Abstract
Lactase is a member of the β-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose. However, extracellular lactase production was still restricted to the process of cell lysis. In this study, lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-d-galactopyranoside. The purified enzyme was identified as a neutral lactase with an optimum pH of 9. To facilitate extracellular production of lactase, a putative mannoprotein KLLA0_E01057g of K. lactis was knocked out. It could effectively promote cell wall degradation and lactase production after lyticase treatment, which showed potential on other extracellular enzyme preparation. After optimizing the fermentation conditions, the lactase yield from mannoprotein-deficient K. lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.