Part:BBa_K4224000

Description

Enzyme beta-galactosidase (lactase) (E.C. 3.2.1.23) catalyzes the hydrolysis of ß(1–3) and ß(1–4) galactosyl bonds in oligo- and disaccharides but it also catalyzes the reverse reaction of the hydrolysis, often called transglycosylation. Based on sequence homology, beta-galactosidases have been classified as members of GH1, GH2, GH35, and GH42 of the GH-A superfamily of glycoside hydrolase [13]. Beta-Galactosidase from Aspergillus oryzae (Ao-ß-gal) belongs to GH 35 family. The amino acid sequence of Ao-ß-gal includes 1005 residues with an average molecular mass of 110kDa [9].
Beta-Galactosidase is mostly known for its ability to hydrolyze milk sugar lactose [1,2]. Lactose is a disaccharide and consists of one galactose and one glucose linked together with a ß(1–4) bond. Hence, a common example of the industrial utilization of beta-galactosidase is the manufacture low-lactose or lactose-free dairy products. Aspergillus oryzae is one of the most widely-used fungal sources of commercial beta-galactosidase, as A. oryzae beta-galactosidase showed great advantages of extracellular expression with higher culture yield, less contaminated proteins, and easier downstream processing.
The low-lactose or lactose-free dairy products are important to people who suffer from lactose intolerance. Since lactase hydrolyses lactose into glucose and galactose, when the expression of lactase declines due to genetic disorders or natural ageing, the amount of lactose people can tolerate will decrease. This results in lactose malabsorption which leads up to several symptoms, such as abdominal pain, diarrhoea and nausea. For humans, lactose intolerance is a worldwide problem. It is estimated that 70% of the world’s adult population suffers from different degrees of lactose intolerance [3]. To allow people with lactose intolerance to enjoy dairy products, our project aimed to express lacA in Pichia pastoris to produce lactose-free dairy products.

Protocols

Heterlogous expression in Pichia pastoris X33

1. Inoculate a positive-transformed colony in 30 ml BMGY medium in a 100-ml flask. Grow at 28°C to 30°C in a shaking incubator (250 to 300 rpm) until the culture reaches an OD600 = 2 to 6 log-phase growth, 16 to 18 hr.
2. Harvest cells by centrifuging at 1,000 × g for 5 minutes at room temperature. To induce expression, decant supernatant and resuspend cell pellet in 100 ml BMMY medium. Add 1 ml of 100% methanol (HPLC grade) directly to culture flasks to reach a final concentration of 1%(vol/vol).
3. Continue culturing in 28°C to 30°C in a shaking incubator (250 to 300 rpm) for about 5 days. Add 100% methanol to a final concentration of 1% methanol (HPLC grade) every 24 hr to maintain induction. Be sure to check the volume of the culture and add methanol (HPLC grade) accordingly.

Enzyme Activity Assay

The optimum pH of A.oryzae beta-galactosidase is 5.0.

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The pH stability range of A.oryzae beta-galactosidase is between 3.5 and 8.0.

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The optimum reaction temperature of beta galactosidase is 60 °C. Activity of beta-galactosidase loses about 2/3 if the reaction temperature is 70°C.

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In the thermal stability experiment, there was basically no loss of enzyme activity when beta-galactosidase was treated at 30-60°C for 30 minutes, and 50% loss of enzyme activity when the enzyme was treated at 70°C, indicating that beta-galactosidase had high thermal stability.

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