Key Laboratory of Enzyme Engineering of Agricultural Microbiology

Zhengzhou, China

Key Laboratory of Enzyme Engineering of Agricultural Microbiology

Zhengzhou, China

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Su L.,Henan Agricultural University | Yang L.,Henan Agricultural University | Huang S.,CAS Qingdao Institute of Bioenergy and Bioprocess Technology | Li Y.,Henan Agricultural University | And 8 more authors.
Applied Biochemistry and Biotechnology | Year: 2016

Termites are well recognized for their thriving on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling microbiota; however, the effects of diet changes on termite gut microbiota are poorly understood, especially for the lower termites. In this study, we employed high-throughput 454 pyrosequencing of 16S V1–V3 amplicons to compare gut microbiotas of Tsaitermes ampliceps fed with lignin-rich and lignin-poor cellulose diets after a 2-week-feeding period. As a result, the majority of bacterial taxa were shared across the treatments with different diets, but their relative abundances were modified. In particular, the relative abundance was reduced for Spirochaetes and it was increased for Proteobacteria and Bacteroides by feeding the lignin-poor diet. The evenness of gut microbiota exhibited a significant difference in response to the diet type (filter paper diets < corn stover diets < wood diets), while their richness was constant, which may be related to the lower recalcitrance of this biomass to degradation. These results have important implications for sampling and analysis strategies to probe the lignocellulose degradation features of termite gut microbiota and suggest that the dietary lignocellulose composition could cause shifting rapidly in the termite gut microbiota. © 2016 Springer Science+Business Media New York


Wang F.,Henan Agricultural University | Wang F.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Yin S.,Henan Agricultural University | Yin S.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | And 6 more authors.
Nongye Gongcheng Xuebao/Transactions of the Chinese Society of Agricultural Engineering | Year: 2012

To enhance the pretreatment effect for production of cellulose ethanol, the influences of water immersion and CaO treatments before steam explosion on the structure destruction of corn stalks and enzymatic saccharification were studied. Field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were involved in this study. The results showed that compared to the control (corn stalks pretreated with steam explosion), corn stalk immersed with 30% water (mass ratio of water to corn stalk was 30: 100) for 5 days, or treated with 2% CaO (mass ratio of CaO to corn stalk is 2: 100) for 3days, or treated with 2% CaO and 30% water for 1 day before steam explosion enhanced the lignin degradation rate from 20.7% to 27.8%, 35.1% and 30.9%, respectively; the concentrations of reducing sugars in the three pretreatments were 3.81, 3.59 and 3.46 g/100 mL respectively; and the sugar yields in the three pretreatments were 42.2%, 39.8% and 38.3% and increased by 23.7%, 16.6% and 12.3% respectively compared with the control. Pretreatment with 30% water immersion for 5 days and 2% CaO for 3 days intensified the destruction of surface structures and the degradation of lignin, the relative crystallinity of which increased by 47.0% and 54.5% respectively compared to the control (42.6%). Pretreatment with 30% water or 2% CaO exhibited high efficiency and sugar yields. It is benefit for the promotion of this technology with low-price and non-contaminating reagents.


Liu L.,Henan Agricultural University | Chen L.,Henan Agricultural University | Chen L.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Tian H.,Henan Agricultural University | And 2 more authors.
Process Biochemistry | Year: 2012

Signal peptide (SP) prediction is used, but not known correct or not. The Aspergillus niger GH10 xylanase (XynB) SP is predicted to be 1-19 (Met 1-Ser19) residue, differing from the Penicillium simplicissimum xylanase 1-25 residue SP. To determine the real SP, two types of XynB, XynΔ19 and XynΔ25, were constructed by respectively deleting the 19 (Met1-Ser19) or 25 (Met1-Arg 25) residues. The XynΔ25 had 10 °C higher Topt and 21.6-times longer thermostability than the XynΔ19 (46 vs. 36 °C and 47.6 vs. 2.2 m). When the kinetics were assayed, the XynΔ25 had ∼2.1-times higher Vmax and higher binding-affinity for xylan than the XynΔ19 (53.7 vs. 25.3 μmol/ml/m and 2.43 vs. 2.96 mg/ml). Thus, the XynB real SP is the 1-25 and not predicted 1-19 residues. The extra six N-terminal residues (Glu20Pro21Ile22Glu 23Pro24Arg25) drastically interfered with the XynΔ19 thermal activity, thermostability, and catalytic efficiency. © 2012 Elsevier Ltd.


Xu W.,Henan Agricultural University | Liu Y.,Henan Agricultural University | Ye Y.,Henan Agricultural University | Liu M.,Henan Agricultural University | And 4 more authors.
Biotechnology Letters | Year: 2016

Objective: The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. Results: A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. Conclusion: C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location. © 2016 Springer Science+Business Media Dordrecht


Chai R.,Henan Agricultural University | Chai R.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Zhang G.,Henan Agricultural University | Zhang G.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | And 8 more authors.
Fungal Biology | Year: 2013

Liposome-mediated transformation is common for cells with no cell wall, but has very limited usage in cells with walls, such as bacteria, fungi, and plants. In this study, we developed a procedure to introduce DNA into mycelium of filamentous fungi, Rhizopus nigricans LH 21 and Pleurotus ostreatus TD 300, by liposome-mediation but with no protoplast preparation. The DNA was transformed into R. nigricans via plasmid pEGFP-C1 and into P. ostreatus via 7.2kb linear DNA. The mycelia were ground in 0.6M mannitol without any grinding aids or glass powder for 15min to make mycelial fragments suspension; the suspension was mixed with a mixture of the DNA and Lipofectamine 2000, and placed on ice for 30min; 100μL of the transformation solution was plated on potato dextrose agar (PDA) plate and cultivated at 28°C for transformant screening. The plasmid and the linear DNA were confirmed to be integrated into the host chromosome, proving the success of transformation. The transformation efficiencies were similar to those of electroporation-mediated protoplast transformation (EMPT) of R. nigricans or PEG/CaCl2-mediated protoplast transformation (PMT) of P. ostreatus, respectively. The results showed that our procedure was effective, fast, and simple transformation method for filamentous fungi. © 2013.


Chen S.,Henan Agricultural University | Chen S.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Qiu C.,Henan Agricultural University | Qiu C.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | And 12 more authors.
Fungal Ecology | Year: 2013

The mechanism of casing soil stimulating the primordium formation of Agaricus bisporus is not well understood so far. Our results showed that 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (AcdS)-producing bacteria were abundant in the casing soil of A. bisporus and accounted for up to 20 % of total culturable bacteria. A. bisporus produced ACC and ethylene. The supplement of methionine increased the ACC concentrations within the hyphae, and aminooxyacetic acid displayed an opposite effect. Methionine and ACC promoted the ethylene production while CoCl2 suppressed the production. The AcdS-producing bacterial strain Pseudomonas putida UW4 co-cultured with A. bisporus could attach to hyphae, stimulate the hyphal growth, and reduce the ethylene production of A. bisporus. Added in sterilized casing soil, it induced the primordium formation of A. bisporus. In comparison, its AcdS-deficient mutant UW4-AcdS- displayed the opposite effects. These results indicated that the inhibitor to the primordium formation of A. bisporus was ethylene; the AcdS-producing bacteria within the casing layer cleaved ACC, lowered the ethylene level in mushroom hyphae, and relieved the inhibition of ethylene. This is a new model of the synergism between bacteria and fungi. © 2012 Elsevier Ltd and The British Mycological Society.


Chai R.,Henan Agricultural University | Chai R.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Qiu C.,Henan Agricultural University | Qiu C.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | And 10 more authors.
PLoS ONE | Year: 2013

Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi. © 2013 Chai et al.


Song A.,Henan Agricultural University | Song A.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Feng X.,Henan Agricultural University | Feng X.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | And 6 more authors.
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2013

To evaluate the ability of microbial mix-culture fermenting syngas into ethanol, we studied the microbial mix-cultures A-fm 4, G-fm 4, Lp-fm 4 and B-fm 4 obtained by enrichment and compared with Clostridium autoethanogenum DSM10061 with 10% and 25% inoculation size. The results show that, with 10% inoculation size, the ethanol production of A-fm 4, G-fm 4, Lp-fm 4, B-fm 4 and C. autoethanogenum were 349.15, 232.16, 104.25, 79.90 and 26.99 mg/L respectively. With 25% inoculation size, the ethanol production were 485.81, 472.73, 348.58, 272.52 and 242.15 mg/L respectively. Higher inoculation size will increase the production of ethanol. The tested mix-culture exhibited a significant yield advantage compared with the maximum production of C. autoethanogenum reported in the literature (259.64 mg/L). This research provided a practical method to improve ethanol production from syngas. © 2013 Chin J Biotech, All rights reserved.


Su L.-J.,Henan Agricultural University | Liu Y.-Q.,Henan Agricultural University | Liu H.,Henan Agricultural University | Wang Y.,Henan Agricultural University | And 6 more authors.
Genetics and Molecular Research | Year: 2015

Tsaitermes ampliceps (lower termites) and Mironasutitermes shangchengensis (higher termites) are highly eusocial insects that thrive on recalcitrant lignocellulosic diets through nutritional symbioses with gut dwelling prokaryotes and eukaryotes. We used denaturing gradient gel electrophoresis and a 16S rRNA clone library to investigate i) how microbial communities adapt to lignocellulosic diets with different cellulose and lignin content, ii) the differences in the dominant gut microbial communities of the 2 types of termites. The results indicated that gut microbiota composition in T. ampliceps was profoundly affected by 2-week diet shifts. Comparison of these changes indicated that Bacteroidetes and Spirochaetes act in cellulose degradation, while Firmicutes were responsible for lignin degradation. Additionally, Proteobacteria consistently participated in energy production and balanced the gut environment. Bacteroidetes may function without hindgut protozoans in higher termites. The diversity of enteric microorganisms in M. shangchengensis was higher than that in T. ampliceps, possibly because of the more complicated survival mechanisms of higher termites. © FUNPEC-RP.


Liu L.,Henan Agricultural University | Sun X.,Henan Agricultural University | Yan P.,Henan Agricultural University | Wang L.,Henan Agricultural University | And 3 more authors.
PLoS ONE | Year: 2012

The Aspergillus niger xylanase (Xyn) was used as a model to investigate impacts of un-structured residues on GH11 family enzyme, because the β-jelly roll structure has five residues (Ser1Ala2Gly3Ile4Asn5) at N-terminus and two residues (Ser183Ser184) at C-terminus that do not form to helix or strand. The N- or/and C-terminal residues were respectively deleted to construct three mutants. The optimal temperatures of XynΔN, XynΔC, and XynΔNC were 46, 50, and 46°C, and the thermostabilities were 15.7, 73.9, 15.5 min at 50°C, respectively, compared to 48°C and 33.9 min for the Xyn. After kinetic analysis, the substrate-binding affinities for birch-wood xylan decreased in the order XynΔC>Xyn>XynΔNC>XynΔN, while the Kcat values increased in the order XynΔC

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