Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from Aspergillus usamii E001

Wu, Minchen; Wang, Junqing; Zhang, Huimin; Tang, Cun-Duo; Gao, Jinhu; Tan, Zhiyong

doi:10.1007/s11274-010-0525-1

Cited by 21 publications

(27 citation statements)

References 18 publications

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“…Aspergillus usamii E001, isolated by and preserved in the lab of biochemistry and molecular biology, School of Medicine and Pharmaceutics, Jiangnan University, China as reported previously [23], was used as the source for total RNA and genomic DNA extraction. E. coli JM109 and pUCm-T (Sangon, Shanghai, China) were used as hostvector system for gene cloning and DNA sequencing.…”

Section: Strains Vectors and Mediamentioning

confidence: 99%

“…The total RNA was extracted from the mycelia by using onestep method according to the specification of TRIzol Kit (Sangon). Extraction of the genomic DNA from the mycelia of A. usamii E001 was performed according to the method as reported previously [23].…”

Section: Total Rna and Genomic Dna Extractionmentioning

confidence: 99%

“…Since then, the demands for cellulases have increased dramatically, covering a wide range of industrial fields [2,9,15]. Recently, cellulases together with hemicellulases accounted for approximately 20% of the global industrial enzyme market [23], and will continue to grow as new types of cellulases are identified and characterized.…”

Section: Introductionmentioning

confidence: 99%

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Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris

Shi

Yin

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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Using 3' and 5' rapid amplification of cDNA ends methods, the full-length cDNA sequence encoding an endo-1,4-β-glucanase of Aspergillus usamii E001 (abbreviated as AuCel12A) was amplified from the total RNA. The clone cDNA sequence of the gene encoding the AuCel12A, named as Aucel12A, is 1,027 bp in length harboring 5' and 3' non-coding regions, as well as a 720 bp of open reading frame that encodes a 16-aa signal peptide, and a 223-aa mature AuCel12A with a theoretical M.W. of 24,294 Da, a calculated pI of 4.15, and one putative N-glycosylation site. The complete DNA sequence of the gene Aucel12A was amplified from the genomic DNA of A. usamii E001 by using the conventional PCR and pUCm-T vector-mediated PCR initially developed in our lab. The clone DNA sequence is 1,576 bp in length, consisting of a 5' flanking regulatory region, three exons, and two introns with sizes of 50 and 66 bp. The cDNA fragment encoding the mature AuCel12A was expressed in a fully active form in Pichia pastoris. One P. pastoris transformant expressing the highest recombinant AuCel12A (rAuCel12A) activity, labeled as P. pastoris GSCel2-1, was chosen for subsequent studies. Integration of the Aucel12A into P. pastoris genome was confirmed by PCR analysis using 5'- and 3'-AOX1 primers. SDS-PAGE and enzyme activity assays demonstrated that the rAuCel12A, a glycosylated protein with an apparent M.W. of 27.0 kDa and a carbohydrate content of 4.82%, was secreted into the culture medium. The purified rAuCel12A displayed the highest activity at pH 5.0 and 60°C. It was highly stable at a pH range of 3.5-7.0, and at a temperature of 55°C or below. Its activity was not significantly affected by an array of metal ions and EDTA, but inhibited by Ag⁺, Hg²⁺ and Fe²⁺. The K(m) and V(max) of the rAuCel12A, towards carboxymethylcellulose-Na (CMC-Na) at pH 5.0 and 50°C were 4.85 mg/ml and 160.5 U/mg, respectively.

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Section: Strains Vectors and Mediamentioning

confidence: 99%

Section: Total Rna and Genomic Dna Extractionmentioning

confidence: 99%

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Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris

Shi

Yin

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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“…Subsequently, the genes encoding the AuXyn11A and AuXyn11B were cloned and expressed in Pichia pastoris (Wu et al 2010;Zhou et al 2009). Recently, other two family 11 xylanase genes encoding AuXyn11C and AuXyn11D were also cloned from the same strain of A. usamii.…”

Section: Introductionmentioning

confidence: 99%

Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii

Wang

Zhang

et al. 2011

Biotechnol Lett

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A full-length cDNA sequence, encoding a novel endo-1,4-β-D: -xylanase (AuXyn10A) of Aspergillus usamii, was obtained by using rapid amplification of cDNA ends (RACE) methods and cloned into the pUCm-T vector, followed by DNA sequencing. The cDNA gene, designated as Auxyn10A, is 1,235 bp in length harboring 5'- and 3'-non-encoding regions, as well as an ORF of 984 bp that encodes a 19-aa signal peptide, a 6-aa propeptide and a 302-aa mature peptide with a calculated MW of 32,756 Da. The AuXyn10A displays high similarity to the xylanases of Aspergillus niger, Aspergillus kawachii and Aspergillus niger, members of the glycoside hydrolase family 10. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/on-line programs based on the crystal structure of Penicillium simplicissimum xylanase (1B30_A) from the family 10. The complete DNA gene was cloned from the genomic DNA of A. usamii using conventional PCR and hairpin structure-mediated PCR techniques. The DNA gene is 2,255 bp in length, containing a 510 bp of 5'-flanking promoter region and a 1,745 bp of downstream fragment that consists of ten exons and nine short introns ranging from 52 to 62 bp.

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“…The leukocytes were washed two times with 0.9% normal saline (pH 7.4). Total RNA was extracted by using an RNA Extraction kit (Sangon) according to the method reported previously (12).…”

Section: Introductionmentioning

confidence: 99%

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

Wei

Tang

et al. 2012

Mol Med Report

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Abstract. hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacⅠ and transformed into Pichia pastoris GS115 (his4, Mut + ) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His + , Mut + ) with multiple hly copies. One transformant resistant to 4.0 mg/ ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol. IntroductionHuman lysozyme (EC 3.2.1.17) is a bacteriolytic enzyme that is widely distributed in a variety of tissues and body fluids (1). It hydrolyzes preferentially the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine residues that occur in the peptidoglycan cell wall structures of certain microorganisms, particularly those of Gram-positive bacteria, and therefore appears to have a role in host defense (2). The hLY comprises 130 amino acid residues and belongs to the group of lysozymes called c-type lysozymes (3). Due to its bacteriolytic activity, it is considered to be useful for medical and food uses. There is a reemergence of uses of lysozyme in various clinical trials. The addition of lysozyme to baby formulas was proposed (4), and lysozyme had also been shown to inhibit the growth of HIV-1 in vitro. Of note, the anti-HIV activity of lysozyme is independent of its muramidase activity (5). In addition, findings of previous studies have shown that it is effective in inactivating spores of Bacillus cereus in cheese by causing high hydrostatic pressure following the addition of lysozyme (6).Currently, hLY is mainly extracted from human milk and placenta, which is restricted by many factors, such as the lack of raw materials and high cost of purification. Therefore, the hen egg-white lysozyme is the most readily available form of commercial lysozyme, since it is relatively inexpensive compared...

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Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from Aspergillus usamii E001

Cited by 21 publications

References 18 publications

Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris

Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris

Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

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