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Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal …


Biology Articles » Biochemistry » Carbohydrate Biochemistry » Role of the linker region in the expression of Rhizopus oryzae glucoamylase » Tables

Tables
- Role of the linker region in the expression of Rhizopus oryzae glucoamylase

Table 1

Amino acid sequences of the linker region from fungal GAs. Potential glycosylation sites in the linker sequences were predicted by the NetNGlyc and NetOGlyc programs. The predicted N-glycosylation sites are underlined and the putative O-glycosylation sites are labeled in bold.

GA*
Position (aa)
Linker sequence
RoGA
132–167
SKPTTTTATTTTTTAPSTSTTTRPSSSEPATFPTGN
AnGA
494–531
ATGGTTTTATPTGSGSVTSTSKTTATASKTSTSTSSTS
HrGA
500–504
NVTSS
HgGA
490–508
SKQTPNPSAAPSPSPYPTA
McGA
129–177
SVTTTTTTAPTTTTSGGSSTTTGGSTTTATSVPTGVPSGFPTGNSTISS
NcGA
494–519
ATATATSFPANLTPASTTVTPPTQTG

*GenBank or GenPept accession numbers are as follows: Ro, Rhizopus oryzae (from Simpson Biotech); An, Aspergillus niger (AAP04499); Hr, Hormoconis resinae (Q03045); Hg, Humicola grisea (AAA33386); Mc, Mucor circinelloides (AAN85206); and Nc, Neurospora crassa (P14804).

Lin et al. BMC Biochemistry 2007 8:9   doi:10.1186/1471-2091-8-9

Table 2

Comparison between calculated and experimental molecular weights of GAs. The molecular masses were calculated from the deduced amino acid compositions using the ExPASy Molecular Biology Server. The experimental molecular masses were obtained from mass spectrometry or estimated from SDS-PAGE.

Protein
Calculated mass (kDa)
Apparent mass (kDa) (mass spectrometry/SDS-PAGE)
full-length GA
62.4
ND*/78
T165A
62.4
ND*/78
N167D
62.3
ND*/76
GAΔ168–604
15.2
20.7/27
GAΔ132–604
11.7
12.0/14
GAΔ26–131
50.7
ND*/69
GAΔ26–145
49.4
59.9 to 61.1/60
GAΔ26–160
47.9
55.5 to 56.6/55

*ND, not determined. GAΔ26–145 displayed multiple mass spectral peaks ranging from 59.9 to 61.1 kDa. GAΔ26–160 displayed multiple mass spectral peaks ranging from 55.5 to 56.6 kDa.

Lin et al. BMC Biochemistry 2007 8:9   doi:10.1186/1471-2091-8-9

Table 3

Specific activity of wild-type RoGA and truncated mutants. Specific enzyme activities were evaluated per micromole of protein.

Enzyme
Specific activity (103 U/μmol)*
full-length RoGA
4.58 ± 0.16
GAΔ26–131
4.88 ± 0.04
GAΔ26–145
4.05 ± 0.06
GAΔ26–160
3.73 ± 0.33

*The data are expressed as the means ± SD.

Lin et al. BMC Biochemistry 2007 8:9   doi:10.1186/1471-2091-8-9

Table 4

Oligonucleotide primers used for the construction of plasmids. Primers were used in the amplification reactions to generate various DNA fragments or mutations constructs.

Name
Nucleotide sequence*
F-RoGA
5'-TTCGAATTCATGCAATTATTCAATTTG-3'
R-RoGA
5'-TTCGAATTCTTAAGCGGCAGGTGCACC-3'
R-GAΔ168–604
5'-TTGAATTCTCAGTTACCAGTTGGGA-3'
R-GAΔ132–604
5'-TTGAATTCTCATGTAGATACTTGGT-3'
F-PS1
5'-CGTAGTTTTTCAAGTTCTTAG-3'
R-PS1
5'-TCCTTACCTTCCAATAATTC-3'
F-GAΔ132–167
5'-CAAGTATCTACATCTACAATCTCC-3'
R-GAΔ132–167
5'-AGGAGATTGTAGATGTAGATACTTG-3'
F-GAΔ26–131
5'-TTGCTTGTTTCTGCTTCCAAGCCCACTACT-3'
R-GAΔ26–131
5'-AGTAGTGGGCTTGGAAGCAGAAACAAGCAA-3'
F-GAΔ26–145
5'-AGAAGTAGAAGGGGCAGCAGAAACAAGCAA-3'
R-GAΔ26–145
5'-TTGCTTGTTTCTGCTGCCCCTTCTACTTCT-3'
F-GAΔ26–160
5'-TTGCTTGTTTCTGCTGCTACTTTCCCAACT-3'
R-GAΔ26–160
5'-AGTTGGGAAAGTAGCAGCAGAAACAAGCAA-3'
F-GAΔ26–167
5'-TTGCTTGTTTCTGCTTCTACAATCTCCTCA-3'
R-GAΔ26–167
5'-TGAGGAGATTGTAGAAGCAGAAACAAGCAA-3'
F-T165A
5'-CCAGCTGGTAACTCTACAATCTCCTCA
R-T165A
5'-GGAGATTGTAGAGTTACCAGCTGG
F-N167D
5'-ACTGGTGACTCTACAATCTCCTCATGGATT-3'
R-N167D
5'-GGAGATTGTAGAGTCACCAGTTGGGAAAGT-3'

*Underlined letters indicate EcoRI sites. Bold letters indicate mutated nucleotides.

Lin et al. BMC Biochemistry 2007 8:9   doi:10.1186/1471-2091-8-9


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