Study on growth inhibition of psychrotrophs in foods on the basis of properties of a psychrotrophic enzyme

根据一种嗜冷酶的特性，研究了食品中嗜冷菌的生长抑制条件。从典型的嗜冷菌荧光假单胞菌中纯化了一种嗜冷型丙氨酸消旋酶，并对其进行了表征。在30℃以上的温度下进行热处理，并用低浓度的有机溶剂和变性剂孵育，使酶容易失活。这些处理也抑制了嗜冷菌的生长。对该酶变性过程的分析表明，吡哆醛5′-磷酸（PLP）对维持该耐热酶的二级结构起重要作用。我们克隆并在大肠杆菌SOLR中表达了嗜冷解糖芽孢杆菌的嗜冷性丙氨酸消旋酶基因。该酶从携带质粒pYOK3 （6.3kbp）的克隆细胞提取液中纯化至均匀性，并对其进行了表征。该酶在0ºC时也表现出较高的催化活性，但在35ºC以上的温度下热处理和低浓度的有机溶剂和变性剂孵育容易失活。测定PLP的km值和在PLP存在下的稳定性表明，该酶可能在高温下底物外消旋过程中失去PLP。PLP与酶蛋白的解离可能与三级结构的改变以及随后的失活有关。添加低浓度的乙醇可有效抑制嗜冷菌的生长。

We examined the conditions for grwoth inhibition of psychrotrophs in foods on the basis of properties of a psychrotrophic enzyme. A psychrotrophic alanine racemase from a typical psychrotroph, Pseudomonas fluorescens, was purified to homogeneity and characterized. The enzyme was easily inactivated by heat-treating at over 30ﾟC and by incubating with low concentration of organic solvents and denaturants. These treatments also suppressed the growth of the psychrotroph. Analysis of the denaturation process of the enzyme suggest that pyridoxal 5'-phosphate (PLP) plays an important role in maintaining the secondary structure of the thermolabile enzyme. We cloned and expressed the psychrotrophic alanine racemase gene from Bacillus psychrosaccharolyticus into E.coli SOLR.The enzyme was purified to homogeneity from the cell extract of a clone carrying the plasmid pYOK3 (6.3kbp) and characterized. The enzyme from B.psychrosaccharolyticus shows a high catalytic activity even at 0ﾟC,but is easily inactivated by heat-treating at over 35ﾟC and by incubating with low concentration of organic solvents and denaturants. Determination of km value for PLP and stability in the presence of PLP suggests that the enzyme probably looses PLP during the racemization of the substrate at high temperature. Dissociation of PLP from the enzyme protein may be related to the change of the tertiary structure followed by the inactivation. Growth of psychrotrophs was easily suppressed by addition of low concentration of ethanol.