Thermophilic ß-mannanases from bacteria: production, resources, structural features and bioengineering strategies.

ß-mannanases are pivotal enzymes that cleave the mannan backbone to release short chain mannooligosaccharides, which have tremendous biotechnological applications including food/feed, prebiotics and biofuel production. Due to the high temperature conditions in many industrial applications, thermophilic mannanases seem to have great potential to overcome the thermal impediments. Thus, structural analysis of thermostable ß-mannanases is extremely important, as it could open up new avenues for genetic engineering, and protein engineering of these enzymes with enhanced properties and catalytic efficiencies. Under this scope, the present review provides a state-of-the-art discussion on the thermophilic ß-mannanases from bacterial origin, their production, engineering and structural characterization. It covers broad insights into various molecular biology techniques such as gene mutagenesis, heterologous gene expression, and protein engineering, that are employed to improve the catalytic efficiency and thermostability of bacterial mannanases for potential industrial applications. Further, the bottlenecks associated with mannanase production and process optimization are also discussed. Finally, future research related to bioengineering of mannanases with novel protein expression systems for commercial applications are also elaborated.

Characterization of Thermotoga neapolitana Alcohol Dehydrogenases in the Ethanol Fermentation Pathway.

Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pHopt of 8.5 and Topt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pHopt of 7.0 and Topt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively.

Modifying Thermostability and Reusability of Hyperthermophilic Mannanase by Immobilization on Glutaraldehyde Cross-Linked Chitosan Beads.

In the current study, the purified ß-mannanase (Man/Cel5B) from Thermotoga maritima was immobilized on glutaraldehyde cross-linked chitosan beads. The immobilization of Man/Cel5B on chitosan beads was confirmed by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. After immobilization, the protein loading efficiency and immobilization yield were found to be 73.3% and 71.8%, respectively. The optimum pH for both free and immobilized enzymes was found to be pH 5.5. However, the optimum temperature of immobilized Man/Cel5B increased by 10 °C, from 85 °C (free Man/Cel5B) to 95 °C (Immobilized). The half-life of free and immobilized enzymes was found to be 7 h and 9 h, respectively, at 85 °C owing to the higher thermostability of immobilized Man/Cel5B. The increase in thermostability was also demonstrated by an increase in the energy of deactivation (209 kJmol-1) for immobilized enzyme compared to its native form (92 kJmol-1), at 85 °C. Furthermore, the immobilized Man/Cel5B displayed good operational stability as it retained 54% of its original activity after 15 repeated catalytic reactions concerning its free form.

Man/Cel5B, a Bifunctional Enzyme Having the Highest Mannanase Activity in the Hyperthermic Environment.

Thermotoga maritima (Tma) contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in Tma genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5-8. The K m and V max of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.

A novel bifunctional aldehyde/alcohol dehydrogenase catalyzing reduction of acetyl-CoA to ethanol at temperatures up to 95 °C.

Hyperthermophilic Thermotoga spp. are excellent candidates for the biosynthesis of cellulosic ethanol producing strains because they can grow optimally at 80 °C with ability to degrade and utilize cellulosic biomass. In T. neapolitana (Tne), a putative iron-containing alcohol dehydrogenase was, for the first time, revealed to be a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that catalyzed both reactions from acetyl-coenzyme A (ac-CoA) to acetaldehyde (ac-ald), and from ac-ald to ethanol, while the putative aldehyde dehydrogenase (Aldh) exhibited only CoA-independent activity that oxidizes ac-ald to acetic acid. The biochemical properties of Fe-AAdh were characterized, and bioinformatics were analyzed. Fe-AAdh exhibited the highest activities for the reductions of ac-CoA and acetaldehyde at 80-85 °C, pH 7.54, and had a 1-h half-life at about 92 °C. The Fe-AAdh gene is highly conserved in Thermotoga spp., Pyrococcus furiosus and Thermococcus kodakarensis, indicating the existence of a fermentation pathway from ac-CoA to ethanol via acetaldehyde as the intermediate in hyperthermophiles.

Green synthesis of Ag and Pd nanoparticles for water pollutants treatment.

Silver (Ag) and palladium (Pd) nanoparticles were synthesized via a green synthesis route, which was mediated with the extract of Daucus carota leaves. The morphological, crystalline and structural nature of the synthesized nanoparticles was characterized by UV-Vis spectrophotometer, and TEM, XRD and FT-IR analyses. High antibacterial activities of the prepared Ag and Pd nanoparticles were observed towards different water-borne pathogens of Klebsiella pneumonia, Vibrio cholera and Escherichia coli. The catalytic efficiency of the prepared nanoparticles for the removal of rhodamine 6G (Rh-6G) dye was also evaluated. Nearly 98% of the Rh-6G dye was decolorized by the synthesized Pd nanoparticles within 2 min, and the synthesized Ag nanoparticles took 30 min for 89.4% decolorization. This work provided greener nanocatalysts for pollutant treatment and demonstrated the power of green biosynthesis for metallic nanoparticles.

Efficient xylan-to-sugar biotransformation using an engineered xylanase in hyperthermic environment.

Hyperthermophilic xylanases play a critical role in bioconversion from xylan to sugar in the process of biomass utilization. The discovery of new or improvement of existing xylanases based on directed evolution is expected to be an effective approach to meet the increasing demand of thermostable xylanases. In this work, a xylanase B gene (CTN_0623) from Thermotoga neapolitana (Tne) was cloned and xylanase B (herein named TnexlnB) was solubly expressed in E. coli with a high amount using a heat shock vector pHsh. TnexlnB showed the highest endo-ß-1,4-xylan hydrolase activity at 75 °C and pH 6.0. Additionally, 1 mM Mg2+, Ba2+ and Ca2+ improved the activity of TnexlnB by 31%, 37%, and 53%, respectively. The optimal temperature reached 85 °C by site-directed mutation at the last three helices of TnexlnB. Km and Vmax towards birchwood xylan were determined for both wide type and the best mutant, as follow: 1.09 mg/mL, 191.76 U/mg and 0.29 mg/mL, 376.42 U/mg, respectively. Further characterization highlighted good thermal stability (>80% of enzymatic activity after 1 h at 90 °C) for the best mutant, which made this enzyme suitable for biomass degradation at high temperature.

Protein engineering to enhance keratinolytic protease activity and excretion in Escherichia coli and its scale-up fermentation for high extracellular yield.

Escherichia coli is one kind of the simple and excellent biosystem to overexpress heterologous enzymes, such as keratinolytic protease, an excellent enzyme to hydrolyze keratin substrate for broad industrial application. However, protein expression in E. coli frequently faces some problems such as inactive and inclusion body formation. This work described a series of protein engineering strategies of N-terminal propeptide replacement and site-directed mutagenesis to modify this enzyme activity and production. Site-directed mutagenesis (S180G/Y215S) on N-terminal propeptide altered mutant contributed to the highest specific activity (4725 ± 65 U/mg, more than 1300 U/mg improvement than wild-type enzyme). This comprehensive mutation also achieved 2.5-fold improvement of extracellular enzyme yield in shake-flask level. The fermentation strategies about optimizing glycerol feeding and inducing point in scale-up bioreactor resulted in tremendous leakage of keratinolytic protease (954 mg/L extracellular yield within 48 h, about 9.26-fold higher than the original shake-flask level) as well as cell lysis. Although this proposed strategy faces a major challenge to maintain cell integrity or viability, it still exists the opportunity to realize other enzymes extracellular expression in E. coli system and simplify downstream processing to meet the industrial application.

Development and Use of a Novel Random Mutagenesis Method: In Situ Error-Prone PCR (is-epPCR).

Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.

Efficient production of (2)H, (13)C, (15)N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds.

BACKGROUND: In order to use most modern methods of NMR spectroscopy to study protein structure and dynamics, isotope-enriched protein samples are essential. Especially for larger proteins (>20 kDa), perdeuterated and Ile (δ1), Leu, and Val methyl-protonated protein samples are required for suppressing nuclear relaxation to provide improved spectral quality, allowing key backbone and side chain resonance assignments needed for protein structure and dynamics studies. Escherichia coli and Pichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. The P. pastoris system can be used to produce (13)C, (15)N-enriched and even (2)H,(13)C, (15)N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (δ1), Leu and Val methyl-protonated groups in P. pastoris are still unavailable. Glycosylation heterogeneity also provides challenges to NMR studies. E. coli expression systems are efficient for overexpressing perdeuterated and Ile (δ1), Leu, Val methyl-protonated protein samples, but are generally not successful for producing secreted eukaryotic proteins with native disulfide bonds. RESULTS: The 33 kDa protein-Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using both P. pastoris and E. coli BL21 trxB (DE3) systems. Samples produced from both systems exhibit identical native disulfide bond formation and similar 2D NMR spectra, indicating similar native protein folding. The yield of (13)C, (15)N-enriched r27RCL produced using P. pastoris was 1.7 times higher that obtained using E. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced in P. pastoris exhibit O-glycosylation, while the protein samples produced in E. coli were not glycosylated. The specific activity of r27RCL from P. pastoris was ~1.4 times higher than that produced in E. coli. CONCLUSIONS: These data demonstrate efficient production of (2)H, (13)C, (15)N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic protein r27RCL with native disulfides using the E. coli BL21 trxB (DE3) system. For certain NMR studies, particularly efforts for resonance assignments, structural studies, and dynamic studies, E. coli provides a cost-effective system for producing isotope-enriched RCL. It should also be potential for producing other (2)H, (13)C, (15)N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic proteins with native disulfide bonds.

Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris.

BACKGROUND: The methylotrophic yeast, Pichia pastoris, is widely used as a useful experimental tool in protein engineering and production. It is common for proteins expressed in P. pastoris to exhibit N-glycosylation. In recent years, glycosylation studies in P. pastoris have attracted increasing attention from scholars. Rhizopus chinensis lipase (RCL) is one of the most important industrial lipases, and it has four potential N-linked glycosylation sites. The aim of the present study was to determine whether RCL undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in RCL expression and function. RESULTS: In this study, we demonstrated that RCL expressed in Pichia pastoris was N-glycosylated at the sites N-14, N-48 and N-60. The majority of the sites N-14 and N-60 were glycosylated, but the glycosylation degree of the site N-48 was only a very small portion. The glycan on N-60 played a key role in the expression and secretion of RCL. RT-PCR results showed that the mRNA level of proRCLCN60Q remained unchanged even though the protein secretion was hampered. Although the N-glycan on N-14 had no effect on the secretion of RCL, this glycan was beneficial for the lipase catalytic activity. On the other hand, the little amount of N-glycan on N-48 had no effect both on the secretion and activity of RCL in P. pastoris. Moreover, the thermostability analysis of RCL revealed that the lipase with more N-glycan was more thermostable. CONCLUSIONS: RCL was N-glycosylated when expressed in P. pastoris. The N-glycans of RCL on the different sites had different functions for the secretion and enzymatic properties of the lipase. Our report may also provide theoretical support for the improvement of enzyme expression and stability based on the N-linked glycosylation modification to meet the future needs of the biotechnological industry.

Streptomyces violaceoruber phospholipase A2: expression in Pichia pastoris, properties, and application in oil degumming.

The phospholipase A2 (PLA2) from Streptomyces violaceoruber was successfully expressed in the methylotrophic yeast Pichia pastoris GS115 under the control of AOX1 promoter for the first time. The maximum activity of the recombinant PLA2 (rPLA2) reached 34.7 ± 0.2 U/mL, and specific activity was 170 ± 4 U/mg after purification. On the sodium dodecyl sulfate polyacrylamide gel electrophoresis of the culture supernatants, three bands of 21, 18, and 14.3 kDa were observed. The peptide mass fingerprinting analysis showed that all of these three bands were rPLA2 from S. violaceoruber. By the treatment with Endo H and PNGase F, it indicated that the rPLA2 occurred N-glycosylation. The enzymatic properties of this enzyme were determined. The rPLA2 exhibited a lower optimum pH (pH = 6.0) compared to the wild-type enzyme, which was a desirable property in the application of oil degumming. In the enzymatic degumming process, the phosphorus content decreased from 261.77 ± 3.51 mg/kg to 20.74 ± 0.23 mg/kg, which is very promising for the industrial application.

Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase.

Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris.

Efficient secretion of lipase r27RCL in Pichia pastoris by enhancing the disulfide bond formation pathway in the endoplasmic reticulum.

The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500 U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30 % higher enzyme yields (16,200 U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39 g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8 h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space-time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.

High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production.

BACKGROUND: Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called "China wood oil" is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications. RESULTS: The prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C. CONCLUSIONS: This is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase from R. oryzae as an economic catalyst make this study a promising one for biodiesel applications.

Impact of gene dosage on the production of lipase from Rhizopus chinensis CCTCC M201021 in Pichia pastoris.

In this work, the high-level expression of the lipase r27RCL was achieved by optimization of the lipase gene copy number in the host strain Pichia pastoris. The copy number of the lipase gene proRCL from Rhizopus chinensis CCTCC M201021 was quantified by real-time quantitative polymerase chain reaction and a range of Mut(+) P. pastoris strains carrying one, three, five, and six copies of proRCL were obtained. The maximum lipase activity was achieved at 12,500 U/mL by the five-copy recombinant strain after 96 h of methanol induction in the 7-L fermenter. However, the enzyme activity of the six-copy recombinant strain decreased remarkably. By transcription analysis of proRCL, ERO1, and PDI, it suggested that unfolded protein response seemed to be triggered in the highest copy recombinant strain after 24 h. Thus, elaborate optimization of foreign gene dosage was very important for the high-level expression of foreign proteins in P. pastoris.

[Increasing activity of Rhizopus chinensis CCTCC M201021 lipase by directed evolution-error prone PCR].

Directed evolution strategy (error-prone PCR) was conducted to improve the activity of lipase from Rhizopus chinensis CCTCC M201021. Through two rounds of ep-PCR and pNPP top agar screening, two optimum mutant strains 1-11 and 2-28 were obtained with 2 and 4 fold of enzyme activity higher than that of parent strain, respectively. DNA sequencing of mutant lipase 2-28 revealed four amino acid substitutions: A129S, K161R, A230T, K322R. According to the simulated protein structure of Rhizopus chinensis lipase, A129S, K161R, A230T were located on the surface of the protein. A230T substitution improved the stability of the alpha-helix loop. K322R, near the catalytic center of lipase, located at a loop, formed a salt-bridge with a nearby aspartic acid (negative charged). Electrostatic force pulled the loop to the opposite direction of the substrate channel and made it easier for substrate to enter the lipase catalytic domain. Purified lipase was characterized and the result showed that Km of 2-28 lipase decreased by 10% compared with Km of the parent lipase, and Kcat was 2.75 fold improved than that of the original lipase.