High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis.

Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious. Here, a pair of plasmids, pUB-MazF and pUB'-EX1, were firstly constructed. pUB-MazF is a thermosensitive, self-replicable plasmid. It was able to efficiently cure from the host cell through induced expression of an endoribonuclease MazF, which is lethal to the host cell. pUB'-EX1 is a nonreplicative and integrative plasmid. Its replication was dependent on the thermosensitive replicase produced by pUB-MazF. Transformation of pUB'-EX1 into the B. licheniformis BL-UBM harboring pUB-MazF resulted in both plasmids coexisting in the host cell. At an elevated temperature, and in the presence of isopropyl-1-thio-ß-d-galactopyranoside and kanamycin, curing of the pUB-MazF and multiple-copy integration of pUB'-EX1 occurred, simultaneously. Through this procedure, genetically stable recombinants integrated multiple copies of amyS, from Geobacillus stearothermophilus ATCC 31195 were facilely obtained. The genetic stability of the recombinants was verified by repeated subculturing and shaking flask fermentations. The production of α-amylase by recombinant BLiS-002, harboring five copies of amyS, in a 50-l bioreactor reached 50 753 U/ml after 72 hr fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species.

Exploitation of ammonia-inducible promoters for enzyme overexpression in Bacillus licheniformis.

Ammonium hydroxide is conventionally used as an alkaline reagent and cost-effective nitrogen source in enzyme manufacturing processes. However, few ammonia-inducible enzyme expression systems have been described thus far. In this study, genomic-wide transcriptional changes in Bacillus licheniformis CBBD302 cultivated in media supplemented with ammonia were analyzed, resulting in identification of 1443 differently expressed genes, of which 859 genes were upregulated and 584 downregulated. Subsequently, the nucleotide sequences of ammonia-inducible promoters were analyzed and their functionally-mediated expression of amyL, encoding an α-amylase, was shown. TRNA_RS39005 (copA), TRNA_RS41250 (sacA), TRNA_RS23130 (pdpX), TRNA_RS42535 (ald), TRNA_RS31535 (plp), and TRNA_RS23240 (dfp) were selected out of the 859 upregulated genes and each showed higher transcription levels (FPKM values) in the presence of ammonia and glucose than that of the control. The promoters, PcopA from copA, PsacA from sacA, PpdpX from pdpX, Pald from ald, and Pplp from plp, except Pdfp from dfp, were able to mediate amyL expression and were significantly induced by ammonia. The highest enzyme expression level was mediated by Pplp and represented 23% more α-amylase activity after induction by ammonia in a 5-L fermenter. In conclusion, B. licheniformis possesses glucose-independent ammonia-inducible promoters, which can be used to mediate enzyme expression and therefore enhance the enzyme yield in fermentations conventionally fed with ammonia for pH adjustment and nitrogen supply.

Production, characteristics and applications of phytase from a rhizosphere isolated Enterobacter sp. ACSS.

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40-80 °C) and pH (2.0-6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K m, V max, K cat, and K cat/K m of the pure phytase were 0.21 mM, 131.58 nmol mg(-1) s(-1), 1.64 × 10(3) s(-1), and 7.81 × 10(6) M(-1) s(-1), respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca(+2), Mg(+2) and Mn(+2), but inhibited by Zn(+2), Cu(+2), Fe(+2), Pb(+2), Ba(+2) and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.

Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations.

Thermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65kDa. Our study reports that chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/ß domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350K.

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications.

Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of chitinases, of which chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanuginosus and its industrial application.

Purification and Characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant.

An extracellular endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 mutant was purified to homogeneity by gel filtration chromatography and showed a specific activity of 119 U/mg. The optimum pH and temperature of the purified enzyme were found to be 6.0 and 50 °C, respectively. The enzyme was stable up to 60 °C, retaining 60% of residual activity for 30 min, but inactivated rapidly above 60 °C. The enzyme was found to be stable at pH=6-9 when it retained 100% of its residual activity. The Lineweaver-Burk plot showed that the apparent Km and vmax values of the inulinase when using inulin as a substrate were 1.15 mg/mL and 0.15 µM/min, respectively, whereas the kcat value was found to be 0.145 min-1. The calculated catalytic efficiency of the enzyme was found to be 0.126 (mg·min)/mL. The purified inulinase can be used in the production of high fructose syrups.

Biodegradation of glycerol using bacterial isolates from soil under aerobic conditions.

Glycerol, a non-biodegradable by-product during biodiesel production is a major concern to the emerging biodiesel industry. Many microbes in natural environments have the ability to utilize glycerol as a sole carbon and energy source. The focus of this study was to screen for microorganisms from soil, capable of glycerol utilization and its conversion to value added products such as ethanol and 1,3-propanediol (1,3-PDO). Twelve bacterial isolates were screened for glycerol utilization ability in shake flask fermentations using M9 media supplemented with analytical grade glycerol (30 g/L) at various pH values (6, 7 and 8) and temperatures (30°C, 35°C and 40°C). Among these, six bacterial isolates (SM1, SM3, SM4, SM5, SM7 and SM8) with high glycerol degradation efficiency (>80%) were selected for further analysis. Highest level of 1,3-PDO production (15 g/L) was observed with isolate SM7 at pH 7 and 30°C, while superior ethanol production (14 g/L) was achieved by isolate SM9 at pH 8 and 35°C, at a glycerol concentration of 30 g/L. The selected strains were further evaluated for their bioconversion efficiency at elevated glycerol concentrations (50-110 g/L). Maximum 1,3-PDO production (46 g/L and 35 g/L) was achieved at a glycerol concentration of 70 g/L by isolates SM4 and SM7 respectively, with high glycerol degradation efficiency (>90). Three isolates (SM4, SM5 and SM7) also showed greater glycerol tolerance (up to 110 g/L). The isolates SM4 and SM7 were identified as Klebsiella pneumoniae and SM5 as Enterobacter aerogenes by 16S rDNA analysis. These novel isolates with greater glycerol tolerance could be used for the biodegradation of glycerol waste generated from the biodiesel industry into value-added commercial products.

Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol.

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.

Enhanced fructooligosaccharides and inulinase production by a Xanthomonas campestris pv. phaseoli KM 24 mutant.

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 +/- 0.03 U mL(-1)) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 +/- 0.03 U mL(-1) after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L(-1) h(-1)) and specific (119,025 U g(-1) h(-1)) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L(-1) h(-1) and specific productivity of 72 g g(-1) h(-1) FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.

A unique xylose reductase from Thermomyces lanuginosus: Effect of lignocellulosic substrates and inhibitors and applicability in lignocellulosic bioconversion.

In this study, the xylose reductase gene (XRTL) from Thermomyces lanuginosus SSBP was expressed in Pichia pastoris GS115 and Saccharomyces cerevisiae Y294. The purified 39.2 kDa monomeric enzyme was optimally active at pH 6.5 and 50 °C and showed activity over a wide range of temperatures (30-70 °C) and pH (4.0-9.0), with a half-life of 1386 min at 50 °C. The enzyme preferred NADPH as cofactor and showed broad substrate specificity. The enzyme was inhibited by Cu2+, Fe2+ and Zn2+, while ferulic acid was found to be the most potent lignocellulosic inhibitor. Recombinant S. cerevisiae with the XRTL gene showed 34% higher xylitol production than the control strain. XRTL can therefore be used in a cell-free xylitol production process or as part of a pathway for utilization of xylose from lignocellulosic waste.

Concomitant production of chitosan and lipids from a newly isolated Mucor circinelloides ZSKP for biodiesel production.

A newly-isolated oleaginous fungus Mucor circinelloides ZSKP concurrently yielded 21.4% lipids and 11.2% chitosan per gram of biomass. Parameters affecting the co-production were identified using Plackett-Burman design and were statistically optimized using Response Surface Methodology, which resulted in a 3-fold improvement in lipid production. The lipid profile showed a high content of unsaturated fatty acids including oleic (37%), linolenic (14%) and linoleic acids (19%), while palmitic acid was the major saturated fatty acid (21%). A comparative study to evaluate the efficacy of enzymatic (lipase) and chemical treatments for biodiesel production from fungal lipids and sunflower oil revealed enhanced production of biodiesel from fungal lipids. Synthesized biodiesel from M. circinelloides ZSKP satisfied standard specifications and had a higher cetane number (56), lower kinematic viscosity (4.6 mm2/s) and lower acid number (0.03) compared to sunflower oil. Results suggest Mucor circinelloides ZSKP is a promising candidate for implementation of the biorefinery concept.

Comparative biocontrol ability of chitinases from bacteria and recombinant chitinases from the thermophilic fungus Thermomyces lanuginosus.

Microbial chitinases (EC 3.2.1.14) are known to hydrolyse the chitinous gut epithelium of insects and cell walls of many fungi. In this study, seven chitinases from different bacteria and fungi were produced, characterized and their biocontrol abilities against graminaceous stem borers Eldana saccharina, Chilo partellus and Sesamia calamistis were assessed. All chitinases were stable over broad ranges of pH and temperature, however, recombinant fungal chitinases were more acid-stable than the bacterial counterparts. Chitinases from the thermophilic filamentous fungi Thermomyces lanuginosus SSBP (Chit1) and from Bacillus licheniformis (Chit lic) caused 70% and 80% mortality, respectively, in second instar larvae of E. saccharina. Six of the seven partially-purified microbial chitinases inhibited Aspergillus niger, A. flavus, A. alliaceus, A. ochraceus, Fusarium verticillioides and Mucor sp. Overall, microbial chitinases show promise as biocontrol agents of fungi and stalk-boring lepidopterans.

Directed evolution of the thermostable xylanase from Thermomyces lanuginosus.

The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability.

Thermo-acid-stable phytase-mediated enhancement of bioethanol production using Colocasia esculenta.

Phytase production by the thermophilic mould Thermomyces lanuginosus SSBP was enhanced 8.56-fold in submerged fermentation, which was further improved in fed-batch cultivations. The protein was purified to homogeneity using ammonium sulphate precipitation, Resource Q anion exchange and Superdex gel-filtration chromatography, with an overall purification of 24.7-fold and a yield of 5.16%. The purified 49kDa protein was optimally active at 55°C and pH 5.0, and was stable between 50 and 90°C from pH 3.0-6.0, with a half-life of 138.6min at 70°C. It was moderately stimulated by Ba+2 and Mg+2. The enzyme reduced phytate content in Colocasia esculenta starch (from 1.43mg/g to 0.05mg/g) that resulted in an improvement in the availability of fermentable sugars with a concomitant reduction in viscosity and 1.59-fold improvement in ethanol production. Thermo-acid-stable phytase from T. lanuginosus SSBP could be of major biotechnological interest, especially due to its robustness and wide applicability.

Amylase production in solid state fermentation by the thermophilic fungus Thermomyces lanuginosus.

The production of extracellular amylase by the thermophilic fungus Thermomyces lanuginosus was studied in solid state fermentation (SSF). Solid substrates such as wheat bran, molasses bran, rice bran, maize meal, millet cereal, wheat flakes, barley bran, crushed maize, corncobs and crushed wheat were studied for enzyme production. Growth on wheat bran gave the highest amylase activity. The maximum enzyme activity obtained was 534 U/g of wheat bran under optimum conditions of an incubation period of 120 h, an incubation temperature of 50 degrees C, an initial moisture content of 90%, a pH of 6.0, an inoculum level of 10% (v/w), a salt solution concentration of 1.5:10 (v/w) and a ratio of substrate weight to flask volume of 1:100 with soluble starch (1% w/w) and peptone (1% w/w) as supplements.

Creation of thermostable and alkaline stable xylanase variants by DNA shuffling.

Mutant xylanases, G41 and G53, were generated by random mutagenesis of Thermomyces lanuginosus xylanase DSM 5826 (xynA) in a previous study. Incubation at 90 min showed that G41 had 75% activity at 80 °C and G53 had 93% activity at pH 10. In order to create xylanase variants possessing both thermal and alkaline stability in a single enzyme, G41 and G53 served as templates for DNA shuffling using the StEP recombination method. One of the resulting StEP recombinants, S340, retained 54% stability at 80 °C and 60% stability at pH 10 with three resulting amino acid mutations. Another StEP recombinant, S325, displayed 85% stability at 80 °C and 60% stability at pH 10 and DNA sequencing showed that it inherited mutations from both parents. All thermostable variants displayed an increase in arginine content with poor enzyme activity. Thus, the StEP recombination method successfully recombined mutations into two xylanases that were more robust than their parent counterparts. Additionally, the 3D-models of the wild type T. lanuginosus xynA (xyl_ext) and its variants, G41 and S325, were predicted using I-TASSER and then subjected to molecular dynamics (MD) simulations at 300 K for a deeper understanding of their structural features. The results from the predicted 3D models show clearly the presence of α-helical regions in the N-terminal residues of the xyl_ext, G41 and S325. Moreover, the MD analysis suggests that the presence of additional residues (1-31) and point mutation induces slight structural changes with the stability of the protein being evenly distributed over the whole structure.

Xylanase Superproducer: Genome Sequence of a Compost-Loving Thermophilic Fungus, Thermomyces lanuginosus Strain SSBP.

We report here the draft genome sequence of Thermomyces lanuginosus strain SSBP, which was isolated from soil in South Africa. This fungus produces the largest amount of xylanase ever reported in the literature.

High level expression of a recombinant xylanase by Pichia pastoris NC38 in a 5 L fermenter and its efficiency in biobleaching of bagasse pulp.

A genetically modified XynA gene from Thermomyces lanuginosus was expressed in Pichia pastoris under the control of GAP promoter. P. pastoris expressed greater levels of xylanase (160 IU ml(-1)) on BMGY medium without zeocin after 56 h. The xylanase production by recombinant P. pastoris was scaled up in a 5L fermenter containing 1% glycerol and the highest xylanase production of 139 IU ml(-1) was observed after 72 h. Further studies carried out in fermenter under controlled pH (5.5) yielded a maximum xylanase production of 177 IU ml(-1) after 72 h. The biobleaching efficacy of crude xylanase was also evaluated on bagasse pulp and a brightness of 47.4% was observed with 50 IU of crude xylanase used per gram of pulp, which was 2.1 points higher in brightness than the untreated samples. Reducing sugars (24.8 mg g(-1)) and UV absorbing lignin-derived compounds values were considerably higher with xylanase treated samples.

Error-prone PCR of a fungal xylanase for improvement of its alkaline and thermal stability.

Random mutagenesis was used to improve the alkaline and thermal stability of the xylanase (XynA) from Thermomyces lanuginosus. Error-prone PCR reactions were carried out; the PCR products were cloned into Escherichia coli and a library of 960 clones was selected on xylan-containing agar plates. The crude filtrates of positive xylanase producers were screened at 80 degrees C and tested separately at pH 10 for alkaline tolerance. The native XynA lost 80% activity after 90 min at 80 degrees C and lost 70% activity at pH 10. Conversely, the most thermostable variant, G41, retained 75% activity after 90 min at 80 degrees C and the best alkali-stable variant, G53, retained 93% activity at pH 10. Sequence analysis revealed four amino acid substitutions in G41 and a single substitution in G53. These variants, therefore, have improved thermal and alkaline stability and are ideal candidates for DNA shuffling experiments to produce a robust xylanase for industrial application.

Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli.

The alkaline stability of the xylanase from Thermomyces lanuginosus was further improved by directed evolution using error-prone PCR mutagenesis. Positive clones were selected by their ability to produce zones of clearing on pH 9 and 12 xylan agar plates. Variant NC38 was able to withstand harsh alkaline conditions retaining 84% activity after exposure at pH 10 for 90 min at 60 degrees C, while the parent enzyme had 22% activity after 60 min. The alkaline stable variant NC38 was cloned into pBGP1 under the control GAP promoter and pET22b(+) for expression in Pichia pastoris and Escherichia coli BL21, respectively. Best extracellular expression of the recombinant xylanase was observed in P. pastoris (261.7+/-0.61 U ml(-1)) whereas intracellular activity was observed in E. coli (47.9+/-0.28 U ml(-1)) was low. Total activity obtained in P. pastoris was 545-fold higher than E. coli. The mutated alkaline stable xylanase from P. pastoris was secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.