Jianfang Li

Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris

A cDNA fragment encoding a mature neutral endoglucanase with 366 amino acids was cloned from Volvariella volvacea WX32. Online analysis of amino acid sequence homology showed that the endoglucanase, designated as NCel5A, belongs to glycoside hydrolase family 5. The recombinant plasmid, pPIC9K-ncel5A, was transformed into Pichia pastoris GS115 by electroporation. Screening of multiple copies of the gene ncel5A in transformants was performed on YPD plates containing geneticin G418. One transformant expressing the highest recombinant NCel5A (rNCel5A) activity, numbered as GSNCel4-3, was chosen for optimizing expression conditions. In optimized conditions, the expressed rNCel5A activity was up to 4612 U/ml. SDS-PAGE and enzyme activity assays demonstrated that the rNCel5A, a glycosylated protein with an M.W. of about 42 kDa, was extracellularly expressed in P. pastoris. The rNCel5A showed the highest activity at pH 7.5 and 55°C and was stable at a broad pH range of 6.0-9.0 and at a temperature of 55°C or below.

Cloning and Functional Expression of an Acidophilic β-Mannanase Gene (Anman5A) from Aspergillus niger LW-1 in Pichia pastoris

A cDNA fragment of the Anman5A, a gene that encodes an acidophilic β-mannanase of Aspergillus niger LW-1 (abbreviated as AnMan5A), was cloned and functionally expressed in Pichia pastoris . Homology alignment of amino acid sequences verified that the AnMan5A belongs to the glycoside hydrolase (GH) family 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay demonstrated that the recombinant AnMan5A (reAnMan5A), a N-glycosylated protein with an apparent molecular weight of 52.0 kDa, was secreted into the medium. The highest reAnMan5A activity expressed by one P. pastoris transformant, labeled as GSAnMan4-12, reached 29.0 units/mL. The purified reAnMan5A displayed the highest activity at pH 3.5 and 70 °C. It was stable at a pH range of 3.0-7.0 and at a temperature of 60 °C or below. Its activity was not significantly affected by an array of metal ions and ethylenediaminetetraacetic acid (EDTA). The K(m) and V(max) of the reAnMan5A, toward locust bean gum, were 1.10 mg/mL and 266.7 units/mg, respectively.

Determinants for the improved thermostability of a mesophilic family 11 xylanase predicted by computational methods

BackgroundXylanases have drawn much attention owing to possessing great potential in various industrial applications. However, the applicability of xylanases, exemplified by the production of bioethanol and xylooligosaccharides (XOSs), was bottlenecked by their low stabilities at higher temperatures. The main purpose of this work was to improve the thermostability of AuXyn11A, a mesophilic glycoside hydrolase (GH) family 11 xylanase from Aspergillus usamii E001, by N-terminus replacement.ResultsA hybrid xylanase with high thermostability, named AEXynM, was predicted by computational methods, and constructed by substituting the N-terminal 33 amino acids of AuXyn11A with the corresponding 38 ones of Ev Xyn11TS, a hyperthermostable family 11 xylanase. Two AuXyn11A- and AEXynM-encoding genes, Auxyn11A and AExynM, were then highly expressed in Pichia pastoris GS115, respectively. The specific activities of two recombinant xylanases (reAuXyn11A and reAEXynM) were 10,437 and 9,529 U mg-1. The temperature optimum and stability of reAEXynM reached 70 and 75°C, respectively, much higher than those (50 and 45°C) of reAuXyn11A. The melting temperature (Tm) of reAEXynM, measured using the Protein Thermal Shift (PTS) method, increased by 34.0°C as compared with that of reAuXyn11A. Analyzed by HPLC, xylobiose and xylotriose as the major hydrolytic products were excised from corncob xylan by reAEXynM. Additionally, three single mutant genes from AExynM (AExynMC5T, AExynMP9S, and AExynMH14N) were constructed by site-directed mutagenesis as designed theoretically, and expressed in P. pastoris GS115, respectively. The thermostabilities of three recombinant mutants clearly decreased as compared with that of reAEXynM, which demonstrated that the three amino acids (Cys5, Pro9, and His14) in the replaced N-terminus contributed mainly to the high thermostability of AEXynM.ConclusionsThis work highly enhanced the thermostability of AuXyn11A by N-terminus replacement, and further verified, by site-directed mutagenesis, that Cys5, Pro9, and His14 contributed mainly to the improved thermostability. It will provide an effective strategy for improving the thermostabilities of other enzymes.

Engineering hyperthermostability into a mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N-terminus substitution.

A mesophilic xylanase from Aspergillus oryzae CICC40186 (abbreviated to AoXyn11A) belongs to glycoside hydrolase family 11. The thermostability of AoXyn11A was significantly improved by substituting its N‐terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11TS. The suitable N‐terminus of AoXyn11A to be replaced was selected by the comparison of B‐factors between AoXyn11A and EvXyn11TS, which were generated and calculated after a 15 ns molecular dynamic (MD) simulation process. Then, the predicted hybrid xylanase (designated AEx11A) was modeled, and subjected to a 2 ns MD simulation process for calculating its total energy value. The N‐terminus substitution was confirmed by comparing the total energy value of AEx11A with that of AoXyn11A. Based on the in silico design, the AEx11A was constructed and expressed in Pichia pastoris GS115. After 72 h of methanol induction, the recombinant AEx11A (reAEx11A) activity reached 82.2 U/mL. The apparent temperature optimum of reAEx11A was 80°C, much higher than that of reAoXyn11A. Its half‐life was 197‐fold longer than that of reAoXyn11A at 70°C. Compared with reAoXyn11A, the reAEx11A displayed a slight alteration in Km but a decrease in Vmax. Biotechnol. Bioeng. 2013; 110: 1028–1038.

Fusing a carbohydrate-binding module into the Aspergillus usamii β-mannanase to improve its thermostability and cellulose-binding capacity by in silico design.

The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 β-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion β-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K m and a slight alteration in V max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.

Enhanced thermostability of a mesophilic xylanase by N-terminal replacement designed by molecular dynamics simulation.

BACKGROUND Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement. RESULTS The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11(TS) , a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn11⁵⁷, was predicted by molecular dynamics (MD) simulation. An NhXyn11⁵⁷-encoding gene, Nhxyn11⁵⁷, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn11⁵⁷ (re-NhXyn11⁵⁷) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn11⁵⁷ were 5.5 and at a range of 4.0-8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²⁺ and Ag⁺. CONCLUSION This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn11⁵⁷ a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes.

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 (18,50 1U/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 Hg(2+). 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.

Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii

The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (k cat/K m) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.

A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity

A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5–9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg2+ and Fe3+, but not significantly affected by EDTA or the other metal ions such as Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Mn2+, Cu2+, and Fe2+. PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe256 plays an important role in conferring the substrate selectivity. Phe256 projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the Oγ of Ser145, which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries.

Optimized expression, purification and characterization of a family 11 xylanase (AuXyn11A) from Aspergillus usamii E001 in Pichia pastoris

BACKGROUND Xylanases have attracted much attention because of their potential applications. Unfortunately, the commercialization of xylanases is limited by their low catalytic activities. The aim of this study was to improve the activity of a xylanase by optimization of the expression conditions and to investigate its characterization. RESULTS The activity of recombinant AuXyn11A (reAuXyn11A), a family 11 xylanase from Aspergillus usamii E001 expressed in Pichia pastoris GS115, reached 912.6 U mL⁻¹ under the optimized conditions, which was 2.14 times as high as that expressed using the standard protocol. After the endogenous 18-aa propeptide had been processed in P. pastoris, reAuXyn11A (188-aa mature peptide) was secreted and purified with a specific activity of 22 714 U mg⁻¹. It displayed maximum activity at pH 5 and 50 °C and was stable in the pH range 4-8 and at a temperature of 45 °C or below. Its activity was not significantly affected by most metal ions and EDTA. Xylooligosaccharides ranging from xylobiose (X2) to xylohexaose (X6) were produced from insoluble corncob xylan by reAuXyn11A. CONCLUSION Its high specific activity and good enzymatic properties suggest that reAuXyn11A is a potential candidate for applications in industrial processes.