Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi di Bari, Bari, Italy.
J Appl Microbiol. 2010 Nov;109(5):1763-74. doi: 10.1111/j.1365-2672.2010.04804.x. Epub 2010 Jul 21.
To screen the glutamate dehydrogenase (GDH) activity of nonstarter lactic acid bacteria (NSLAB) and to determine the effects of temperature, pH and NaCl values used for cheese ripening on enzyme activity and expression of GDH gene.
A subcellular fractionation protocol and specific enzyme assays were used. The effect of temperature, pH and NaCl on enzyme activity was evaluated. The expression of GDH gene was monitored by real-time PCR. One selected strain was also used as adjunct starter for cheese making to evaluate the catabolism of free amino acids and the production of volatile organic compounds (VOC) during cheese ripening. The cytoplasm fraction of all strains showed in vitro NADP-dependent GDH activity. NADP-GDH activity was markedly strain dependent and varied according to the interactions between temperature, pH and NaCl. Lactobacillus plantarum DPPMA49 showed the highest NADP-GDH activity under temperature, pH and NaCl values found during cheese ripening. RT-PCR analysis revealed that GDH expression of Lact. plantarum DPPMA49 was down-expressed by low temperature (<13°C) and over-expressed by NaCl (1·87-5·62%). According to NADP-GDH activity, the highest level of VOC (alcohols, aldehydes, miscellaneous and carboxylic acids) was found in cheeses made with DPPMA49.
The results of this study may be considered as an example of the influence of temperature, pH and NaCl on enzyme activity and expression of functional genes, such as GDH, in cheese-related bacteria.
It focuses on the phenotypic and molecular characterization of the NADP-GDH in lactobacilli under cheese-ripening conditions. The findings of this study contribute to the knowledge about enzymes involved in the catabolism of amino acids, to be used as an important selection trait for cheese strains.
筛选非发酵性乳酸菌（NSLAB）的谷氨酸脱氢酶（GDH）活性，并确定奶酪成熟过程中使用的温度、pH 值和 NaCl 值对酶活性和 GDH 基因表达的影响。
使用亚细胞分级分离方案和特定的酶测定法。评估了温度、pH 值和 NaCl 对酶活性的影响。通过实时 PCR 监测 GDH 基因的表达。还选择了一株菌作为干酪制作的辅助发酵剂，以评估在奶酪成熟过程中游离氨基酸的分解代谢和挥发性有机化合物（VOC）的产生。所有菌株的细胞质部分均显示体外 NADP 依赖性 GDH 活性。NADP-GDH 活性明显依赖于菌株，并且根据温度、pH 值和 NaCl 之间的相互作用而变化。在奶酪成熟过程中发现的温度、pH 值和 NaCl 值下，植物乳杆菌 DPPMA49 表现出最高的 NADP-GDH 活性。RT-PCR 分析显示，植物乳杆菌 DPPMA49 的 GDH 表达在低温（<13°C）下下调，在 NaCl（1.87-5.62%）下上调。根据 NADP-GDH 活性，在使用 DPPMA49 制成的干酪中发现 VOC（醇类、醛类、杂类和羧酸类）的含量最高。
本研究结果可被视为温度、pH 值和 NaCl 对奶酪相关细菌中酶活性和功能基因（如 GDH）表达影响的一个例子。
它侧重于 NADP-GDH 在奶酪成熟条件下乳杆菌的表型和分子特征。本研究的结果有助于了解参与氨基酸分解代谢的酶，可作为奶酪菌株的重要选择特征。
