scholarly journals Expression and Characterization ofGeobacillus stearothermophilusSR74 Recombinantα-Amylase inPichia pastoris

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Sivasangkary Gandhi ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Siti Nurbaya Oslan
Keyword(s):  
Specific Activity ◽  
Thermophilic Bacteria ◽  
Recombinant Expression ◽  
Expression System ◽  
Alcohol Oxidase ◽  
Product Yield ◽  
Inducible Promoter ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Gene Encoding

Geobacillus stearothermophilusSR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactiveα-amylase. Increased production and commercialization of thermostableα-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostableα-amylase inG. stearothermophilusSR74 was amplified, sequenced, and subcloned intoP. pastorisGS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinantα-amylase SR74 achieved in shake flask was 28.6 U mL−1at 120 h after induction. The recombinant 59 kDaα-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH ofα-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostableα-amylase SR74 fromG. stearothermophilusSR74 would be beneficial for industrial applications, especially in liquefying saccrification.

A novel Swollenin from Talaromyces leycettanus JCM12802 exhibits cellulase activity and enhanced cellulase hydrolysis towards various substrates

2020 ◽  
Author(s):  
Honghai Zhang ◽  
Yuan Wang ◽  
Roman Brunecky ◽  
Bin Yao ◽  
Xiangming Xie ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Functional Diversity ◽  
Specific Activity ◽  
Biomass Conversion ◽  
Hydrolytic Enzymes ◽  
Cellulase Activity ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Carbohydrate Binding ◽  
Cell Wall Polysaccharides

Abstract Background Swollenins are present in some fungal species involved in the biodegradation of cellulosic substrates. They appear to promote a rearrangement in the network of non-covalent interactions between the cell wall polysaccharides, thus making it more accessible for degradation by hydrolytic enzymes. Here, we have reported a detailed characterization of a recombinant swollenin with respect to its disruptive activity on cellulosic substrates and synergistic effect with cellulases. Results In the present study, a novel swollenin gene Tlswo consisting of an open reading frame encoding 503 amino acids was identified from Talaromyces leycettanus JCM12802 and successfully expressed in Trichoderma reesei and Pichia pastoris. Similar to other fungal swollenins, TlSWO contained a N-terminal family 1 carbohydrate binding module (CBM1) followed by a Ser/Thr rich linker connected to expansin-like domain which includes a family 45 endoglucanase-like domain and group-2 grass pollen allergen domain. TlSWO demonstrated disruptive activity on Avicel and displayed a high synergistic effect with cellobiohydrolases, enhancing its hydrolytic performance up to 132%. The activity of TlSWO on various substrates and biomass was also examined. It was shown that TlSWO could release reducing sugars from lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of TlSWO towards the substates above is 9.0 ± 0.100 U/mg, 8.9 ± 0.100U/mg, 2.3 ± 0.002 U/mg and 0.79 ± 0.002 U/mg respectively. Moreover, TlSWO exhibits maximum activity at pH 4.0 and 50 ℃. Conclusion This study reported on a novel swollenin with highly efficient for biomass conversion. It also reveals the functional diversity of swollenin with activity on various substrates. Although the exact mechanism of swollenin catalytic action activity still remains unknown, the functional diversity of TlSWO makes it a good candidate for industrial applications.

scholarly journals Cloning and Expression of theγ-Polyglutamic Acid Synthetase GenepgsBCA inBacillus subtilisWB600

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Biaosheng Lin ◽  
Zhijuan Li ◽  
Huixia Zhang ◽  
Jiangwen Wu ◽  
Maochun Luo
Keyword(s):  
Bacillus Subtilis ◽  
Fermentation Broth ◽  
Recombinant Expression ◽  
Product Yield ◽  
Industrial Applications ◽  
Cloning And Expression ◽  
Polyglutamic Acid ◽  
Recombinant Bacteria ◽  
Expression Product ◽  
Clone And Express

To clone and express theγ-polyglutamic acid (γ-PGA) synthetase genepgsBCA inBacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA intoBacillus subtilisWB600.PgsBCAwas expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesizeγ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher.γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates ofγ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of theγ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing theγ-PGA synthetase genepgsBCAinB. subtilissystem has potential industrial applications.

scholarly journals Molecular expression of a recombinant thermostable bacterial amylase from Geobacillus stearothermophilus SR74 using methanol-free Meyerozyma guilliermondii strain SO yeast system

BioResources ◽  
2020 ◽  
Vol 15 (2) ◽  
pp. 3161-3172
Author(s):  
Nurul S. M. Nasir ◽  
Chor T. Leow ◽  
Siti N. H. Oslan ◽  
Abu B. Salleh ◽  
Siti N. Oslan
Keyword(s):  
Expression System ◽  
Alcohol Oxidase ◽  
Industrial Applications ◽  
Geobacillus Stearothermophilus ◽  
Fermentation Time ◽  
Meyerozyma Guilliermondii ◽  
Pichia Pastoris Expression System ◽  
Pastoris Expression System ◽  
Molecular Expression ◽  
External Inducer

α-Amylase, which was isolated from Geobacillus stearothermophilus SR74, has shown its potential to be used in industrial applications. However, its expression in the Pichia pastoris expression system with the alcohol oxidase 1 promoter (PAOX1) requires high methanol consumption and is time-consuming. This study aimed to express SR74 α-amylase in an alternative yeast system, using Meyerozyma guilliermondii strain SO, which was isolated from a spoiled orange (SO) under the regulation of a formaldehyde dehydrogenase promoter (PFLD). Qualitative screening showed that strain SO possessed a native amylase grown on YPD-starch plate at 30 °C. The recombinant SR74 α-amylase was further quantified and validated using the Western blot test. It was confirmed that SR74 α-amylase was expressed by strain SO extracellularly with a size of 59 kDa. Optimization in a shake flask showed that the recombinant SR74 α-amylase, which was regulated by PFLD, was successfully produced (26 U/mL) without any external inducer in the YPT medium after 24 h of cultivation. In conclusion, strain SO was able to produce SR74 amylase without methanol in one-fifth the fermentation time of P. pastoris. Further optimization of the expression may be done to improve the yield, as this methanol-free host is still underexplored.

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

2021 ◽  
Vol 9 ◽  
Author(s):  
Beenish Sadaqat ◽  
Chong Sha ◽  
Parveen Fatemeh Rupani ◽  
Hongcheng Wang ◽  
Wanbing Zuo ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Methyl Cellulose ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Mannanase Activity ◽  
Encoding Protein ◽  
Layer Chromatography ◽  
Carboxy Methyl

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 Km and Vmax 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.

scholarly journals Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Shengyue Ji ◽  
Weili Li ◽  
Abdul Rasheed Baloch ◽  
Meng Wang ◽  
Hengxin Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Fusion Gene ◽  
Expression System ◽  
Inducible Promoter ◽  
Clinical Applications ◽  
Cell Factory ◽  
Efficient Production ◽  
Microbial Cell Factory ◽  
Gene Encoding ◽  
Cecropin A

Abstract The efficient production of antimicrobial peptides (AMPs) for clinical applications has attracted the attention of the scientific community. To develop a novel microbial cell factory for the efficient biosynthesis of a cecropin A-melittin mutant (CAM-W), a recombinant Bacillus subtilis WB700 expression system was genetically modified with a novel vector, including a fusion gene encoding CAM-W, the autoprotease EDDIE and the signal peptide SacB under the control of the maltose-inducible promoter P glv . A total of 159 mg of CAM-W was obtained from 1 L of fermentation supernatant. The purified CAM-W showed a consistent size with the expected molecular weight of 3.2 kDa. Our findings suggest that this novel expression system can be used as a powerful tool for the efficient production of CAM-W.

scholarly journals High-Level Heterologous Expression of Endo-1,4-β-Xylanase from Penicillium citrinum in Pichia pastoris X-33 Directed through Codon Optimization and Optimized Expression

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3515 ◽  
Author(s):  
Chanika Ouephanit ◽  
Nassapat Boonvitthya ◽  
Sophie Bozonnet ◽  
Warawut Chulalaksananukul
Keyword(s):  
Pichia Pastoris ◽  
Xylanase Activity ◽  
Alcohol Oxidase ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Gene Encoding ◽  
Original Activity ◽  
Native Strain ◽  
Wide Range ◽  
High Level

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0–9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s−1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.

scholarly journals Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum

2003 ◽  
Vol 69 (8) ◽  
pp. 4985-4988 ◽  
Author(s):  
Laurence Girbal ◽  
Isabelle Mortier-Barrière ◽  
Frédéric Raynaud ◽  
Céline Rouanet ◽  
Christian Croux ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Acetobutylicum ◽  
Time Course ◽  
Specific Activity ◽  
Expression System ◽  
Regulatory System ◽  
Inducible Promoter ◽  
Reporter System ◽  
Inducible Gene

ABSTRACT A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system.

Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme

2002 ◽  
Vol 362 (2) ◽  
pp. 223-229 ◽  
Author(s):  
Benjamin ADAMS ◽  
David J. LOWE ◽  
Andrew T. SMITH ◽  
Claudio SCAZZOCCHIO ◽  
Stephane DEMAIS ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Aspergillus Nidulans ◽  
Specific Activity ◽  
Recombinant Expression ◽  
Expression System ◽  
Complex Mixture ◽  
Xanthine Dehydrogenase ◽  
Cell System ◽  
Post Translational Modification ◽  
Enzyme Molecules

Recent crystal structures of xanthine dehydrogenase, xanthine oxidase and related enzymes have paved the way for a detailed structural and functional analysis of these enzymes. One problem encountered when working with these proteins, especially with recombinant protein, is that the preparations tend to be heterogeneous, with only a fraction of the enzyme molecules being active. This is due to the incompleteness of post-translational modification, which for this protein is a complex, and incompletely understood, process involving incorporation of the Mo and Fe/S centres. The enzyme has been expressed previously in both Drosophila and insect cells using baculovirus. The insect cell system has been exploited by Iwasaki et al. [Iwasaki, Okamoto, Nishino, Mizushima and Hori (2000) J. Biochem (Tokyo) 127, 771–778], but, for the rat enzyme, yields a complex mixture of enzyme forms, containing around 10% of functional enzyme. The expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans is described. The purified protein has been analysed both functionally and spectroscopically. Its specific activity is indistinguishable from that of the enzyme purified from fruit flies [Doyle, Burke, Chovnick, Dutton, Whittle and Bray (1996) Eur. J. Biochem. 239, 782–795], and it appears to be more active than recombinant xanthine dehydrogenase produced with the baculovirus system. EPR spectra of the recombinant Drosophila enzyme are reported, including parameters for the Fe/S centres. Only a very weak ‘Fe/SIII’ signal (g1,2,3, 2.057, 1.930, 1.858) was observed, in contrast to the strong analogous signal reported for the enzyme from baculovirus. Since this signal appears to be associated with incomplete post-translational modification, this is consistent with relatively more complete cofactor incorporation in the Aspergillus-produced enzyme. Thus we have developed a recombinant expression system for D. melanogaster xanthine dehydrogenase, which can be used for the production of site-specific mutations of this enzyme.

scholarly journals TrzN from Arthrobacter aurescens TC1 Is a Zinc Amidohydrolase

2006 ◽  
Vol 188 (16) ◽  
pp. 5859-5864 ◽  
Author(s):  
Nir Shapir ◽  
Charlotte Pedersen ◽  
Omer Gil ◽  
Lisa Strong ◽  
Jennifer Seffernick ◽  
...  
Keyword(s):  
Sulfonic Acid ◽  
Specific Activity ◽  
Expression System ◽  
Maximum Activity ◽  
Visible Absorption ◽  
Metal Chelator ◽  
Arthrobacter Aurescens ◽  
Spectrum Characteristic ◽  
Amidohydrolase Superfamily ◽  
Iron Manganese

ABSTRACT TrzN, the broad-specificity triazine hydrolase from Arthrobacter and Nocardioides spp., is reportedly in the amidohydrolase superfamily of metalloenzymes, but previous studies suggested that a metal was not required for activity. To help resolve that conundrum, a double chaperone expression system was used to produce multimilligram quantities of functionally folded, recombinant TrzN. The TrzN obtained from Escherichia coli (trzN) cells cultured with increasing zinc in the growth medium showed corresponding increases in specific activity, and enzyme obtained from cells grown with 500 μM zinc showed maximum activity. Recombinant TrzN contained 1 mole of Zn per mole of TrzN subunit. Maximally active TrzN was not affected by supplementation with most metals nor by EDTA, consistent with previous observations (E. Topp, W. M. Mulbry, H. Zhu, S. M. Nour, and D. Cuppels, Appl. Environ. Microbiol. 66:3134-3141, 2000) which had led to the conclusion that TrzN is not a metalloenzyme. Fully active native TrzN showed a loss of greater than 90% of enzyme activity and bound zinc when treated with the metal chelator 8-hydroxyquinoline-5-sulfonic acid. While exogenously added zinc or cobalt restored activity to metal-depleted TrzN, cobalt supported lower activity than did zinc. Iron, manganese, nickel, and copper did not support TrzN activity. Both Zn- and Co-TrzN showed different relative activities with different s-triazine substrates. Co-TrzN showed a visible absorption spectrum characteristic of other members of the amidohydrolase superfamily replaced with cobalt.

scholarly journals Autocloning and Amplification of LIP2 inYarrowia lipolytica

2000 ◽  
Vol 66 (8) ◽  
pp. 3283-3289 ◽  
Author(s):  
Georges Pignède ◽  
Hui-Jie Wang ◽  
Franck Fudalej ◽  
Michel Seman ◽  
Claude Gaillardin ◽  
...  
Keyword(s):  
Waste Treatment ◽  
Pancreatic Insufficiency ◽  
Expression System ◽  
Single Copy ◽  
Industrial Applications ◽  
Extracellular Lipase ◽  
Gene Encoding ◽  
Bacterial Dna ◽  
High Level ◽  
Active Lipase

ABSTRACT We synthesized a Yarrowia lipolytica strain overproducing lipase for industrial applications by using long terminal repeat (ζ) of the Y. lipolytica retrotransposon Ylt1 and an allele of URA3 with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying aNotI-NotI cassette which contains a defectiveURA3 allele, a polylinker sequence, and the ζ region for targeting to multiple sites in the genome of the recipient. We inserted the LIP2 gene (encoding extracellular lipase) under the control of the strong POX2 promoter into JMP3 to generate JMP6. The pHSS6 region was removed by NotI digestion prior to transformation. Two Y. lipolytica strains transformed with the JMP6 LIP2 cassette had a mean of 10 integrated copies devoid of the Escherichia coli region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the LIP2 promoter. This work provides a new expression system in Y. lipolytica that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.

Sign in / Sign up

Export Citation Format

Share Document