Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China.
Protein Pept Lett. 2021;28(2):221-228. doi: 10.2174/0929866527666200813201242.
β-galactosidases are enzymes that are utilized to hydrolyze lactose into galactose and glucose, and are is widely used in the food industry.
We describe the recombinant expression of an unstudied, heterodimeric β-galactosidase originating from Lactobacillus brevis ATCC 367 in Escherichia coli. Furthermore, six different constructs, in which the two protein subunits were fused with different peptide linkers, were also investigated.
The heterodimeric subunits of the β-galactosidase were cloned in expressed in various expression constructs, by using either two vectors for the independent expression of each subunit, or using a single Duet vector for the co-expression of the two subunits.
The co-expression in two independent expression vectors only resulted in low β-galactosidase activities, whereas the co-expression in a single Duet vector of the independent and fused subunits increased the β-galactosidase activity significantly. The recombinant β-galactosidase showed comparable hydrolyzing properties towards lactose, N-acetyllactosamine, and pNP-β-D-galactoside.
The usability of the recombinant L. brevis β-galactosidase was further demonstrated by the hydrolysis of human, bovine, and goat milk samples. The herein presented fused β-galactosidase constructs may be of interest for analytical research as well as in food- and biotechnological applications.
β-半乳糖苷酶是一种能够将乳糖水解为半乳糖和葡萄糖的酶，在食品工业中被广泛应用。
我们描述了来源于短乳杆菌 ATCC 367 的一种未研究过的杂二聚体β-半乳糖苷酶在大肠杆菌中的重组表达。此外，我们还研究了六种不同的构建体，其中两个蛋白质亚基通过不同的肽接头融合。
通过使用两个载体分别表达每个亚基，或者使用单个 Duet 载体共表达两个亚基，将β-半乳糖苷酶的杂二聚体亚基克隆并在不同的表达构建体中表达。
在两个独立的表达载体中进行共表达仅导致β-半乳糖苷酶活性较低，而在单个 Duet 载体中共表达独立和融合的亚基则显著提高了β-半乳糖苷酶的活性。重组β-半乳糖苷酶对乳糖、N-乙酰乳糖胺和 pNP-β-D-半乳糖苷具有相似的水解性质。
重组 L. brevis β-半乳糖苷酶在水解人乳、牛乳和羊乳样品中的应用进一步证明了其可用性。本文提出的融合β-半乳糖苷酶构建体可能对分析研究以及食品和生物技术应用具有重要意义。
