EFFECT OF KOJIC ACID ON THE OXIDATION OF o-DIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid is a very effective inhibitor of mushroom tyrosinase as judged by its effect on the rate of pigmented products formation and on the rate of oxygen uptake when different o-dihydroxyphenols are oxidized by the enzyme. In addition to the ability of kojic acid to inhibit the enzyme per se, the data show that kojic acid can change the spectrum of some pigmented products formed in its absence, probably due to the ability of some o-quinones formed enzymatically to oxidize kojic acid to a yellow product(s). These findings call for caution in the use of kojic acid as an inhibitor of enzymatic browning in tissues and processed foods that are not yellow originally.     

EFFECT OF KOJIC ACID ON THE OXIDATION OF TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid [5-hydroxy-2-(hydroxymethyl)-4-pyrone] inhibited effectively the rate of pigment formation during the oxidation of pyrogallol, 2, 3,4-THAP (2, 3,4-trihydroxyacetophenone) and 2, 4,5-THBP (2, 4,5-trihydroxybutyrophenone) by tyrosinase. On the other hand, kojic acid had a synergistic effect on the rate of methyl gallate and n-propyl gallate oxidation to pigmented product(s) (λ_max= 360 nm and λ_max= 380 nm, respectively). However, kojic acid inhibited effectively the rate of oxygen uptake when each of the above trihydroxyphenols was oxidized by tyrosinase. These results suggest that kojic acid inhibits tyrosinase per se (probably due to its ability to bind copper at the active site of the enzyme) and that it exerts only an apparent stimulatory effect during the formation of pigmented product (s) from methyl gallate and n-propyl gallate. Proof for the latter was obtained by a time-course experiment of kojic acid addition and examination of the spectra of pigmented product(s) formed in the absence versus presence of kojic acid, which suggested that the o-quinone of n-propyl gallate and the o-quinone of methyl gallate can each convert kojic acid to a yellow product(s) absorbing at the 360–380 nm region.     

KOJIC ACID CONVERSION TO A YELLOW PRODUCT(S) BY THE HEMOGLOBLIN/H2O2 SYSTEM1

Kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one; also named 5-hydroxy-2-(hydroxymethyl)-γ-pyrone) in the presence of hydrogen peroxide, but not in its absence, can be oxidized by hemoglobin (Hb) to a yellow product(s). The yellow product(s) formed is characterized by a peak at 370–380 nm and is fluorescent. The relationship between the rate of oxidation of kojic acid and various concentrations of hemoglobin and of H₂O₂ is described. The changes with time in the spectrum of product(s) obtained when kojic acid is acted upon by the Hb/H₂O₂ system and the relationship between the various concentrations of hemoglobin, of kojic acid and of H₂O₂ on the spectrum of the “final yellow product(s)” are shown.     

KOJIC ACID CONVERSION TO A YELLOW PRODUCT(S) VIA THE HORSERADISH PEROXIDASE/H2O2 SYSTEM1

Horseradish peroxidase in the presence of hydrogen peroxide oxidizes kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) to a yellow product(s). The yellow product(s) formed has a major absorbance peak at 375 nm and is fluorescent. The relationships between, and effects of, various concentrations of horseradish peroxidase, kojic acid and hydrogen peroxide on the rate of oxidation of kojic acid to the yellow product(s) are described. The observation that the oxidation of kojic acid to the yellow product(s) occurs best in the presence of very low concentrations of hydrogen peroxide, relative to that of kojic acid, suggests that kojic acid is a poor hydrogen donor (AH₂) for horseradish peroxidase.     

曲酸与阿魏酸对酪氨酸酶的抑制作用研究

本实验采用Lineweaver-Burk双倒数法探讨了曲酸与阿魏酸对蘑菇酪氨酸酶催化L-DOPA氧化的抑制作用并推测其抑制机理。通过对曲酸与阿魏酸的IC50与抑制常数KI的测定可知,曲酸对酪氨酸酶的抑制作用明显高于阿魏酸。分别采用分光光度法和ImageJ图像分析与处理软件定量地测定曲酸与阿魏酸对虾血淋巴和苹果褐变的抑制作用。曲酸对虾血淋巴褐变的抑制作用优于阿魏酸;对于苹果褐变,阿魏酸的抑制作用则高于曲酸。     

p-HYDROXYPHENYLPROPIONIC ACID (PHPPA) AND 3,4-DIHYDROXYPHENYLPROPIONIC ACID (3,4-DPPA) AS SUBSTRATES FOR MUSHROOM TYROSINASE

p-Hydroxyphenylpropionic acid (PHPPA) and 3,4- dihydroxyphenylpropionic acid (3,4-DPPA) serve as substrates for tyrosinase. The Km value of 3,4-DPPA for tyrosinase is 1.3 mM. The yellow o-quinone of 3,4-DPPA (4-carboxyethyl-o-benzoquinone) (λ_max= 400nm), is detected initially and it is then converted to a red product(s) (λ_max= 480±10 nm), the o-quinone of 6,7-dihydroxy 3-dihydrocumarin (dihydroesculetin). When the concentration of the latter is relatively high, it polymerizes to a final brown product(s), characterized by an ill-defined spectrum.
H₂O₂ shortens the lag period of PHPPA hydroxylation, hastens the conversion of the yellow o-quinone of 3,4-DPPA to the red o-quinone of dihydroesculetin, and prevents the polymerization of the latter to the final brown product(s).
The relatively unstable o-quinone of 3,4-DPPA interacts with amines such as hydroxylamine (NH₂OH), p-aminosalicylic acid (PASA) and p-aminobenzoic acid (PABA), forming relatively stable final product(s) characterized by different spectra from those formed in their absence.
Acetohydroxamic acid (AHA) and salicylhydroxamic acid (SHAM) each has an effect on the spectrum of product(s) obtained when 3,4-DPPA is oxidized by tyrosinase, indicating that these hydroxamic acids derivatives interact with the o-quinone of 3,4-DPPA. The spectrum of the final product(s) was also different when 3,4-DPPA was oxidized by tyrosinase in the presence of benzenesulfinic acid than in its absence, suggesting the formation of a stable phenylsulfonyl derivative.     

曲酸对南美白对虾酚氧化酶活性和结构的影响

为研究曲酸对南美白对虾酚氧化酶(phenol oxidase,PO)活性和结构的影响,以南美白对虾头胸部分离纯化出的PO为原料,研究不同浓度曲酸(0、0.1、0.3、0.5、0.7、0.9、1.1、1.3、1.5 mmol/L)对PO活性的抑制,确定曲酸作用PO的抑制类型;通过测定不同浓度曲酸(0、0.1、0.3、0.5、0.7、0.9、1.1 mmol/L)处理的PO蛋白内源荧光光谱、表面疏水性和二级结构类型含量的变化,研究曲酸对PO空间结构的影响。结果表明:随着作用PO曲酸浓度的增加,PO的剩余酶活逐渐减少,当曲酸浓度达到1.5 mmol/L时,PO的剩余酶活仅为9.8%,参与的反应体系褐变指数变化值为3.02,抑褐变效果明显;曲酸能够有效的抑制PO活性,曲酸作用PO的抑制类型属于竞争性抑制剂;随着作用PO曲酸浓度的增加,PO的内源荧光强度不断降低,最大发射波长发生红移,PO蛋白表面疏水性先增加后降低;随着作用PO曲酸浓度的增加,二级结构类型α-螺旋和β-折叠含量降低,无规则卷曲和β-转角增加。可以推测,曲酸与PO结合后,酶蛋白空间结构发生了改变。     

曲酸对冷鲜鸭肉中优势腐败菌的抑制作用及其抑菌机理

以冷鲜鸭肉中优势腐败菌假单胞菌、气单胞菌、热杀索丝菌和具有致腐能力的病原微生物沙门氏菌为研究对象,采用抑菌圈直径、液体生长曲线和最小抑菌浓度评价曲酸的抑菌作用,分析曲酸对细胞膜和菌株胞内物质的影响,探索曲酸的抑菌机理。结果表明：曲酸对4 株菌株均具有良好的抑制作用,其中热杀索丝菌对曲酸处理敏感,最小抑菌质量浓度为0.5 mg/mL,液体体系下低质量浓度曲酸溶液处理即能完全抑制其生长;随着曲酸质量浓度增加,沙门氏菌、假单胞菌和气单胞菌的抑菌圈直径增加,对假单胞菌和气单胞菌的抑制尤为明显,其抑菌圈直径增加1 倍以上;2.0 mg/mL曲酸处理在液体体系中能完全抑制上述3 株菌株的生长。曲酸处理能够提高菌株细胞膜疏水性1～2 倍,降低细胞膜表面的选择渗透性和破坏细胞膜结构,菌株培养液中Ca2＋、K＋、Mg2＋离子浓度增加;曲酸处理还可破坏呼吸链脱氢酶、促使β-半乳糖苷酶和碱性磷酸酶等内容物质泄漏、菌体细胞膜破裂形成孔洞。同时,曲酸处理引起菌株胞外蛋白质含量显著上升（P＜0.05）,胞内分子质量大于66.4 kDa和小于20 kDa的蛋白质减少。整体来看,曲酸抑制4 株菌株的机理基本一致,主要是通过促进疏水物质暴露、破坏菌株相关酶和改变细胞膜通透性,同时干扰菌株蛋白质新陈代谢,从而抑制菌株的增殖。     

曲酸及其衍生物的研究进展

曲酸作为一种新型的食品和化妆品添加剂,吸引了众多食品化学、医药、生物学家的研究兴趣。文中介绍了曲酸的结构、化学性质,并对曲酸生产的菌株、发酵方法、抗菌防腐、护色保鲜及其安全性能作了综述。     

EFFECT OF BENZENESULFINIC ACID ON THE OXIDATION OF o-DIHYDROXY - AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

Benzenesulfinic acid inhibits the rate of oxidation of different o-dihydroxy-phenols and trihydroxyphenols by tyrosinase when assayed spectrophotometrical-ly but barely has an effect when assayed polarographically. Benzenesulfinic acid is a much more effective inhibitor of the rate of oxidation to pigmented product(s) by tyrosinase of o-dihydroxyphenols than of trihydroxyphenols. The spectrum of the final product(s) formed by the oxidation of most of the tested substrates was different in the absence versus presence of benzenesulfinic acid suggesting that the latter traps the o-quinones forming phenylsulfonyl derivatives in the reaction.     

EFFECT OF BENZOIC ACID AND SOME OF ITS DERIVATIVES ON THE RATE OF DL-DOPA OXIDATION BY MUSHROOM TYROSINASE1

Vanillic acid and salicylic acid inhibited the rate of dihydroxyphenylalanine (DL-DOPA) oxidation to dopachrome (γ_max=475 nm) by tyrosinase at all concentrations tested. Benzoic acid and p-hydroxybenzoic acid (PHBA), at relatively low concentrations, slightly stimulated the rate of DL-DOPA oxidation, whereas at higher concentrations each inhibited the reaction. p-Hydroxybenzoic acid methyl ester (PHBAME), at relatively low concentrations, had a pronounced synergistic effect on the reaction, whereas at relatively high concentrations it inhibited the rate of DL-DOPA oxidation. The synergistic effect of 1.6–6.6 mM PHBAME on the rate of DL-DOPA oxidation to dopachrome was found to be only an apparent effect due to the ability of PHBAME to be hydroxylated very slowly by tyrosinase to a yellow pigmented product(s) with DL-DOPA serving as a reductant (AH₂) for the hydroxylation reaction, thus hastening the conversion of PHBAME to pigmented product(s). Vanillic acid, salicylic acid, benzoic acid and PHBA could not be hydroxylated by tyrosinase.     

EFFECT OF ACETOHYDROXAMIC ACID (AHA) AND SALICYLHYDROXAMIC ACID (SHAM) ON THE OXIDATION OF o-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

Acetohydroxamic acid (AHA) and salicylhydroxamic acid (SHAM) each inhibited the rate of oxidation of different o-dihydroxy- and trihydroxyphenols by tyrosinase when assayed spectrophotometrically or polarographically. SHAM was a much more effective inhibitor than AHA. Spectral changes occurring during the oxidation of different o-dihydroxyphenols by tyrosinase in the presence of AHA or SHAM were different than the spectral changes occurring in their absence. AHA and SHAM also had an effect on the spectrum of the final product(s) formed when different o-dihydroxyphenols were oxidized by the enzyme, suggesting that AHA and SHAM conjugate with the o-quinones formed. A lack of an effect of AHA and SHAM on the spectrum of product(s) formed when trihydroxyphenols were oxidized by tyrosinase suggest that AHA and SHAM do not conjugate with the o-quinones derived from trihydroxyphenols.     

曲酸生产菌的复合诱变选育

以生产菌株米曲霉（Aspergillus oryzae）w-56为出发菌株,经2次紫外线、2次60Co多重复合诱变处理,选育获得曲酸生产菌UR28,生产发酵周期由原来的130h缩短至65h,曲酸产量由原来的36g/L,提高到68g/L,比出发菌株提高了88.9%。实验证明采用复合诱变,能有效改变菌株对诱变因素的敏感性,提高突变率,逐步提高突变株的产酸水平。     

食品中曲酸安全性评价研究进展

曲酸是许多曲霉和青霉真菌所产生的一种常见代谢产物。近年来通过发酵法生产的曲酸作为添加剂,在食品和化妆品中广泛应用。目前,曲酸的安全性引起争议,文中就国外有关食品中曲酸的安全性评价作了较为系统的介绍。     

2,3,4-TRIHYDROXYACETOPHENONE (2,3,4-THAP) AS A SUBSTRATE FOR MUSHROOM TYROSINASE1

2,3,4-Trihydroxyacetophenone (2,3,4-THAP) can serve as a substrate for mushroom tyrosinase with a K_m value of 1.2 mM. The product(s) formed is yellow, characterized by a peak at 420–430 nm. A lag period in 2,3,4-THAP oxidation to the yellow product(s) in the presence of ascorbate indicates that the initial product(s) is an o-quinone of 2,3,4-THAP. An oxime, characterized by a broad peak at 510–650 nm, is the likely product formed between o-quinone of 2,3,4-THAP and NH₂OH. The ?_m of the o-quinone of 2,3,4-THAP was estimated to be 1.6 × 10⁴ M^?1 cm^?1 at 425 nm. During relatively long incubation periods, the peak of the yellow product(s) shifts from 420–425 nm to 430–440 nm; the solution remains yellow and transparent for at least a week and no precipitate is formed. The final yellow product(s) is probably a low molecular weight polymer of THAP-o-quinone (dimer, tetramer, etc).     

EFFECT OF HYDROXYLAMINE, p-AMINOBENZOIC ACID AND p-AMINOSALICYLIC ACID ON THE OXIDATION OF O-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

The effect of hydroxylamine (NH₂OH), p-aminobenzoic acid (PABA) and p-aminosalicylic acid (PASA) on the spectrum of the final product (s) formed when o-dihydroxy- and trihydroxyphenols were oxidized by tyrosinase was examined. New pigmented product(s), probably oximes, were formed by the interaction of NH₂OH with the o-quinones of 4-methyl catechol, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylpropionic acid (3,4-DPPA) but not with the o-quinones of catechol or protocatechuic acid. Interaction of PABA or PASA with the o-quinones of catechol, 4-methyl catechol, protocatechuic acid, DOPAC and 3,4-DPPA also yielded pigmented oximes. The interaction of the o-quinones of trihydroxyphenols with NH₂OH, PABA or PASA had little effect on the spectrum of the final product (s), suggesting that oximes are not formed in these reactions.     

p-HYDROXYPHENYLACETIC ACID AND-3,4-DIHYDROXYPHENYLACETIC ACID AS SUBSTRATES FOR MUSHROOM TYROSINASE

The hydroxylation of p-hydroxyphenylacetic acid (PHPAC) and the oxidation of 3,4-dihydroxyphenylacetic acid (DOPAC) by mushroom tyrosinase are illustrated. DOPAC quinone (4-carboxy-methyl-o-benzoquinone (λ_max= 400±10 nm) is the initial pigmented product formed when DOPAC is oxidized by the enzyme. DOPAC quinone is very unstable, and, once formed, is converted rapidly to further oxidation product(s).
The relationships between the rate of p-dihydroxyphenylacetic acid hydroxylation and 3,4-dihydroxyphenylacetic acid oxidation as a function of various concentrations of each substrate and of mushroom tyrosinase, are described.
The effect of the addition of various chemicals that can potentially conjugate otherwise affect DOPAC quinone was studied The Km value of 3,4-dihydroxyphenylacetic acid for mushroom tyrosinase was estimated to be 4.0 mM.     

对羟基肉桂酸对酪氨酸酶催化反应的抑制机理

本文研究了对羟基肉桂酸(HCA)对酪氨酸酶催化单酚底物L-酪氨酸和催化二酚底物L-多巴的抑制能力,并利用紫外-可见光谱、荧光光谱以及分子对接技术探究了其抑制机理。结果表明对羟基肉桂酸对酪氨酸酶催化单酚底物L-酪氨酸比催化二酚底物L-多巴具有更强的抑制作用,半抑制浓度分别为0.096 mmol/L和0.500 mmol/L;紫外-可见分析发现对羟基肉桂酸能与Cu2+发生螯合,使光谱发生明显红移。进一步通过荧光光谱分析得到,对羟基肉桂酸在酪氨酸酶溶液中并没有出现荧光淬灭反而随着对羟基肉桂酸浓度的增大荧光强度变强,说明对羟基肉桂酸被酪氨酸酶催化氧化成对应的醌类物质。利用分子对接技术揭示了对羟基肉桂酸通过氢键和疏水作用竞争性地占据了单酚和二酚底物的空间位置,并与酪氨酸酶中双核铜离子螯合,从而抑制酪氨酸酶催化L-酪氨酸和L-多巴氧化的活性机理。     

Effects of kojic acid on changes in the microstructure and myofibrillar protein of duck meat during superchilled storage

This study investigated the effect of 0.8% (m/v) kojic acid treatment on changes in the microstructure and myofibrillar protein of duck meat covered with oxygen-permeable polyvinylchloride (PVC) film (9 ± 0.5 µM) during superchilled storage (−1.65 ± 0.5°C). The superchilled samples exhibited wider gaps between muscle fibers at 5 weeks storage compared with kojic acid–treated groups. Based on the variation of water status, the water-holding capacity decreased significantly (p < 0.05), and bound water and immobilized water were gradually converted into free water during superchilled storage. For kojic acid–treated samples, however, no major changes were observed with respect to muscle structure, water status, and protein degradation at 6 weeks. The 0.8% (m/v) kojic acid treatment increased the water-holding capacity, reduced carbonyl content and protein degradation, and decreased the α-helix contents loss of myofibrillar proteins. Kojic acid treatment effectively protected myofibrillar protein structure from being destroyed during superchilled storage, suggesting that this method was a good way to reduce protein oxidation and prolonged its shelf life.     

Functional glucosylation of kojic acid and daidzein with the Eucalyptus membrane-associated UDP-glucosyltransferase reaction system

Kojic acid was glucosylated to kojic acid 5- and 7-O-beta-D-glucopyranosides by cultured cells of Eucalyptus perriniana. The glucosylation was also performed effectively by the enzymatic reaction system using membrane-associated UDP-glucosyltransferase from the cells with addition of UDP-glucose. The production of the 5-O-D-glucoside occurred exclusively prior to that of the 7-O-glucoside in both systems. In the same manner, daidzein was converted to daidzin (daidzein 7-O-beta-D-glucopyranoside) by the enzymatic system. Here, we propose methods for the structural modification of phytoestrogen-related substances by enzymatic beta-glucosylation.