Hildebrandt Piotr, Wanarska Marta, Kur Józef
Department of Microbiology, Chemical Faculty, Gdańsk University of Technology, Narutowicza 11/12, 80-952 Gdańsk, Poland.
BMC Microbiol. 2009 Jul 27;9:151. doi: 10.1186/1471-2180-9-151.
The development of a new cold-active beta-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production.
In this article, we present a new beta-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this beta-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium Arthrobacter sp. 32c, sequenced, cloned, expressed in Escherichia coli and Pichia pastoris, purified and characterized. 27 mg of beta-D-galactosidase was purified from 1 L of culture with the use of an intracellular E. coli expression system. The protein was also produced extracellularly by P. pastoris in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of P. pastoris culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50 degrees C, 60% of the maximum activity of the enzyme was determined at 25 degrees C and 15% of the maximum activity was detected at 0 degrees C.
The properties of Arthrobacter sp. 32cbeta-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.
新型冷活性β-D-半乳糖苷酶以及能够高效发酵乳糖的微生物的开发具有很高的生物技术价值，特别是对于低温下去除牛奶和乳制品中的乳糖以及用于同时生产生物乙醇的奶酪乳清生物修复过程。
在本文中，我们展示了一种新型β-D-半乳糖苷酶，作为上述生物技术过程中应用的候选酶。编码这种β-D-半乳糖苷酶的基因已从南极细菌节杆菌属32c的基因组DNA文库中分离出来，进行了测序、克隆，在大肠杆菌和毕赤酵母中表达，纯化并表征。使用细胞内大肠杆菌表达系统从1升培养物中纯化出27毫克β-D-半乳糖苷酶。该蛋白也由毕赤酵母大量胞外产生，对于AOX1和组成型系统，分别从1升毕赤酵母培养物中获得约137毫克和97毫克纯化酶。通过一步法或快速两步法（包括蛋白质沉淀和亲和色谱）将酶纯化至电泳纯。该酶被发现是一种同源三聚体蛋白，每个单体由695个氨基酸残基组成。虽然该酶的最大活性在pH 6.5和50℃下测定，但在25℃下测定到该酶最大活性的60%，在0℃下检测到最大活性的15%。
节杆菌属32cβ-D-半乳糖苷酶的特性表明，这种酶可用于低成本地将乳制品中的乳糖工业转化为半乳糖和葡萄糖，并且可能是从基于乳糖的原料生产乙醇的一个有趣替代方案。
