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

根据一种嗜冷酶的性质，我们研究了在食品中抑制嗜冷菌的条件。对典型嗜冷菌荧光假单胞菌的耐冷性丙氨酸外消旋酶进行了纯化和性质研究。该酶在30ﾟC以上的温度和低浓度的有机溶剂和变性剂中孵育后易失活。这些治疗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)在维持该酶的二级结构中起着重要作用。我们克隆了嗜冷性丙氨酸消旋酶基因，并在大肠杆菌SOLR中进行了表达，从携带pYOK3(6.3kbp)的克隆的细胞提取液中纯化了酶，并对其进行了鉴定。该酶在0ﾟC下仍表现出较高的催化活性，但在35ﾟC以上的热处理以及低浓度有机溶剂和变性剂的孵育下易失活。对PLP的Km值和在PLP存在下的稳定性的测定表明，该酶可能在高温下底物的消旋过程中失去了PLP。PLP从酶蛋白中解离可能与三级结构的改变以及随后的失活有关。低浓度乙醇的加入很容易抑制嗜冷菌的生长。

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.