Krewinkel Manuel, Kaiser Jana, Merz Michael, Rentschler Eva, Kuschel Beatrice, Hinrichs Jörg, Fischer Lutz
Department of Biotechnology and Enzyme Science, University of Hohenheim, 70599 Stuttgart, Germany.
Department of Soft Matter Science and Dairy Technology, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany.
J Dairy Sci. 2015 Jun;98(6):3665-78. doi: 10.3168/jds.2015-9411. Epub 2015 Apr 8.
A selected number of enzymes have recently been assigned to the emerging class of cellobiose 2-epimerases (CE). All CE convert lactose to the rare sugar epilactose, which is regarded as a new prebiotic. Within this study, the gene products of 2 potential CE genes originating from the mesophilic bacteria Cellulosilyticum lentocellum and Dysgonomonas gadei were recombinantly produced in Escherichia coli and purified by chromatography. The enzymes have been identified as novel CE by sequence analysis and biochemical characterizations. The biochemical characterizations included the determination of the molecular weight, the substrate spectrum, and the kinetic parameters, as well as the pH and temperature profiles in buffer and food matrices. Both identified CE epimerize cellobiose and lactose into the C2 epimerization products glucosylmannose and epilactose, respectively. The epimerization activity for lactose was maximal at pH 8.0 or 7.5 and 40°C in defined buffer systems for the CE from C. lentocellum and the CE from D. gadei, respectively. In addition, biotransformations of the foodstuff milk ultrafiltrate containing lactose were demonstrated. The CE from D. gadei was produced in a stirred-tank reactor (12 L) and purified using an automatic system. Enzyme production and purification in this scale indicates that a future upscaling of CE production is possible. The bioconversions of lactose in milk ultrafiltrate were carried out either in a batch process or in a continuously operated enzyme membrane reactor (EMR) process. Both processes ran at an industrially relevant low temperature of 8°C to reduce undesirable microbial growth. The enzyme was reasonably active at the low process temperature because the CE originated from a mesophilic organism. An epilactose yield of 29.9% was achieved in the batch process within 28 h of operation time. In the continuous EMR process, the epilactose yield in the product stream was lower, at 18.5%. However, the enzyme productivity was approximately 6 times higher because the continuous epilactose formation was carried out for about 6 d without further addition of biocatalyst. Within this time, 24g of epilactose in 2.8 L of permeate were produced. The batch and the EMR process showed that the milk ultrafiltrate, which is a sidestream of the milk protein production, might be upgraded to a dairy product of higher value by the enzymatic in situ production of epilactose.
最近，一些特定的酶被归类到新出现的纤维二糖2-表异构酶（CE）类别中。所有的CE都能将乳糖转化为稀有糖表乳糖，而表乳糖被视为一种新型益生元。在本研究中，源自嗜温细菌迟缓纤维素分解菌（Cellulosilyticum lentocellum）和加氏 Dysgonomonas gadei的2个潜在CE基因的基因产物在大肠杆菌中进行重组表达，并通过色谱法进行纯化。通过序列分析和生化特性鉴定，这些酶被确定为新型CE。生化特性鉴定包括分子量、底物谱、动力学参数的测定，以及在缓冲液和食品基质中的pH和温度曲线。两种鉴定出的CE分别将纤维二糖和乳糖表异构化为C2表异构化产物葡萄糖基甘露糖和表乳糖。在特定缓冲系统中，迟缓纤维素分解菌的CE和加氏Dysgonomonas gadei的CE对乳糖的表异构化活性分别在pH 8.0或7.5以及40°C时达到最大值。此外，还展示了对含乳糖的食品原料牛奶超滤物的生物转化。加氏Dysgonomonas gadei的CE在搅拌罐反应器（12 L）中生产，并使用自动系统进行纯化。这种规模的酶生产和纯化表明，未来CE生产扩大规模是可行的。牛奶超滤物中乳糖的生物转化在分批过程或连续运行的酶膜反应器（EMR）过程中进行。两个过程均在工业相关的低温8°C下运行，以减少不良微生物生长。由于该CE源自嗜温生物，因此在低温工艺温度下仍具有相当的活性。在分批过程中，运行28小时后表乳糖产率达到29.9%。在连续EMR过程中，产物流中的表乳糖产率较低，为18.5%。然而，酶的生产率约高6倍，因为连续表乳糖形成过程持续约6天，无需进一步添加生物催化剂。在此期间，在2.8 L渗透物中产生了24 g表乳糖。分批和EMR过程表明，作为牛奶蛋白生产侧流的牛奶超滤物，通过原位酶促生产表乳糖，有可能升级为更高价值的乳制品。
