Comparative Analysis of Bacterial Expression Systems for Keratinase Production

To explore a better expression system for the production of keratinase, the keratinase gene from Bacillus licheniformis BBE11-1 was expressed in Escherichia coli, Bacillus subtilis, and Pichia pastoris. The corresponding recombinant keratinases were named ker E, ker B, and ker P, respectively. All recombinant keratinases had an optimal pH at 10 although the pH stability of ker E and ker P was higher than that of ker E. The optimal temperature and thermostability of ker P were enhanced compared with those of ker E and ker B. The recombinant keratinases were inhibited by Mn²⁺ but experienced little influence from other metal ions. Furthermore, all recombinant keratinases could retain almost 80 % activity after treatment with 1 M hydrogen peroxide for 5 h. Under optimized conditions in a 3-L fermenter, the maximum keratinase activities obtained from recombinant B. subtilis and P. pastoris were 3,010 and 1,050 U/mL, respectively. This maximum keratinase activity from B. subtilis is the highest activity ever reported for any bacterial strain. These results indicate that B. subtilis is the ideal host for keratinase production, with potential applications in textile processing and feed supplements.     

Production of XynX, a Large Multimodular Protein of Clostridium thermocellum, by Protease-Deficient Bacillus subtilis Strains

XynX of Clostridium thermocellum is a large, multimodular xylanase of 116?kDa. An Escherichia coli transformant carrying the entire xynX produced three active truncated xylanase species of 105, 85, and 64?kDa intracellularly. The Bacillus subtilis WB700 transformant with the xynX, a strain deficient in seven proteases including Vpr, secreted two active truncated xylanase species of 65 and 44?kDa. The B. subtilis WB800 transformant with xynX, a strain deficient in eight proteases including Vpr and WprA, secreted more active enzymes, 8.46?U?ml^?1, mostly in the form of 105 and 85?kDa, than the WB700 transformant, 6.93?U?ml^?1. This indicates that the additional deletion of wprA enabled the WB800 to secrete XynX in its intact form. B. subtilis WB800 produced more total enzyme activity than E. coli (1,692?±?274 U vs. 141.9?±?27.1 U), and, more importantly, secreted almost all the enzyme activity. The results suggest the potential use of B. subtilis WB800 as a host system for the production of large multimodular proteins.     

Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.     

Cloning, Expression, and Characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris

The β-mannanase gene (1,029 nucleotide) from Bacillus subtilis MAFIC-S11, encoding a polypeptide of 342 amino acids, was cloned and expressed in Pichia pastoris. To increase its expression, the β-mannanase gene was optimized for codon usage (mannS) and fused downstream to a sequence-encoding modified α-factor signal peptide. The expression level was improved by 2-fold. This recombinant enzyme (mannS) showed its highest activity of 24,600 U/mL after 144-h fermentation. The optimal temperature and pH of mannS were 50 °C and 6.0, respectively, and its specific activity was 3,706 U/mg. The kinetic parameters V _max and K _m were determined as 20,000 U/mg and 8 mg/mL, respectively, representing the highest ever expression level of β-mannanase reported in P. pastoris. In addition, the enzyme exhibited much higher binding activity to chitin, chitosan, Avicel, and mannan. The superior catalytic properties of mannS suggested great potential as an effective additive in animal feed industry.     

Genome Mining for New α-Amylase and Glucoamylase Encoding Sequences and High Level Expression of a Glucoamylase from Talaromyces stipitatus for Potential Raw Starch Hydrolysis

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD₆₀₀?>?200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ～8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71–87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.     

A Recombinant Highly Thermostable β-Mannanase (ReTMan26) from Thermophilic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (TBS2) Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and Its pH and Temperature Stability

A gene encoding a highly thermostable β-mannanase from a thermophilic Bacillus subtilis (TBS2) was successfully expressed in Pichia pastoris. The maximum activity of the recombinant thermostable β-mannanase (ReTMan26) was 5435 U/mL, which was obtained by high-density, fed-batch cultivation after 168-h induction with methanol in a 50-L bioreactor. The protein yield reached 3.29 mg/mL, and the protein had a molecular weight of ~42 kDa. After fermentation, ReTMan26 was purified using a 10-kDa cut-off membrane and Sephadex G-75 column. The pH and temperature optima of purified ReTMan26 were pH 6.0 and 60 °C, respectively, and the enzyme was stable at pH 2.0–8.0 and was active at 20–100 °C. HPLC analysis of the products of locust bean gum hydrolysis showed that the mannan-oligosaccharide content was 62.5%. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min, which was higher than any other β-mannanase reported up to now, suggesting its potential for industrial applications.     

Cloning of LicB from Clostridium thermocellum and its efficient secretive expression of thermostable β-1,3-1,4-glucanase

β-1,3-1,4-glucanase is a widely used enzyme in brewing and in animal feed processing. To produce the bacterial enzyme at an industrial scale, the enzyme should be able to be secreted from microbial cells into fermentation broth and be stable in different conditions. In this study, the LicB gene encoding β-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum was secretively expressed in a secretive strain, Bacillus subtilis WB800, with eight extracellular protease deletion which made LicB expressed obviously and reached 1.18 U/g cell mass. The secreted β-1,3-1,4-glucanase was found to be active from 40 °C to 80 °C and achieved the optimal activity at 80 °C. The enzyme also has a wide pH range (pH 4–11). The most common metal ions and chemicals were found to be inert on its activity. The property of LicB-encoded β-1,3-1,4-glucanase and its efficient secretive expression makes it a potential enzyme for industrial production and application.     

Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi’s L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO₄ of 0.5 mM to the medium, and shaking off while cell mass reached to OD_600nm of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2′-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.     

Layer-by-Layer Self-Assembly Immobilization of Catalases on Wool Fabrics

A new immobilization strategy of catalases on natural fibers was reported in this paper. Catalase (CAT) from Bacillus subtilis was assembled into multiple layers together with poly(diallyldimethylammonium chloride) (PDDA) on wool fabrics via layer-by-layer (LBL) electrostatic self-assembly deposition. The mechanism and structural evaluation of LBL electrostatic self-assembly were studied in terms of scanning electron microscopy (SEM), surface zeta potential, and apparent color depth (K/S). The SEM pictures showed obvious deposits absorbed on the wool surfaces after LBL self-assembly. The surface zeta potential and dyeing depth of CAT/PDDA-assembled wool fabrics presented a regular layer-by-layer alternating trend along with the change of deposited materials, revealing the multilayer structure of the wool fiber immobilized catalases. The V _max values were found to be 2,500?±?238 U/mg protein for the free catalase and 1,000?±?102 U/mg protein for the immobilized catalase. The K _m value of free catalase (11.25?±?2.3 mM) was found to be lower than that of the immobilized catalase (222.2?±?36.5 mM). The immobilized catalase remained high enzymatic activity and showed a measureable amount of reusability, which proved that LBL electrostatic self-assembly deposition is a promising approach to immobilize catalases.     

Engineering <Emphasis Type="Italic">Pichia pastoris</Emphasis> for Efficient Production of a Novel Bifunctional <Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis> Invertebrate-Type Lysozyme

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of >?99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na⁺. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.     

Production Optimization and Characterization of Recombinant Cutinases from Thermobifida fusca sp. NRRL B-8184

Two genes, cut1 and cut2, of Thermobifida fusca NRRL B-8184 with cutin-hydrolyzing activity were cloned and expressed in Escherichia coli BL21 (DE3) separately. Enhanced expression was achieved after screening of six different media, optimization of the culture conditions and medium components. Among the screened media, modified Terrific Broth was found to be the best for maximum production of recombinant cutinases in E. coli BL21 (DE3). Under optimal conditions, the production of recombinant Cut1 and Cut2 (cutinases) were found to be 318?±?0.73 and 316?±?0.90 U/ml, respectively. The production of recombinant cutinases was increased by 11-fold as compared with T. fusca NRRL B-8184 wild-type strain. Both the recombinant cutinases were purified to homogeneity. They were found to be thermostable, organic solvent, and surfactant tolerant. Both the cutinase were active in a broad range of temperature (40–80 °C) and pH (6.8–9) with optimum activity at pH 8.0 and 55 °C.     

High Level Production of β-Galactosidase Exhibiting Excellent Milk-Lactose Degradation Ability from Aspergillus oryzae by Codon and Fermentation Optimization

A β-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL^?1 with o-nitrophenyl-β-d-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L^?1 in which the target protein (6.24 g L^?1) occupied approximately 86 %. The recombinant β-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the β-galactosidase may have a good application potential in whey and milk industries.     

Expression, High Cell Density Culture and Purification of Recombinant EC-SOD in Escherichia coli

Superoxide dismutase (SOD) catalyzes the dismutation of the biologically toxic superoxide anion into oxygen and hydrogen peroxide and is deployed by the immune system to kill invading microorganisms. Extracellular SOD (EC-SOD) is a copper- and zinc-containing glycoprotein found predominantly in the soluble extracellular compartment that consists of ～30-kDa subunits. Here, we purified recombinant EC-SOD3 (rEC-SOD) from Escherichia coli BL21(DE3) expressing a pET-SOD3-1 construct. Cells were cultured by high-density fed-batch fermentation to a final OD₆₀₀ of 51.8, yielding a final dry cell weight of 17.6 g/L. rEC-SOD, which was expressed as an inclusion body, comprised 48.7% of total protein. rEC-SOD was refolded by a simple dilution refolding method and purified by cation-exchange and reverse-phase chromatography. The highly purified rEC-SOD thus obtained was a mixture of monomers and dimers, both of which were active. The molecular weights of monomeric and dimeric rEC-SOD were 25,255 and 50,514 Da, respectively. The purified rEC-SOD had 4.3 EU/mg of endotoxin and the solubility of rEC-SOD was more than 80% between pH 7 and 10. In 2 L of fed-batch fermentation, 60 mg of EC-SOD (99.9% purity) could be produced and total activity was 330.24 U. The process established in this report, involving high-cell-density fermentation, simple dilution refolding, and purification with ion-exchange and reverse-phase chromatography, represents a commercially viable process for producing rEC-SOD.     

Acetaldehyde Detoxification Using Resting Cells of Recombinant Escherichia coli Overexpressing Acetaldehyde Dehydrogenase

Acetaldehyde dehydrogenase (E.C. 1.2.1.10) plays a key role in the acetaldehyde detoxification. The recombinant Escherichia coli cells producing acetaldehyde dehydrogenase (ist-ALDH) were applied as whole-cell biocatalysts for biodegradation of acetaldehyde. Response surface methodology (RSM) was employed to enhance the production of recombinant ist-ALDH. Under the optimum culture conditions containing 20.68 h post-induction time, 126.75 mL medium volume and 3 % (v/v) inoculum level, the maximum ist-ALDH activity reached 496.65?±?0.81 U/mL, resulting in 12.5-fold increment after optimization. Furthermore, the optimum temperature and pH for the catalytic activity of wet cells were 40 °C and pH 9.5, respectively. The biocatalytic activity was improved 80 % by permeabilizing the recombinant cells with 0.075 % (v/v) Triton X-100. When using 2 mmol/L NAD⁺ as coenzyme, the permeabilized cells could catalyze 98 % of acetaldehyde within 15 min. The results indicated that the recombinant E. coli with high productivity of ist-ALDH might be highly efficient and easy-to-make biocatalysts for acetaldehyde detoxification.     

Lipase Production by Botryosphaeria ribis EC-01 on Soybean and Castorbean Meals: Optimization, Immobilization, and Application for Biodiesel Production

The effects of soybean and castorbean meals were evaluated separately, and in combinations at different ratios, as substrates for lipase production by Botryosphaeria ribis EC-01 in submerged fermentation using only distilled water. The addition of glycerol analytical grade (AG) and glycerol crude (CG) to soybean and castorbean meals separately and in combination, were also examined for lipase production. Glycerol-AG increased enzyme production, whereas glycerol-CG decreased it. A 2⁴ factorial design was developed to determine the best concentrations of soybean meal, castorbean meal, glycerol-AG, and KH₂PO₄ to optimize lipase production by B. ribis EC-01. Soybean meal and glycerol-AG had a significant effect on lipase production, whereas castorbean meal did not. A second treatment (2² factorial design central composite) was developed, and optimal lipase production (4,820 U/g of dry solids content (ds)) was obtained when B. ribis EC-01 was grown on 0.5 % (w/v) soybean meal and 5.2 % (v/v) glycerol in distilled water, which was in agreement with the predicted value (4,892 U/g ds) calculated by the model. The unitary cost of lipase production determined under the optimized conditions developed ranged from US$0.42 to 0.44 based on nutrient costs. The fungal lipase was immobilized onto Celite and showed high thermal stability and was used for transesterification of soybean oil in methanol (1:3) resulting in 36 % of fatty acyl alkyl ester content. The apparent K _m and V _max were determined and were 1.86 mM and 14.29 μmol min^?1 mg^?1, respectively.     

Efficient Production of (S)-Naproxen with (R)-Substrate Recycling Using an Overexpressed Carboxylesterase BsE-NP01

An (S)-enantioselective esterase from Bacillus subtilis ECU0554, named BsE-NP01, has been cloned and over-expressed in a heterologous host Escherichia coli BL21. BsE-NP01 was shown to be a carboxylesterase with a molecular mass of about 32 kDa, and temperature and pH optima at 50 °C and 8.5, respectively. It could catalyze the selective hydrolysis of the (S)-enantiomer of racemic naproxen methyl ester, giving optically pure (S)-naproxen with 98% enantiomeric excess. A mechanic-grinding approach to substrate dispersion was also reported, which was considered to be an alternative to take the place of deleterious surfactants such as Tween-80, with improved performance of the hydrolysis reaction. Batch production of (S)-naproxen was repeatedly carried out in a solid-water biphasic system at 2-L scale, achieving an average total yield of about 85% after ten runs with complete recycling of (R)-substrate.

Cloning of Thermostable Cellulase Genes of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and Their Secretive Expression in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.     

Bioethanol Production from Soybean Residue via Separate Hydrolysis and Fermentation

Bioethanol was produced using polysaccharide from soybean residue as biomass by separate hydrolysis and fermentation (SHF). This study focused on pretreatment, enzyme saccharification, and fermentation. Pretreatment to obtain monosaccharide was carried out with 20% (w/v) soybean residue slurry and 270 mmol/L H₂SO₄ at 121 °C for 60 min. More monosaccharide was obtained from enzymatic hydrolysis with a 16 U/mL mixture of commercial enzymes C-Tec 2 and Viscozyme L at 45 °C for 48 h. Ethanol fermentation with 20% (w/v) soybean residue hydrolysate was performed using wild-type and Saccharomyces cerevisiae KCCM 1129 adapted to high concentrations of galactose, using a flask and 5-L fermenter. When the wild type of S. cerevisiae was used, an ethanol production of 20.8 g/L with an ethanol yield of 0.31 g/g consumed glucose was obtained. Ethanol productions of 33.9 and 31.6 g/L with ethanol yield of 0.49 g/g consumed glucose and 0.47 g/g consumed glucose were obtained in a flask and a 5-L fermenter, respectively, using S. cerevisiae adapted to a high concentration of galactose. Therefore, adapted S. cerevisiae to galactose could enhance the overall ethanol fermentation yields compared to the wild-type one.     

Mutation Breeding of Lycopene-Producing Strain Blakeslea Trispora by a Novel Atmospheric and Room Temperature Plasma (ARTP)

To improve the fermentation efficiency of lycopene, a plasma jet, driven by an active helium atom supplied with atmospheric and room temperature plasma (ARTP) biological breeding system, was used as a new method to generate mutations in Blakeslea trispora (?). After several rounds of screening, a mutant A5 with high concentration of lycopene and dry biomass was isolated, which showed a maximum lycopene concentration (26.4?±?0.2 mg/g dry biomass) which was 55 % higher than the parent strain (16.9?±?0.3 mg/g dry biomass) in the production of lycopene. Compared with parent strain, B. trispora A5 required less dissolved oxygen (10 % less than that of parent strain) to reach maximum concentration in a 5-L stirred tank reactor batch fermentation.