Part:BBa_K1360003

endo-1,4-beta-mannosidase ManA1

This part is the coding sequence for a thermostable mannosidase, which specifically degrades mannan.

Usage and Biology

The ManA1 enzyme produced by this part can be used in pulp bleaching. It presents high activity under a wide range of temperature (55℃-99℃) and pH conditions (3-10.5). The optimum working condition is pH = 5.0, temperature = 75.4℃.

Design considerations

This part is only a coding sequence without expression regulators, and is compatible with RFC10 standards, and the codon is optimized for E.coli expression.

Lab Archives

We use DNS to test the enzymatic activity. DNS is a reagent detecting reductive saccharides. When mannan or xylan is degraded into mannose or xylose, DNS color get dark. When DNS color darker, the enzymatic activity higher.

In 2014, team Tongji carried out DNS enzymatic experiment of xynB, arfB and manA1 on one plate, so that you can get conclusion in a very straightforward way.

We carried out temperature test, pH test and synergy test on one plate.

The result of temperature test.

The result of pH test.

As for synergy test, we add three enzymes at different ratio.

Firstly, we recorded DNS color when there is only one enzyme.

And then, we supposed they have no synergistic effect, and simulate the RGB color.

Lastly, we compared the real DNS color with the non-synergy supposed DNS color.

As you can see, the real DNS color is darker, so we think they can work in synergy.

We also collect the absorbance data of the DNS. In this DNS method, OD540 corresponds to reductive saccharide concentration, but due to equipment limitation, we used OD570 for plate reader data.

Extra

In 2014, team Tongji's project Decolor Four.

The reason why these three hemicellulase can reduce chlorine consumption is because they break down hemicellulose which connects lignin and cellulose. In this way, bleaching detergent can "get into" the pulp easier, and less chlorine can make the same effect. This benefits environmental protection.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 552
  Illegal BamHI site found at 1170
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 544
  Illegal SapI.rc site found at 459