Team:UiOslo Norway/Basic Part

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Basic parts

BBa_K2711000 - Glucanase

Source: Cellulosimicrobium cellulans

β-Glucan is a water-soluble polysaccharide that consists of glucose units. In yeast cell walls, glucose monomers are linked in a branched structure via β-(1→3) and β-(1→6) glycosidic bonds [1]. β-1,3-Glucanases (EC 3.2.1.39 and EC 3.2.1.6) catalyze the hydrolysis of glucans containing β-1,3-linked glucose monomers, one of the most abundant polysaccharides present in the yeast cell wall [2]. Bacterial endo-1,3-β-glucanases catalyze the hydrolysis of (1 → 3)-beta-D-glucosidic linkages in (1 → 3)-beta-D-glucans. These enzymes have a β-sandwich architecture and belong to the glycoside hydrolase family 16 (GH16) [3].

The UiOslo_Norway 2018 team used the sequence of endo-1,3-β-glucanase from Cellulosimicrobium cellulans (previously knows as Arthrobacter Luteus) to enable targeted lysis of yeast cells. This glucanase is also used in the commercially available product Zymolyase, among other components, which is a lytic enzyme for the digestion of yeast cell wall glucan. In our case we wanted to specifically lyse Candida albicans cells in a vaginal sample which will also contain for example bacteria and skin cells. The glucanase was synthesized by IDT after the sequence was codon optimized.

Glucanase Glucanase vector

A 3D-render of our BioBrick Glucanase. Adapted in Pymol from Oda et al.[3]
Vector generated from the parts registry.

Our glucanase has activity comparable to a 100-1000 fold dilution of commercially available Zymolyase (2000 U/μl). Further details on the activity of our glucanase may be found on our Results.

Sources
  1. Deepak Mudgil, in Dietary Fiber for the Prevention of Cardiovascular Disease, 2017
  2. Salazar et al., 2001. Overproduction, Purification, and Characterization of β-1,3- Glucanase Type II in Escherichia coli. Protein Expression and Purification 23, 219–225.
  3. Oda et al., 2017. Structural and thermodynamic characterization of endo-1,3-β-glucanase: Insights into the substrate recognition mechanism. BBA - Proteins and Proteomics 1866 (2018) 415–425.