Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa an...
Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, $pYES2M{\alpha}$-egIV, and $pYES2M{\alpha}$-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants $IpYES2M{\alpha}$-xegIV was higher than that of transformant IpYES2-xegIV or $IpYES2M{\alpha}$-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that $MF{\alpha}$ signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of $35^{\circ}C$ to $65^{\circ}C$. The optimal reaction condition for EGIV enzyme activity was at the temperature of $55^{\circ}C$, pH of 5.0, 0.75 mM $Ba^{2+}$, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant $IpYES2M{\alpha}$-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.
Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, $pYES2M{\alpha}$-egIV, and $pYES2M{\alpha}$-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants $IpYES2M{\alpha}$-xegIV was higher than that of transformant IpYES2-xegIV or $IpYES2M{\alpha}$-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that $MF{\alpha}$ signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of $35^{\circ}C$ to $65^{\circ}C$. The optimal reaction condition for EGIV enzyme activity was at the temperature of $55^{\circ}C$, pH of 5.0, 0.75 mM $Ba^{2+}$, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant $IpYES2M{\alpha}$-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.
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제안 방법
To analyze the decomposition ability of the recombinant EGIV enzyme to different substrates, bran, corn stalk, rice straw, cotton straw, filter paper, CMC and MCC were used in this study. Samples of the reaction solution were collected then used to measure EGIV enzyme activity.
To study expression of gene egIV in transformants, the yeast transformants were cultured in SC-U medium containing 2% galactose and were collected at inducing for 12, 24, 36, 48, and 60 h, respectively. Total RNA (15 µg) was fractionated on a formaldehyde agarose gel (1%) and blotted onto a Nylon membrane.
대상 데이터
0 program. The conserved domain of the EGIV protein was identified by InterProScan (http://www. ebi.ac.uk/Tools/InterProScan/). The N-glycosylation and O-glycosylation sites were predicted by the NetNGlyc v1.
Con: control, where the CMCase activity was measured under the normal reaction condition without any additional ions. The experiments were performed in triplicate. Different letters above the columns indicate a significant difference as determined by Duncan’s multiple comparisons test (capital letters show significant difference at P<0.
Homology of endoglucanase gene egIV was identified using BlastX against the NCBI non-redundant database. The open reading frame (ORF) was identified using the ORF program (http://www.ncbi. nlm.nih.gov/gorf/gorf.html). The signal peptide was predicted using the SignalP3.
이론/모형
Then, pMD18-T/egIV was transformed into competent cell of E. coli DH5α by using the heat-shock method.
성능/효과
Different letters above the columns indicate a significant difference as determined by Duncan’s multiple comparisons test (capital letters show significant difference at P<0.01, and lowercase letters show significant difference at P<0.05.
In this study, the CMCase activity of transformant IpYEMα-xegIV with MFα signal peptide from S. cerevisiae was 2-fold higher than that of EGIV with native signal peptide (Fig. 4), illustrating that the signal peptide of S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae.
The CMCase activities of the transgenic yeast IpYES2- egIV, IpYES2Mα-egIV, and IpYES2Mα-xegIV showed a peak value at 48 h under galactose induction (Fig. 4).
Multiple sequence alignment of the EGIV protein from different fungal species was carried out using ClustalX. The EGIV amino acid sequence identities between ACD36973 from T. viride AS 3.3711 and other fungal species ranged from 43% to 100%, demonstrating that the amino acid sequences of the EGIV protein from different fungi were not highly conserved. There were two conserved domains containing “QCGG” in CBD and “GNYVLRHE” in CD, respectively (Fig.
The putative catalytic region showed homology with endoglucanase belonging to the glycoside hydrolase family 61 [15]. The EGIV amino acid sequence similarity between T. viride and other fungal species ranged from 43% to 53%, demonstrating that the amino acid sequences of EGIV from different fungi were not highly conserved. However, the EGIV protein from Trichoderma species was much conserved.
The analysis of variance showed that CMCase activity of EGIV was significantly different (F=132.38, df=13, P<0.01) in the presence of different metal ions.
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