mentioned frozen surimi is placed in an air-conditioned room so that the center temperature is not frozen at a temperature of −2 to −5 ° C., and then the sulfite reductase obtained above, the enzyme solution of the raw material or the enzyme of the raw material Powder was added. Simultaneously, after adding 2.5% salt, the surimi was ground with a grinder and then for 20 minutes. Finally, 3% potato starch was added and mixed uniformly. The obtained sample was packed in a case, settled at 25 ° C. for 30 minutes, and then heated at 95 ° C. for 20 minutes. Samples were chilled in running water and stored at 4 ° C. Its properties were measured the next day.
[0043]
Characterization of surimi
Surimi actomyosin was extracted according to the method of Noguchi and Matsumoto (1978). The reactive group of actomyosin was determined according to a modified method of Ito et al. (Itoh (1979)). The gel-forming property was determined by cutting a refined surimi product to a height of 3.0 cm, and measuring the gel strength using a physical property terminator having a 5 mm diameter plug.
[0044]
Preparation of actomyosin from surimi and measurement of SH
Actomyosin (AM) was extracted according to the method of Noguchi and Matsumoto (1978). Actomyosin in 50 mM phosphate buffer (pH 7.0) containing 0.6 M KCL and 6 mM EDTA was mixed with a 0.01 M DTNB solution and incubated at 5 ° C for 1 hour. Reactive SH was determined according to Ito et al. (Itoh (1979)).
[0045]
Measurement of gel strength
The gel strength was measured with a rheometer and expressed as breaking force (g) and deformation (cm) [gel strength (g × cm) = breaking force (g) × deformation (cm)].
[0046]
Statistical analysis
Duncan's multiple range test was used for statistical analysis. For gel strength, ten measurements from each group were analyzed. Significant differences were defined as p <0.05.
[0047]
(E) Example
The following Preparation Examples and Use Examples are illustrated to illustrate the invention in more detail. However, these examples do not limit the invention.
[0048]
Preparation Example 1: Extraction, purification and biochemical properties of sulfite reductase from Escherichia coli
To 1 part by volume of E. coli cells (CCRC11634), 2 parts by volume of 0.1 M potassium phosphate containing 0.5 mM EDTA (pH 7.7) was added. Cells were disrupted and stained using a 30% power sonicator at 4 ° C. for 30 minutes. A microscope was used to observe whether the cells were completely disrupted. The cell buffer mixture was then centrifuged at 5000 × g for 30 minutes. The supernatant was collected. An equal volume of the same phosphate buffer was added to the remaining disrupted cells. The obtained product was pulverized again in the same manner and centrifuged to collect a supernatant crude enzyme solution.
[0049]
Multiple collected supernatants of the crude enzyme solution were mixed. Solid ammonium sulfate was gently added to the crude enzyme with stirring. It was found that the precipitate at a saturation of 30 to 60% had the highest activity. The precipitate at 30-60% saturation was collected, dissolved in 0.1 M phosphate buffer and dialyzed against 0.1 M phosphate buffer (pH 7.7) for 24 hours. All steps were performed at 4 ° C.
[0050]
The dialyzed crude enzyme was subjected to color layer analysis (chromatography) on DEAE Sephacel (2.6 × 20 cm) pre-equilibrated with 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7). After washing with 10 volumes of phosphate buffer, the adsorbed protein was eluted with a linear gradient of 0.1 M sodium chloride in 0.1 M phosphate buffer at a flow rate of 1 mL / min. Multiple 5 mL fractions were collected using a fraction collector. Crude reductase was eluted at a concentration of 0.32 to 0.40 M sodium chloride.
[0051]
Fractions having reductase activity were concentrated by DEAE Sephacel chromatography and equilibrated with 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7) using Amicon ultrafiltration. The obtained sample was applied to a Sephacryl S-300 HR column (2.6 × 90 cm) which had been pre-equilibrated with a 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7). The flow rate was 1 mL / min and 5 or 2.5 mL was collected per fraction. This Sephacryl S-300 HR chromatography was performed three times. Purified enzyme was stored at -70 ° C until use.
[0052]
After the analysis, it was confirmed that the yield and specific activity of the sulfite reductase derived from Escherichia coli were 31.7% and 10.03 units / mg, respectively. In addition, a 579.5-fold purification was achieved (as indicated in Table 1).
[0053]
[Table 1]
Table 1-Summary of production and purification of E. coli-derived sulfite reductase

The biochemical properties of the sulfite reductase derived from Escherichia coli were as follows.
[0054]
(1) Molecular weight
From the calibration curve (shown in FIG. 1) obtained from the standard protein by Sephacryl S-300HR gel filtration column chromatography, the molecular weight of E. coli-derived sulfite reductase was estimated to be 119,000.
[0055]
(2) Optimum pH value and pH stability
The sulfite reductase from E. coli had an optimal pH at 7.7 and was very stable between pH 6.5 and 8.0 (shown in FIG. 2). After standing at 25 ° C. and pH 4.0 to 10.0, the activity of sulfite reductase was measured. It was found that at pH 6.5 to 7.5, 95% or more of the activity remained. At pH 6.5, 73.7% or more of the activity remained, and at pH 8.0, 79.0% or more of the activity remained. Therefore, the stability of the enzyme was found to be better in the neutral state than in the slightly acidic or alkaline state.
[0056]
(3) Optimal temperature and thermal stability
The optimum temperature of the sulfite reductase derived from Escherichia coli was 25 ° C. Regarding thermostability, the enzyme was very stable in the temperature range of 5 to 25 ° C., and 98% or more of the activity remained. At temperatures above 25 ° C., the enzyme was gradually inactivated and was completely inactivated when it reached 50 ° C. (shown in FIG. 3).
[0057]
(4) Effect of inhibitor on enzyme activity
The catalytic activity of sulfite reductase from E. coli was completely inhibited by PCMB and KCN and partially inhibited by NEM, PMSF and IAA.
[0058]
(5) Effect of metal ions on enzyme activity
The catalytic activity of sulfite reductase derived from Escherichia coli is Hg²⁺, Fe²⁺, Fe³⁺, Ca²⁺, Co²⁺, And Cu²⁺Strongly inhibited by Cd²⁺, Zn²⁺, Mn²⁺, And Ba²⁺Received moderate inhibition. However, Na⁺, K⁺And Mg²⁺Did not affect enzyme activity. Hg among metal ions²⁺, Fe²⁺, Cu²⁺, And Zn²⁺Interacts with the sulfhydryl group of the enzyme and inhibits its activity. The results in this section prove that the active site of the enzyme contains a cysteine residue.
[0059]
(6) Effect of reducing agent on enzyme activity
Reducing agents such as glutathione, dithiothreitol, β-mercaptoethanol, and cysteine all improved the activity of sulfite reductase from E. coli.
[0060]
(7) Low temperature storage stability
Enzyme solutions of sulfite reductase from E. coli were stored at 4 ° C and -20 ° C, respectively. Residual enzyme activity was measured at regular time intervals. As a result of the test, it was found that the enzyme activity was still 77.8% after storage at 4 ° C. for 28 days, and that the activity was hardly changed after storage at −20 ° C. for 2 months.
[0061]
Preparation Example 2: Production, purification and biochemical properties of sulfite reductase from Saccharomyces cerevisiae
To one volume of cells of Saccharomyces cerevisiae (CCRC22223) was added 2 volumes of 0.3 M phosphate buffer containing 0.1 mM EDTA (pH 7.3). Cells were disrupted and stained using a 30% power sonicator at 4 ° C. for 30 minutes. A microscope was used to observe whether the cells were completely disrupted. The cell buffer mixture was then centrifuged at 13,000 xg for 60 minutes. The supernatant was collected. An equal volume of the same phosphate buffer was added to the remaining disrupted cells. The disrupted cells were again crushed in the same manner, centrifuged, and the supernatant was collected.
[0062]
Multiple collected supernatants of the crude enzyme solution were mixed. The solid ammonium sulfate was gently added to the crude enzyme solution with stirring. It was found that the ammonium sulfate precipitate at 30 to 60% saturation had the highest activity. The precipitate at 30-60% saturation was dissolved in 0.1 M phosphate buffer (pH 7.7) and dialyzed against the same buffer for 24 hours. All steps were performed at 4 ° C.
[0063]
The dialyzed crude enzyme was chromatographed on DEAE Sephacel (2.6 x 20 cm) pre-equilibrated with 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7). . After washing with 10 volumes of phosphate buffer, the adsorbed protein was eluted with a linear gradient of 0.1 M sodium chloride in 0.1 M phosphate buffer at a flow rate of 1 mL / min. Multiple 5 mL fractions were collected using a fraction collector. Crude reductase was eluted at a concentration of 0.32 to 0.40 M sodium chloride.
[0064]
Fractions having reductase activity were concentrated by DEAE Sephacel chromatography and equilibrated with 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7) using Amicon ultrafiltration. The obtained sample was applied to a Sephacryl S-300 HR column (2.6 × 90 cm) that had been pre-equilibrated with a 0.1 M phosphate buffer containing 0.5 mM EDTA (pH 7.7). The flow rate was 1 mL / min and 5 or 2.5 mL was collected per fraction. This Sephacryl S-300 HR chromatography was performed three times. The purified enzyme was stored at -70 ° C until use until use.
[0065]
After analysis, the yield and specific activity of the sulfite reductase from Saccharomyces cerevisiae were 14.2% and 1.34 units / mg, respectively. Further, purification was carried out by 432.3 times (shown in Table 2).
[0066]
[Table 2]
Table 2-Summary of production and purification of sulfite reductase from Saccharomyces cerevisiae

[0067]
The biochemical properties of the sulfite reductase from Saccharomyces cerevisiae are as follows.
(1) Molecular weight
The molecular weight of the sulfite reductase from Saccharomyces cerevisiae was estimated to be 358,000 based on the calibration curve obtained from the standard protein by Sephacryl S-300HR gel filtration column chromatography.
[0068]
(2) Optimum pH and pH stability
Sulphite reductase from Saccharomyces cerevisiae had an optimal pH at 7.3 and was very stable at pH 6.5 to 7.5.
[0069]
(3) Optimal temperature and thermal stability
The optimum temperature of sulfite reductase from Saccharomyces cerevisiae was 25 ° C. It was very stable in the temperature range from 5 to 30 ° C.
[0070]
(4) Effect of inhibitor on enzyme activity
The catalytic activity of the sulfite reductase from Saccharomyces cerevisiae was completely inhibited by PCMPS, PCMB and KCN and partially inhibited by NEM, PMSF and IAA.
[0071]
(5) Effect of metal ions on enzyme activity
The catalytic activity of sulfite reductase from Saccharomyces cerevisiae is Hg²⁺, Zn²⁺, Pb²⁺, And Cu²⁺Strongly inhibited by Co²⁺, Ni²⁺, Fe²⁺, Mn²⁺And Ba²⁺Was partially inhibited by However, Na⁺, K⁺, Ca²⁺And Mg²⁺Did not affect enzyme activity.
[0072]
(6) Effect of reducing agent on enzyme activity
Reducing agents such as glutathione, dithiothreitol, β-mercaptoethanol, and cysteine all improved the activity of sulfite reductase from Saccharomyces cerevisiae.
[0073]
(7) Low temperature storage stability
Enzyme solutions of sulfite reductase from Saccharomyces cerevisiae were stored at 4 ° C and -20 ° C, respectively. Residual enzyme activity was measured at regular intervals. The test showed that after storage at 4 ° C. for 28 days, the enzyme activity was still 77.8%, and after storage at −20 ° C. for 2 months, the activity was almost unchanged.
[0074]
Use Example 1: Effect of sulfite reductase from Escherichia coli on surimi of freeze-denatured mackerel
The effect of the amount of added sulfite reductase from E. coli added on the number of reactive sulfhydryl groups and gel strength in freeze-denatured mackerel surimi is shown in FIG. Various activity units of sulfite reductase from Escherichia coli were added to the freeze-denatured mackerel surimi. Reactive sulfhydryl groups increased significantly with increasing amounts of added reductase. When 0.03 activity units of sulfite reductase were added per gram of minced meat, the number of reactive sulfhydryl groups in the mackerel surimi was 4.19 × 10^-5From 7.23 × 10^-5Mol / g increased to mince. Addition of more than 0.03 units / g minc of reductase did not show any apparent change in the reactive sulfhydryl groups. A similar trend was observed for changes in gel strength. The gel strength increased from 115.0 g × cm to 235.7 g × cm when 0.03 units / g mince of E. coli-derived sulfite reductase was added. Even when reductase was added in an amount of more than 0.03 units, no apparent change was observed in gel strength.
[0075]
The effect of the reaction time of the sulfite reductase derived from Escherichia coli on the freeze-denatured mackerel surimi is shown in FIG. Sulfite reductase from E. coli at 0.03 activity units / g minc was added. As the reaction time increased, the number of reactive sulfhydryl groups increased significantly. The number of reactive sulfhydryl groups was 4.20 x 10^-57.65 x 10 after 25 minutes incubation from mol / g^-5Mol / g increased to mince. A similar trend was observed for changes in gel strength. The gel strength increased from 109.4 g × cm to 211.2 g × cm and then tended to change slowly.
[0076]
Use Example 2: Effect of sulfite reductase from Saccharomyces cerevisiae on freeze-denatured mackerel surimi
Various activity units of sulfite reductase from Escherichia coli were added to the freeze-denatured mackerel surimi. The number of reactive sulfhydryl groups increased significantly with increasing amounts of added reductase. When 0.5 units / g minced sulfite reductase from Saccharomyces cerevisiae was added, the number of reactive sulfhydryl groups was 2.19 × 10 5 of the control.^-53.48 × 10 from mol / g^-5Mol / g increased to mince. A similar trend was observed for changes in gel strength. As shown in FIG. 6, the gel strength increased from 115.0 g × cm to 369.9 g × cm when 0.5 unit / g minced sulfite reductase derived from Saccharomyces cerevisiae was added.
[0077]
The above examples are given by way of explanation and do not limit the invention, and various changes and modifications can be made without departing from the spirit and scope of the invention.
[Brief description of the drawings]
FIG. 1 shows a calibration curve for determining the molecular weight of sulfite reductase from E. coli using Sephacryl S-300HR gel permeation column chromatography. In the figure, point A indicates thyroglobulin (669 kDa), point B indicates ferritin (440 kDa), point C indicates catalase (232 kDa), point D indicates aldolase (158 kDa), and point E indicates bovine serum albumin (67 kDa).
FIG. 2 shows the effect of pH value on the activity of sulfite reductase from E. coli. In the figure, “〇” indicates pH-dependent activity, and “「 ”indicates stability at the indicated pH.
FIG. 3 shows the effect of temperature on the stability of sulfite reductase from E. coli. In the figure, “Δ” indicates temperature-dependent activity, and “●” indicates stability at the indicated temperature.
FIG. 4 shows the effect of the amount of added E. coli-derived sulfite reductase on the number of reactive sulfhydryl groups and gel strength of freeze-denatured mackerel surimi. In the figure, “●” indicates the number of reactive sulfhydryl groups, and “■” indicates gel strength.
FIG. 5 shows the effect of the time during which the sulfite reductase from Escherichia coli acts on the meat of a freeze-denatured mackerel surimi on the number of reactive sulfhydryl groups and gel strength of the freeze-denatured mackerel surimi. In the figure, “●” indicates the number of reactive sulfhydryl groups, and “■” indicates gel strength.
FIG. 6 shows the effect of the amount of added sulfite reductase from Saccharomyces cerevisiae on the number of reactive sulfhydryl groups and gel strength of mackerel surimi decolorized with ozone. In the figure, “〇” indicates the number of reactive sulfhydryl groups, and “●” indicates gel strength.

Claims (10)

A purified sulfite reductase having the following characteristics:
a. Catalyze the reduction of sulfites to sulfides or regenerate sulfhydryl groups from disulfide groups;
b. In the reduction catalytic reaction, reduced nicotinamide adenine dinucleotide phosphate (NADPH), methyl viologen (MVH) or another donor group acts as an electron donor group;
c. Have a molecular weight of 100,000 to 400,000;
d. Optimal temperature of activity is from 20 ° C to 30 ° C; and e. The optimal pH of the activity is 6.5 to 8.0,
A sulfite reductase characterized by the following: 2. The sulfite reductase according to claim 1, wherein the electron donor is NADPH. 2. The sulfite reductase according to claim 1, wherein the sulfite reductase is derived from Escherichia coli or one of the genus Saccharomyces. The sulfite reductase according to claim 3, wherein one of the genus Saccharomyces is Saccharomyces cerevisiae. A method for producing purified sulfite reductase, comprising purifying sulfite reductase from a crude enzyme solution of Escherichia coli or Saccharomyces using ammonium sulfate fractionation and chromatography. The crude enzyme solution comprises the following steps:
adding a phosphate buffer having a pH of 6.5 to 8.5 to one cell of Escherichia coli or Saccharomyces;
Disrupting the cells using a sonicator at a temperature of 2 to 10 ° C. for 0.1 to 2 hours,
Collecting the supernatant after centrifugation at 5000 × g for 30 minutes,
After suspending the residual debris cells in the same buffer, grinding the debris cells,
After centrifugation as above, collecting the supernatant,
Mixing a plurality of supernatants,
A method for producing the purified sulfite reductase according to claim 5, which is produced by: The ammonium sulfate fraction is subjected to the following steps:
Gently adding ammonium sulfate to the crude enzyme solution produced as claimed in claim 6,
After centrifugation at a speed of 3000 to 15,000 xg for 0.1 to 2 hours, the precipitate at 30 to 60% saturation is collected and the solution of the precipitate is 0.1 M to 0.2 M phosphate buffered. The method for producing purified sulfite reductase according to claim 5, comprising a step of dialyzing the solution (pH 6.5 to 8.5) to obtain a dialysate, wherein all steps are performed at 2 to 10 ° C. The method for producing a sulfite reductase according to claim 5, wherein the chromatography is selected from the group consisting of DEAE Sephacel column chromatography and / or Sephacryl S-300HR column chromatography. A method for regenerating denatured fish protein, wherein the sulfite reductase of claim 1 is applied in solution or powder form in an amount of 0.01 to 0.5 activity units per gram of denatured fish protein. A method that includes: The method for regenerating a denatured fish protein according to claim 9, wherein the time during which the sulfite reductase in a solution or powder form acts on the denatured fish protein is 5 to 40 minutes.

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