Enzymatically produced cellulose
a technology of cellulose and cellulose coating, which is applied in the field of polysaccharides, can solve the problems of high shear, laborious and expensive preparation of this cellulose type, and the activation of mcc requires high shear, so as to increase the viscosity and increase the shear thinning behavior of the aqueous composition
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example 1
Expression and Analysis of a Vibrio ruber Cellodextrin Phosphorylase
[0287]This Example describes expression of a putative Vibrio ruber cellodextrin phosphorylase enzyme in E. coli. Also, this Example demonstrates that this enzyme is indeed a cellodextrin phosphorylase through analysis of enzyme specific activity.
[0288]A putative cellodextrin phosphorylase, VruCdp1 (also referred to herein as “CRC03362-VruCdp1”), was identified in Vibrio ruber DSM14379. The nucleic acid sequence encoding VruCdp1 was predicted based on a genomic sequence, and is presented as SEQ ID NO:1. The amino acid sequence of VruCdp1 encoded by SEQ ID NO:1 is presented as SEQ ID NO:2.
[0289]Putative VruCdp1 cellodextrin phosphorylase was next heterologously expressed in E. coli, as follows. A polynucleotide sequence encoding VruCdp1 was codon-optimized for expression in E. coli. This sequence (SEQ ID NO:3) was inserted into the pET30a (Novagen) expression vector at the NdeI and XhoI sites by Generay (Shanghai, Chi...
example 2
Expression and Analysis of a Ruminococcus Champanellensis Cellodextrin Phosphorylase
[0293]This Example describes expression of a putative Ruminococcus champanellensis cellodextrin phosphorylase enzyme in E. coli. Also, this Example demonstrates that this enzyme is indeed a cellodextrin phosphorylase through analysis of enzyme specific activity.
[0294]A putative cellodextrin phosphorylase, RchCdp1 (also referred to herein as “CRC03359-RchCdp1”), was identified in Ruminococcus champanellensis 18P13. The nucleic acid sequence encoding RchCdp1 (positions 2373141 to 2375537 of GENBANK Accession No. NC_021039.1) is presented as SEQ ID NO:5. The amino acid sequence of RchCdp1 encoded by SEQ ID NO:5 is presented as SEQ ID NO:6.
[0295]Putative RchCdp1 cellodextrin phosphorylase was next heterologously expressed in E. coli, as follows. A polynucleotide sequence encoding RchCdp1 was codon-optimized for expression in E. coli. This sequence (SEQ ID NO:7) was inserted into the pET30a (Novagen) expr...
example 3
Using V. ruber and R. champanellensis Cellodextrin Phosphorylases to Produce Low Molecular Weight, Insoluble Cellulose
[0299]This Example describes using the cellodextrin phosphorylases described in Examples 1 and 2 to produce cellulose when applied in reactions containing G-1-P and cellodextrin.
[0300]A reaction comprising G-1-P and cellobiose in the presence of a V. ruber cellodextrin phosphorylase (VruCdp1, refer to Example 1) produced insoluble polysaccharide. To generate enough insoluble polysaccharide for analysis, a scale-up reaction was conducted by adding 1 g G-1-P, 0.25 g cellobiose, and 400 μg (˜7.4 units) isolated VruCdp1 to a glass bottle containing 80 mL of 25 mM Tris buffer pH 7.0. The reaction was incubated overnight at 37° C. Insoluble polysaccharide product was collected by centrifugation at 3000 rpm for 20 minutes. This material was determined to be low molecular weight cellulose (refer to Example 4 below).
[0301]A reaction comprising G-1-P and cellobiose in the pres...
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