Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation

A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield β-mannanase was obtained through a series of screening. The β-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32 °C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,501 U/g dried koji) of the parent strain LW-1. The purified E-30 β-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS–PAGE. Its optimal pH and temperature were 3.5 and 65 °C, respectively. It was highly stable at a pH range of 3.5–7.0 and at a temperature of 60 °C and below. The kinetic parameters K_m and V_max, toward locust bean gum and at pH 4.8 and 50 °C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The β-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag⁺ and Hg²⁺. In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 β-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50 °C, β-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.     

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

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 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.     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> E001

The full-length cDNA gene that encodes an acidophilic endo-1,4-β- xylanase XynI of Aspergillus usamii E001 was amplified by rapid amplification of cDNA 3′ and 5′ ends (RACE) using the total RNA as template and then cloned onto the pUCm-T vector, followed by sequencing. The cloned cDNA is 881 bp in length including 5′ and 3′ non-encoding regions, as well as a 678 bp of open reading frame (ORF) which encodes an E001 XynI of 188 amino acid residues together with a signal peptide of 37 amino acid residues. The homologies of E001 XynI with xylanases of Aspergillus niger, Aspergillus kawachii, Emericella nidulans and Penicillium funiculosum are 97.8, 92.0, 74.6 and 60.5%, respectively. From a BLAST search result, we concluded that E001 XynI belongs to the glycoside hydrolase family 11. Its three-dimensional structure was predicted using programs based on that of the P. funiculosum xylanase (1TE1B) from the family 11. In addition, the complete DNA gene xynI encoding E001 XynI was cloned from the genomic DNA of A. usamii E001 by conventional PCR and ligation-mediated PCR amplification. The cloned xynI is 1,206 bp in length, composed of a promoter region, a 68 bp of intron and two exons when compared with the cDNA of E001 XynI.     

N端二硫键对11家族木聚糖酶热稳定性的影响

以来源于米曲霉Aspergillus oryzae的11家族常温木聚糖酶AoXyn11A为母本,将其N端替换成同一家族耐热木聚糖酶EvXyn11TS的对应片段,构建出耐热杂合木聚糖酶AEx11A。将AoXyn11A和AEx11A基因分别在毕赤酵母GS115中进行表达并分析比较温度对表达产物酶活性的影响。结果表明,AEx11A的最适温度Topt为75℃,在70℃的半衰期t1/270为197 min,较AoXyn11A(Topt=50℃,t1/270=1.0 min)显著提高。通过对AEx11A结构的同源建模及其与AoXyn11A结构的比对,发现在AEx11A的N端引入了一个二硫键(Cys5–Cys32)。利用定点突变法将其5位的半胱氨酸突变为苏氨酸(C5T),去除该二硫键,以探讨其对AEx11A热稳定性的影响。分析表明,突变酶(AEx11AC5T)的Topt由突变前的75℃降为60℃,其t1/270和t1/280也分别由197 min和25 min缩短为3.0 min和1.0 min。     

人防御素分子生物学研究进展及其应用

人防御素(human defensins)是一类内源性阳离子抗微生物肽,在天然免疫防御中起重要作用,也能增强继承性免疫应答.它具有强烈的广谱抗微生物活性,并且不易对微生物产生耐药性.对人防御素的分子生物学特性方面的研究进展作一综述,重点在于比较和探讨其结构的异同,并对其应用和发展的前景进行展望.     

Cloning and bioinformatics analysis of a novel acidophilic β-mannanase gene,Auman5A,from Aspergillus usamii YL-01-78

The full-length cDNA sequence, which encodes a novel acidophilic β-mannanase (abbreviated as AuMan5A) of Aspergillus usamii YL-01-78, was amplified by 3′ and 5′ rapid amplification of cDNA ends (RACE) using the total RNA as template. The cDNA sequence is 1,427 bp in length, including 5′ and 3′ non-coding regions and an open reading frame (ORF). The ORF encodes a 21-aa signal peptide, a 17-aa propeptide, and a 345-aa mature peptide (AuMan5A) with the calculated M.W. of 37,614 Da and pI of 4.09 and two putative N-glycosylation sites. Online analysis of amino acid sequence homology demonstrated that the AuMan5A belongs to the glycoside hydrolase (GH) family 5. Its three-dimensional structure was predicted using Pred3D Web Server 1.0 based on the crystal structure of the T. reesei RutC-30 β-mannanase (1QNO) from the GH family 5. Furthermore, the complete DNA sequence encoding the AuMan5A, designated as Auman5A, was cloned from the genomic DNA of A. usamii YL-01-78 by the conventional PCR and pUCm-T vector-mediated PCR techniques. The cloned Auman5A is 2,168 bp in length, harboring 5′ and 3′ flanking regulatory regions and the full-length cDNA sequence in which two short introns with 63 and 60 bp are inserted, respectively.     

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

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.     

新型扁桃酸消旋酶的基因挖掘和性能表征

消旋酶是实现手性化合物去消旋化制备光学纯化学品的重要工具,来源于恶臭假单胞菌的扁桃酸消旋酶(MR),是目前唯一可以催化两种构型扁桃酸互相转换的消旋酶。通过基因组数据挖掘获得了9个新的扁桃酸消旋酶基因及活性蛋白,其中来源于放射性土壤杆菌Agrobacterium radiobacter的Ar MR酶对扁桃酸和邻氯扁桃酸具有较高的催化活力,而且该酶的异源表达水平也较理想。ArMR催化扁桃酸消旋反应的最适温度为50℃,最适pH为7.8。该酶在30℃、40℃和50℃下的半衰期分别为70.7、7.2、0.17 h。ArMR对(R)-和(S)-扁桃酸的K_M值分别为1.44 mmol/L和0.81 mmol/L,k_(cat)值分别为410 s~(–1)和218 s~(–1);对(R)-和(S)-邻氯扁桃酸的KM值分别为6.48 mmol/L和6.37 mmol/L,而k_(cat)值为0.22 s~(–1)和0.23 s~(–1)。Mg~(2+)和Mn~(2+)对该酶的活力有促进作用,而Zn~(2+)使其完全失活。新型扁桃酸消旋酶的发现和表征为今后此类酶的深入研究和开发提供了更多资源和数据参考。     

Directed modification of the Aspergillus usamii β-mannanase to improve its substrate affinity by in silico design and site-directed mutagenesis

β-Mannanases (EC 3.2.1.78) can catalyze the cleavage of internal β-1,4-d-mannosidic linkages of mannan backbones, and they have found applications in food, feed, pulp and paper, oil, pharmaceutical and textile industries. Suitable amino acid substitution can promote access to the substrate-binding groove and maintain the substrate therein, which probably improves the substrate affinity and, thus, increases catalytic efficiency of the enzyme. In this study, to improve the substrate affinity of AuMan5A, a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, had its directed modification conducted by in silico design, and followed by site-directed mutagenesis. The mutant genes, Auman5A ^Y111F and Auman5A ^Y115F, were constructed by megaprimer PCR, respectively. Then, Auman5A and its mutant genes were expressed in Pichia pastoris GS115 successfully. The specific activities of purified recombinant β-mannanases (reAuMan5A, reAuMan5A^Y111F and reAuMan5A^Y115F) towards locust bean gum were 152.5, 199.6 and 218.9 U mg^?1, respectively. The two mutants were found to be similar to reAuMan5A regarding temperature and pH characteristics. Nevertheless, the K _m values of reAuMan5A^Y111F and reAuMan5A^Y115F, towards guar gum, decreased to 2.95 ± 0.22 and 2.39 ± 0.33 mg ml^?1 from 4.49 ± 0.07 mg ml^?1 of reAuMan5A, which would make reAuMan5A^Y111F and reAuMan5A^Y115F promising candidates for industrial processes. Structural analysis showed that the two mutants increased their affinity by decreasing the steric conflicts with those more complicated substrates. The results suggested that subtle conformational modification in the substrate-binding groove could substantially alter the substrate affinity of AuMan5A. This study laid a solid foundation for the directed modification of substrate affinities of β-mannanases and other enzymes.