Difference between revisions of "Part:BBa K3979010"

 
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<partinfo>BBa_K3979010 parameters</partinfo>
 
<partinfo>BBa_K3979010 parameters</partinfo>
 
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==Overview==
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<br/>
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The chitinase enzyme hydrolyzes insoluble chitin to its oligo and monomeric components. Chitinase proteins are abundant in microorganisms such as bacteria, which use these enzymes to degrade chitin for nutrition. Chitinases are classified as endochitinase or exochitinase. Endochitinases cleave chitin at internal sites to produce GlcNAc multimers. Exochitinases catalyze the progressive hydrolysis of chitin to produce GlcNAc, chitobiose, or chitotriose. Chitinases are classified into different glycoside hydrolase (GH) families based on their amino acid sequences, such as GH18, GH19, and GH20. The GH18 family contains the majority of bacterial chitinases. Based on amino acid sequence homology of the individual catalytic domains, bacterial GH18 chitinases are classified into three major subfamilies, A, B, and C. Chitinolytic bacteria are found in a variety of habitats and decompose chitin in both aerobic and anaerobic conditions[3]. Amycolatopsis orientalis is known to secrete various chitinolytic enzymes such as chitinase β-N-acetylhexosaminidase and endo-β-glucosaminidase. These enzymes have been shown to catalyze efficient transglycosylation activities. It has been reported that the chitinase was able to degrade cell walls of Rhizopus and Mucor which belong to the order of Mucorales more effectively[2].
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<br/>
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This is the Chitinase A is from Amycolatopsis orientalis strain B-37.
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<br/>
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<br/>
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==Experiments==
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<br/>
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ChiA [BBa_K3979010] is a wild-type chitinase derived from Streptomyces orientalis (also known Amycolatopsis orientalis) strain B-37 . The chitinase was codon optimised for ease of expression in E.coli BL21(DE3) using BOOST. ChiA is an endochitinase capable of hydrolysing and cleaving the (1-4)-beta-linkages in chitin and Chito dextrins. The enzyme has a size of 975 bp and molecular weight of 36.6715 kDa. The approximate PI and extinction coefficient as calculated from ProtParam Tool is 9.39 and 48,150 M<sup>-1</sup> cm<sup>-1</sup> (assuming all cysteine bonds exist).
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The wet lab work plan associated with Streptomyces Chitinase (ST) can be broadly divided into three steps:
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<ul>
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<li>Cloning</li>
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<li>Expression and purification</li>
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<li>Enzyme characterization</li>
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</ul>
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<br/>
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===Cloning of Streptomyces Chitinase (ST)===
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<br/>
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We were able to successfully clone ST into the pET28a vector. The Streptomyces (ST) chitinase construct and corresponding primers were obtained from IDT. Upon receiving the construct and primers, both were dissolved in autoclaved Milli-Q water for a working concentration of 10ng/μL and 100uM respectively, as instructed by the manufacturer. The working stock of the same was 1 ng/μL and 10 uM, respectively.

Revision as of 11:30, 19 October 2021


ChiA is from Amycolatopsis orientalis strain B-37

Chitinase A is from Amycolatopsis orientalis strain B-37. The molecular size and weight of the protein are 33.13 kDa and 975 base pairs.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 63
    Illegal NheI site found at 81
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 7
    Illegal XhoI site found at 970
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 760



Overview


The chitinase enzyme hydrolyzes insoluble chitin to its oligo and monomeric components. Chitinase proteins are abundant in microorganisms such as bacteria, which use these enzymes to degrade chitin for nutrition. Chitinases are classified as endochitinase or exochitinase. Endochitinases cleave chitin at internal sites to produce GlcNAc multimers. Exochitinases catalyze the progressive hydrolysis of chitin to produce GlcNAc, chitobiose, or chitotriose. Chitinases are classified into different glycoside hydrolase (GH) families based on their amino acid sequences, such as GH18, GH19, and GH20. The GH18 family contains the majority of bacterial chitinases. Based on amino acid sequence homology of the individual catalytic domains, bacterial GH18 chitinases are classified into three major subfamilies, A, B, and C. Chitinolytic bacteria are found in a variety of habitats and decompose chitin in both aerobic and anaerobic conditions[3]. Amycolatopsis orientalis is known to secrete various chitinolytic enzymes such as chitinase β-N-acetylhexosaminidase and endo-β-glucosaminidase. These enzymes have been shown to catalyze efficient transglycosylation activities. It has been reported that the chitinase was able to degrade cell walls of Rhizopus and Mucor which belong to the order of Mucorales more effectively[2].

This is the Chitinase A is from Amycolatopsis orientalis strain B-37.

Experiments


ChiA [BBa_K3979010] is a wild-type chitinase derived from Streptomyces orientalis (also known Amycolatopsis orientalis) strain B-37 . The chitinase was codon optimised for ease of expression in E.coli BL21(DE3) using BOOST. ChiA is an endochitinase capable of hydrolysing and cleaving the (1-4)-beta-linkages in chitin and Chito dextrins. The enzyme has a size of 975 bp and molecular weight of 36.6715 kDa. The approximate PI and extinction coefficient as calculated from ProtParam Tool is 9.39 and 48,150 M-1 cm-1 (assuming all cysteine bonds exist).


The wet lab work plan associated with Streptomyces Chitinase (ST) can be broadly divided into three steps:

  • Cloning
  • Expression and purification
  • Enzyme characterization



Cloning of Streptomyces Chitinase (ST)


We were able to successfully clone ST into the pET28a vector. The Streptomyces (ST) chitinase construct and corresponding primers were obtained from IDT. Upon receiving the construct and primers, both were dissolved in autoclaved Milli-Q water for a working concentration of 10ng/μL and 100uM respectively, as instructed by the manufacturer. The working stock of the same was 1 ng/μL and 10 uM, respectively.