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

根据嗜冷酶的特性，研究了抑制食品中嗜冷菌生长的条件。从一个典型的嗜冷菌，荧光假单胞菌，嗜冷丙氨酸消旋酶纯化到同质性和特征。该酶在30 ℃以上热处理和低浓度有机溶剂及变性剂中温育时易于失活。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）在维持该不耐热酶的二级结构中起重要作用。从嗜冷解糖芽孢杆菌（Bacillus psychrosaccharolyticus）中克隆了嗜冷丙氨酸消旋酶基因，并在大肠杆菌SOLR中表达，从携带质粒pYOK 3（6.3kbp）的克隆的细胞提取物中纯化了该酶，并对其进行了鉴定。来自解冷糖B.psychrosaccharolyticus的酶即使在0 ℃下也显示出高催化活性，但在超过35 ℃的热处理和与低浓度的有机溶剂和变性剂一起孵育时容易失活。对PLP的Km值和稳定性的测定表明，在高温下底物外消旋化过程中，酶可能会释放PLP。PLP从酶蛋白中的解离可能与三级结构的变化以及随后的失活有关。低浓度乙醇的加入容易抑制耐冷菌的生长。

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.