Cloning and expression of cellulase gene EG1 from Rhizopus stolonifer var. reflexus TP-02 in Escherichia coli

Tang, Bin; Pan, Haibo; Zhang, Qingqing; Ding, Lixia

doi:10.1016/j.biortech.2009.06.091

Cited by 17 publications

(8 citation statements)

References 12 publications

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“…There may be many problems to express gene from eukaryotic organism in procaryotic organism (Barros and Thomson 1987) especially applying the most common E. coli as the host cell (Akcapinar et al 2011). However, prokaryotic expression system is a mature system; also it is easy to be cultured and high productivity (Tang et al 2009). However, we adopted the plasmid pET-32a, which contained an extra label encoding thioredoxin to help disulfide bond folded correctly.…”

Section: Discussionmentioning

confidence: 99%

“…Some EG gene was further overexpressed in Saccharomyces cerevisiae since S. cerevisiae preliminary post modification ability (Akcapinar et al 2011; Huang et al 2010; Qin et al 2008; Wang and Zhang 2003). Also, some other constituent of cellulase have also been expressed in yeast system to improve its productivity (Barros and Thomson 1987; Fang and Xia 2013; Haan et al 2007; Tang et al 2009; Teng et al 2007). However, discovery novel enzyme is always the eternal theme to enzyme research.…”

Section: Introductionmentioning

confidence: 99%

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Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

et al. 2016

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Endo-1,4-β-d-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting β-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, which had 95.2% similarity to the EG IV from T. viride AS 3.3711. Further, the expression vector pET-32a-EG was constructed and was successfully heterologously expressed in Escherichia coli. The expression product was purified with Ni2+ affinity chromatography and its enzymatic properties were investigated. The SDS-PAGE showed the target protein is 39 kDa, which is consistent with the translated result from the DNA sequence. The kinetic parameter for the expression product was Km = 13.71 mg/mL and Vmax=0.51 μmol/min·mL. The optimal reaction pH and temperature was pH = 7.0 and T = 40 °C, which is similar to the native EG produced by Trichoderma virens ZY-01. It provides the foundation for the endo-1,4-β-d-glucanase further evolution and application.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0282-0) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

et al. 2016

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“…Recently, more and more cellulase genes were cloned in order to use the tremendous resource of cellulose waste that is currently available [12][13][14]22]. In the present study, to avoid hnRNA contamination, which also has poly A structures and introns, and to easily synthesize the EG IV gene from T. viride, we used a PCR-based exon splicing method, rather than traditional method of total RNA isolation followed by RT-PCR.…”

Section: Discussionmentioning

confidence: 98%

Molecular Cloning and Characterization of a Putative cDNA Encoding Endoglucanase IV from Trichoderma Viride and its Expression in Bombyx Mori

Zhang

Liang

et al. 2011

Appl Biochem Biotechnol

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The development of cellulase production technology has greatly contributed to the successful use of cellulosic materials as renewable carbon sources. In this study, a putative endoglucanase IV (EG IV) complementary DNA was cloned from the mycelium of a strain of the filamentous fungus Trichoderma viride using a PCR-based exon-splicing method and expressed in both a silkworm BmN cell line and in silkworm larvae. Western blot analysis detected a band of 42 kDa in BmN cells after infection with a recombinant mBacmid/BmNPV/EG IV baculovirus. Sequence alignment analysis of the T. viride EG IV gene showed two domains that were highly conserved with glycosyl hydrolases and a funga-type cellulose-binding domain. Analysis of variance showed that silkworms infected with recombinant baculoviruses exhibited significantly higher enzyme activity that was 48.84% higher than silkworms infected with blank baculoviruses and 46.61% higher than normal silkworms. The expressed bioactive EG IV was also stable at the pH range from 5.0 to 10.0. The availability of large quantities of bioactive EG IV in silkworm provided a possibility to produce cellulase transgenic silkworm, which express bioactive cellulase specially in its digestive tract and improve its metabolism efficiency of mulberry leaves. Its application in the sericulture industry may be very promising.

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“…However, compared with other microorganism expression systems, such as E. coli and yeast expression systems, which also can express cellulase with high bioactivity [20,21], Fig. 4 Analysis of enzyme activity.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression Characteristics of Trichoderma viride Endoglucanase V in the Silkworm, Bombyx mori L.

Wang

Zhang

et al. 2011

Appl Biochem Biotechnol

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Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and β-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.

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Cloning and expression of cellulase gene EG1 from Rhizopus stolonifer var. reflexus TP-02 in Escherichia coli

Cited by 17 publications

References 12 publications

Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

Molecular Cloning and Characterization of a Putative cDNA Encoding Endoglucanase IV from Trichoderma Viride and its Expression in Bombyx Mori

Heterologous Expression Characteristics of Trichoderma viride Endoglucanase V in the Silkworm, Bombyx mori L.

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