Biased clique shuffling reveals stabilizing mutations in cellulase Cel7A

Renewable fuels produced from biomass‐derived sugars are receiving increasing attention. Lignocellulose‐degrading enzymes derived from fungi are attractive for saccharification of biomass because they can be produced at higher titers and at significantly less cost than those produced by bacteria or archaea. However, their properties can be suboptimal; for example, they are subject to product inhibition and are sensitive to small changes in pH. Furthermore, increased thermostability would be advantageous for saccharification as increased temperature may reduce the opportunity for microbial contamination. We have developed a mutagenesis platform to improve these properties and applied it to increase the operating temperature and thermostability of the fungal glycosyl hydrolase Cel7A. Secretion of Cel7A at titers of 26 mg/L with limited hyperglycosylation was achieved using a Saccharomyces cerevisiae strain with upregulated protein disulfide isomerase, an engineered α‐factor prepro leader, and deletion of a plasma membrane ATPase. Using biased clique shuffling (BCS) of 11 Cel7A genes, we generated a small library (469) rich in activity (86% of the chimeras were active) and identified 51 chimeras with improved thermostability, many of which contained mutations in the loop networks that extend over the enzyme's active site. This BCS library was far superior as a source of active and stable chimeras compared to an equimolar library prepared from the same 11 genes. Biotechnol. Bioeng. 2012; 109: 2710–2719. © 2012 Wiley Periodicals, Inc.     

Alleviating product inhibition in cellulase enzyme Cel7A

Enzymes that degrade cellulose into glucose are one of the most expensive components of processes for converting cellulosic biomass to fuels and chemicals. Cellulase enzyme Cel7A is the most abundant enzyme naturally employed by fungi to depolymerize cellulose, and like other cellulases is inhibited by its product, cellobiose. There is thus great economic incentive for minimizing the detrimental effects of product inhibition on Cel7A. In this work, we experimentally generated 10 previously proposed site‐directed mutant Cel7A enzymes expected to have reduced cellobiose binding energies (the majority of mutations were to alanine). We then tested their resilience to cellobiose as well as their hydrolytic activities on microcrystalline cellulose. Although every mutation tested conferred reduced product inhibition (and abolished it for some), our results confirm a trade‐off between Cel7A tolerance to cellobiose and enzymatic activity: Reduced product inhibition was accompanied by lower overall enzymatic activity on crystalline cellulose for the mutants tested. The tempering effect of mutations on inhibition was nearly constant despite relatively large differences in activities of the mutants. Our work identifies an amino acid in the Cel7A product binding site of interest for further mutational studies, and highlights both the challenge and the opportunity of enzyme engineering toward improving product tolerance in Cel7A. Biotechnol. Bioeng. 2016;113: 330–338. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

The cellulose-binding domain of cellobiohydrolase Cel7A from Trichoderma reesei is also a thermostabilizing domain

The thermostability of cellobiohydrolase I Cel7A from Trichoderma reesei was investigated using dynamic light scattering. While the whole enzyme displayed a melting point of 59 °C, the catalytic domain obtained via papain-catalyzed proteolysis was shown to denature at 51 °C and the cellulose-binding domain (with linker attached) melted at 65-66 °C. This variation in individual melting temperatures is proposed to account for the full retention of binding capacity of Cel7A at 50 °C, along with a loss of catalytic activity observed for the catalytic domain alone. Thus, the cellulose-binding domain of Cel7A acts as a thermostabilizing domain for the enzyme. The effect of reducing agents on the protein melting behavior was also investigated.     

Function of four tryptophan residues on Cel7A catalytic efficiency: Insight into cellulase screening strategy based on natural cellulose or cellulose analogs

There is a high level of conservation of tryptophans within the active site architecture of the cellulase family, whereas the function of the four tryptophans in the catalytic domain of Cel7A is unclear. By mutating four tryptophan residues in the catalytic domain of Cel7A from Penicillium piceum (PpCel7A), the binding affinity between PpCel7A and p-nitrophenol-d -cellobioside (pNPC) was reduced as determined by Michaelis–Menten constants, molecular dynamics simulations, and fluorescence spectroscopy. Furthermore, PpCel7A variants showed a reduced level of cellobiohydrolase (CBH) activity against cellulose analogs or natural cellulose. Therefore, it could be concluded four tryptophan residues in Cel7A played a critical role in substrate binding. Mutagenesis results indicated that the W390 stacking interactions at the −2 site played an essential role in facilitating substrate distortion to the −1 site. As soon as the function was altered, the mutation would inevitably affect the catalytic activity against the natural substrate. Interestingly, no clear relationship was found between the CBH activity of PpCel7A variants against pNPC and Avicel. p-Nitrophenol contains many electrophilic groups that may result in overestimation of the binding constant between tryptophan residues and pNPC in comparison with the natural substrate. Consequently, screening improved cellulase using cellulose analogs would divert attention from the target direction for lignocellulose biorefinery. Clarifying mechanism of catalytic diversity on the natural cellulose or cellulose analogs may give better insight into cellulase screening and selecting strategy.     

Hypocrea jecorina (Trichoderma reesei) Cel7A as a molecular machine: A docking study

Hypocrea jecorina (formerly Trichoderma reesei) Cel7A has a catalytic domain (CD) and a cellulose-binding domain (CBD) separated by a highly glycosylated linker. Very little is known of how the 2 domains interact to degrade crystalline cellulose. Based on the interaction energies and forces on cello-oligosaccharides computationally docked to the CD and CBD, we propose a molecular machine model, where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel. Enzyme-substrate interactions produce the forces required to pull cellulose chains from the surface and also to help the enzyme move on the cellulose chain for processive hydrolysis. The energy to generate these forces is ultimately derived from the chemical energy of glycosidic bond breakage.     

Investigation of an “alternate water supply system” in enzymatic hydrolysis in the processive endocellulase Cel7A from Rasamsonia emersonii by molecular dynamics simulation

Cel7A from Rasamsonia emersonii is one of the processive endocellulases classified under family 7 glycoside hydrolase. Molecular dynamics simulations were carried out to obtain the optimized sliding and hydrolyzing conformations, in which the reducing ends of sugar chains are located on different sites. Hydrogen bonds are investigated to clarify the interactions between protein and substrate in either conformation. Nine hydrogen bonding interactions are identified in the sliding conformation, and six similar interactions are also found correspondingly in the hydrolyzing conformation. In addition, four strong hydrophobic interactions are also determined. The domain cross‐correlation map analysis shows movement correlation of protein including autocorrelation between residues. The root mean square fluctuations analysis represents the various flexibilities of different fragment in the two conformations. Comparing the two conformations reveals the water‐supply mechanism of selective hydrolysis of cellulose in Cel7A. The mechanism can be described as follow. When the reducing end of substrate slides from the unhydrolyzing site (sliding conformation) to the hydrolyzing site (hydrolyzing conformation), His225 is pushed down and rotated, the rotation leads to the movement of Glu209 with the interstrand hydrogen bonding in β‐sheet. It further makes Asp211 close to the hydrolysis center and provides a water molecule bounding on its carboxyl in the previous unhydrolyzing site. After the hydrolysis takes place and the product is excluded from the enzyme, the Asp211 comes back to its initial position. In summary, Asp211 acts as an elevator to transport outer water molecules into the hydrolysis site for every other glycosidic bond.     

Use of the cell wall protein Pir4 as a fusion partner for the expression of Bacillus sp. BP-7 xylanase A in Saccharomyces cerevisiae

Xylanase A from Bacillus sp. BP7, an enzyme with potential applications in biotechnology, was used to test Pir4, a disulfide bound cell wall protein, as a fusion partner for the expression of recombinant proteins in standard or glycosylation-deficient mnn9 strains of Saccharomyces cerevisiae. Five different constructions were carried out, inserting in-frame the coding sequence of xynA gene in that of PIR4, with or without the loss of specific regions of PIR4. Targeting of the xylanase fusion protein to the cell wall was achieved in two of the five constructions, while secretion to the growth medium was the fate of the gene product of one of the constructions. In all three cases localization of the xylanase fusion proteins was confirmed both by Western blot and detection with Pir-specific antibodies and by xylanase activity determination. The cell wall-targeted fusion proteins could be extracted by reducing agents, showing that the inclusion of a recombinant protein of moderate size does not affect the way Pir4 is attached to the cell wall. Also, the construction that leads to the secretion of the fusion protein permitted us to identify a region of Pir4 responsible for cell wall retention. In summary, we have developed a Pir4-based system that allows selective targeting of an active recombinant enzyme to the cell wall or the growth medium. This system may be of general application for the expression of heterologous proteins in S. cerevisiae for surface display and secretion.     

人骨形态发生蛋白7在中国仓鼠卵巢细胞中的表达研究

目的:构建重组人骨形态发生蛋白-7(rhBMP7)表达质粒,并研究其在中国仓鼠卵巢细胞中的表达。方法:将hBMP7重组表达质粒电转到中国仓鼠卵巢细胞(CHO)中,并用DOT-BLOT和ELISA方法分析检测rhBMP7在重组CHO细胞中的表达。结果:hBMP7 cDNA整合到CHO细胞基因组中并被转录。点杂交和ELISA检测证实rhBMP7在CHO细胞中得到表达。结论:hBMP7成功在CHO表达系统中得到表达。     

A graphene screen-printed carbon electrode for real-time measurements of unoccupied active sites in a cellulase

Cellulases hydrolyze cellulose to soluble sugars and this process is utilized in sustainable industries based on lignocellulosic feedstock. Better analytical tools will be necessary to understand basic cellulase mechanisms, and hence deliver rational improvements of the industrial process. In this work we describe a new electrochemical approach to the quantification of the populations of enzyme that are respectively free in the aqueous bulk, adsorbed to the insoluble substrate with an unoccupied active site or threaded with the cellulose strand in the active tunnel. Distinction of these three states appears essential to the identification of the rate-limiting step. The method is based on disposable graphene-modified screen-printed carbon electrodes, and we show how the temporal development in the concentrations of the three enzyme forms can be derived from a combination of the electrochemical data and adsorption measurements. The approach was tested for the cellobiohydrolase Cel7A from Hypocrea jecorina acting on microcrystalline cellulose, and it was found that the threaded enzyme form dominates for this system while adsorbed enzyme with an unoccupied active site constitutes less than 5% of the population.     

Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity

To investigate the function of N-glycosylation of Cel5A (endoglucanase II) from Hypocrea jecorina, two N-glycosylation site deletion Cel5A mutants (rN124D and rN124H) were expressed in Saccharomyces cerevisiae. The weights of these recombinant mutants were 54 kDa, which were lower than that of rCel5A. This result was expected to be attributed to deglycosylation. The enzyme activity of rN124H was greatly reduced to 60.6% compared with rCel5A, whereas rN124D showed slightly lower activity (10%) than that of rCel5A. rN124D and rN124H showed different thermal stabilities compared with the glycosylated rCel5A, especially at lower pH value. Thermal stabilities were reduced and improved for rN124D and rN124H, respectively. Circular dichroism spectroscopy showed that the modification of secondary structure by mutation may be the reason for the change in enzymatic activity and thermal stability. [BMB Reports 2014; 47(5): 256-261]     

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.     

Purification of recombinant human phosphodiesterase 7A expressed in Dictyostelium discoideum

Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis–Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.     

Expression of recombinant HA1 protein for specific detection of influenza A/H1N1/2009 antibodies in human serum

The hemagglutinin genes (HA1 subunit) from human and animal 2009 pandemic H1N1 virus isolates were expressed with a baculovirus vector. Recombinant HA1 (rHA1) protein‐based ELISA was evaluated for detection of specific influenza A(H1N1)pdm09 antibodies in serum samples from vaccinated humans. It was found that rHA1 ELISA consistently differentiated between antibodies recognizing the seasonal influenza H1N1 and pdm09 viruses, with a concordance of 94% as compared to the hemagglutination inhibition test. This study suggests the utility of rHA1 ELISA in serosurveillance.     

The role of BRD7 in embryo development and glucose metabolism

Bromodomain‐containing protein 7 (BRD7) is a member of bromodomain‐containing protein family and its function has been implicated in several diseases. We have previously shown that BRD7 plays a role in metabolic processes. However, the effect of BRD7 deficiency in glucose metabolism and its role in in vivo have not been fully revealed. Here, we report the essential role of BRD7 during embryo development. Mice homozygous for BRD7 led to embryonic lethality at mid‐gestation. Homozygous BRD7 knockout (KO) mice showed retardation in development, and eventually all BRD7 KO embryos died in utero prior to E16.5. Partial knockdown of Brd7 gene displayed mild changes in glucose metabolism.