Fernandes S, Geueke B, Delgado O, Coleman J, Hatti-Kaul R
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Sweden.
Appl Microbiol Biotechnol. 2002 Mar;58(3):313-21. doi: 10.1007/s00253-001-0905-4. Epub 2002 Jan 12.
The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.
β-半乳糖苷酶是从一株分离自南极洲的低温适应型微生物中纯化得到的。该微生物被鉴定为嗜冷性假交替单胞菌属。通过一种快速纯化方案，该酶得以高产率纯化，该方案包括在水两相系统中提取，随后进行疏水相互作用色谱和超滤。以邻硝基苯基-β-D-吡喃半乳糖苷为底物，在pH 9和26℃下测定2分钟时，β-半乳糖苷酶的活性最佳。该酶活性对30℃以上的温度高度敏感，在40℃时无法检测到。阳离子Na⁺、K⁺、Mg²⁺和Mn²⁺激活该酶，而Ca²⁺、Hg²⁺、Cu²⁺和Zn²⁺抑制活性。在0.1%（w/v）聚乙烯亚胺存在下，纯酶在4℃的保质期显著延长。还对纯β-半乳糖苷酶进行了乳糖水解评估。在酶浓度为1 U/ml的缓冲液中，8小时内乳糖水解率超过50%；在0.1%（w/v）聚乙烯亚胺存在下，水解率提高到70%。在牛奶中乳糖水解程度为40 - 50%。该酶可通过不同化学方法固定在琼脂糖上，活性保留率为60 - 70%。固定化酶更稳定，其水解乳糖的能力与可溶性酶相似。
