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 Screening and Characterization of Thermostable Amylase-Producing Bacteria Isolated from Soil Samples of Afdera, Afar Region, and Molecular Detection of Amylase-Coding Gene

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Semira Nureddin Yassin ◽  
Tamene Milkessa Jiru ◽  
Meera Indracanti
Keyword(s):  
Maximum Activity ◽  
Soil Samples ◽  
Effect Of Temperature ◽  
Bacterial Isolates ◽  
Original Activity ◽  
Zone Formation ◽  
Afar Region ◽  
Wide Range ◽  
Thermostable Amylase

Studying thermostable amylase-producing bacteria in extreme environments has a crucial role to overcome different industrial challenges. Afar Region is one of the hottest and salty areas, making it the home of extremophiles. This study aimed at screening and characterizing amylase-producing bacteria isolated from soil samples of Afdera, Afar Region, and detection of their amylase-coding genes. Thus, a total of 49 bacterial isolates were obtained from the collected soil samples. Out of these, three isolates (M2, M8, and M13) were selected on the basis of diameter of the average clear zone formation and time taken to decolorize iodine solution. Based on their morphological and biochemical characteristics, the isolates were identified as genus Bacillus. PCR amplification and detection of the amylase-coding gene confirmed the presence of the amylase gene in the three bacterial isolates. Optimum amylase production time for these isolates was 48 hrs (M13 and M8) and 72 hrs (M2) corresponding to the amylase activity of 0.67 U/mL for M13, 0.74 U/mL for M8, and 0.73 U/mL for M2 with an optimum temperature of 55°C. Studies on the effect of temperature revealed that the crude enzyme had a maximum activity and stability at 75°C, 70°C, and 65°C for isolates M13, M8, and M2, respectively. Additionally, amylase produced from all isolates retained more than 66.41% of their original activity after incubating them at a temperature range from 55 to 80°C for 50 min. Optimum pH for the activity of all crude amylases was in the range from 5 to 9 with a peak activity at pH 8. Their activity decreased significantly by the presence of Zn+2 and Mg2+; however, their activity increased by the presence of Ca+2. Moreover, the three crude amylases were stable (0–3 M) with NaCl concentration. Amylases of this finding with thermophilic and halophilic characteristics offer a wide range of applications in food, brewing, textile, starch, paper, and deterrent industries. Thus, identification of these Bacillus isolates at a molecular level and purification as well as detailed characterization of the types of amylases are recommended for effective utilization in different industries.

scholarly journals Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianrong Wang ◽  
Xiaoming Li ◽  
Hao Chen ◽  
Bilian Lin ◽  
Liangzhong Zhao
Keyword(s):  
Recombinant Strain ◽  
High Efficiency ◽  
Batch Cultivation ◽  
Maximum Activity ◽  
Gene Encoding ◽  
Enzymatic Production ◽  
Bacillus Mojavensis ◽  
Chitosan Oligosaccharides ◽  
High Level

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

scholarly journals Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

2014 ◽  
Vol 2014 ◽  
pp. 1-20 ◽  
Author(s):  
Norsyuhada Alias ◽  
Mu’adz Ahmad Mazian ◽  
Abu Bakar Salleh ◽  
Mahiran Basri ◽  
Raja Noor Zaliha Raja Abd. Rahman
Keyword(s):  
Signal Sequence ◽  
Alcohol Oxidase ◽  
Protease Production ◽  
Protease Gene ◽  
Gene Encoding ◽  
Reading Frame ◽  
Antarctic Yeast ◽  
Glaciozyma Antarctica Pi12 ◽  
Rapid Amplification ◽  
High Level

Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa.

scholarly journals Identification and Characterization of Lactocyclicin Q, a Novel Cyclic Bacteriocin Produced by Lactococcus sp. Strain QU 12

2009 ◽  
Vol 75 (6) ◽  
pp. 1552-1558 ◽  
Author(s):  
Naruhiko Sawa ◽  
Takeshi Zendo ◽  
Junko Kiyofuji ◽  
Koji Fujita ◽  
Kohei Himeno ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Hydrophobic Interaction Chromatography ◽  
Heat Stability ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
High Level

ABSTRACT Lactococcus sp. strain QU 12, which was isolated from cheese, produced a novel cyclic bacteriocin termed lactocyclicin Q. By using cation-exchange chromatography, hydrophobic interaction chromatography, and reverse-phase high-performance liquid chromatography, lactocyclicin Q was purified from culture supernatant, and its molecular mass was determined to be 6,062.8 Da by mass spectrometry. Lactocyclicin Q has been characterized by its unique antimicrobial spectrum, high level of protease resistance, and heat stability compared to other reported bacteriocins of lactic acid bacteria. The amino acid sequence of lactocyclicin Q was determined chemically, and this compound is composed of 61 amino acid residues that have a cyclic structure with linkage between the N and C termini by a peptide bond. It showed no homology to any other antimicrobial peptide, including cyclic bacteriocins. On the basis of the amino acid sequences obtained, the sequence of the gene encoding the prepeptide lactocyclicin Q was obtained. This is the first report of a cyclic bacteriocin purified from a strain belonging to the genus Lactococcus.

scholarly journals High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yihong Lu ◽  
Cheng Fang ◽  
Qinhong Wang ◽  
Yuling Zhou ◽  
Guimin Zhang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Pichia Pastoris ◽  
Paper Industry ◽  
Xylanase Activity ◽  
Multiple Gene ◽  
E Coli ◽  
Gene Insertion ◽  
Alkaline Conditions ◽  
Pulp Properties ◽  
High Level

Abstract In paper industry, xylanases are used to increase the pulp properties in bleaching process as its eco-friendly nature. The xylanases activity is hindered by high temperature and alkaline conditions with high enzyme production cost in the paper industry. Here, XynHB, an alkaline stable xylanase from Bacillus pumilus HBP8 was mutated at N188A to XynHBN188A. Expressed mutant in E. coli showed 1.5-fold higher xylanase activity than XynHB at 60 °C. The mutant expressed in Pichia pastoris was glycosylated, remained stable for 30 min at 60 °C. XynHBN188A optimized based on codon usage bias for P. pastoris (xynHBN188As) showed an increase of 39.5% enzyme activity. The strain Y16 forming the largest hydrolysis halo in the xylan plate was used in shake flask experiments produced an enzyme activity of 6,403 U/ml. The Y16 strain had 9 copies of the recombinant xynHBN188As gene in the genome revealed by qPCR. The enzymatic activity increased to 48,241 U/ml in a 5 L fermentor. Supplement of 15 U/g xylanase enhanced the brightness of paper products by 2% in bleaching experiment, and thereby improved the tensile strength and burst factor by 13% and 6.5%, respectively. XynHBN188As has a great potential in paper industries.

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.

Codon optimization, expression and characterization of Bacillus subtilis MA139 β-1,3-1,4-glucanase in Pichia pastoris

Biologia ◽  
2010 ◽  
Vol 65 (2) ◽  
Author(s):  
Jiayun Qiao ◽  
Bo Zhang ◽  
Yiqun Chen ◽  
Yunhe Cao
Keyword(s):  
Bacillus Subtilis ◽  
Pichia Pastoris ◽  
Codon Bias ◽  
De Novo ◽  
Alcohol Oxidase ◽  
Methanol Induction ◽  
Chemical Reagents ◽  
Prospective Application ◽  
High Level

Abstractβ-1,3-1,4-Glucanase has been broadly used in feed and brewing industries. According to the codon bias of Pichia pastoris, the Bacillus subtilis MA139 β-1,3-1,4-glucanase gene was de novo synthesized and expressed in P. pastoris X-33 strain under the control of the alcohol oxidase 1 promoter. In a 10-L fermentor, the β-1,3-1,4-glucanase was overexpressed with a yield of 15,000 U/mL by methanol induction for 96 h. The recombinant β-1,3-1,4-glucanase exhibited optimal activity at 40°C and pH 6.4. The activity of the recombinant β-1,3-1,4-glucanase was not significantly affected by various metal ions and chemical reagents. To our knowledge, the expression of this β-1,3-1,4-glucanase from Bacillus sp. in P. pastoris is in relatively high level compared to previous reports. These biochemical characteristics suggest that the recombinant β-1,3-1,4-glucanase has a prospective application in feed and brewing industries.

scholarly journals Evaluation of Three Pichia pastoris-Expressed Plasmodium falciparum Merozoite Proteins as a Combination Vaccine against Infection with Blood-Stage Parasites

2005 ◽  
Vol 73 (10) ◽  
pp. 6530-6536 ◽  
Author(s):  
Dongmei Zhang ◽  
Weiqing Pan
Keyword(s):  
Plasmodium Falciparum ◽  
Pichia Pastoris ◽  
Chimeric Protein ◽  
Parasite Growth ◽  
Blood Stage ◽  
Combination Vaccine ◽  
High Yield ◽  
Gene Encoding ◽  
High Level

ABSTRACT Because invasion of erythrocytes by Plasmodium falciparum merozoites involves multiple receptor-ligand interactions, it may be necessary to develop a multivalent malaria vaccine that is comprised of distinct parasite ligands. PfAMA-1, PfMSP1, and PfEBA-175 are merozoite proteins that play important roles in invasion. We have constructed a PfCP-2.9 chimeric protein consisting of PfAMA-1 and PfMSP1 and tested it for immunogenicity in animal models and humans. The F2 subdomain of PfEBA-175 (PfEBA-175II F2) was identified as the binding domain for glycophorin A on erythrocytes. In this study, we used the codon frequencies of the yeast Pichia pastoris to redesign and synthesize a gene encoding the F2 domain. We found that the codon-optimized gene was expressed at a high level in P. pastoris as a soluble protein with a yield of about 300 mg/liter. The expressed protein was able to bind normal erythrocytes but not those treated with neuraminidase or trypsin. Moreover, the protein was recognized by the sera of malaria patients and was highly immunogenic in mice, rabbits, and rhesus monkeys. Immunoglobulin G isolated from both immunized rabbits and monkeys inhibited in vitro parasite growth. Immunization of animals with a combination of PfEBA-175II F2 and PfCP-2.9 did not result in antigen (Ag) competition in animals. Moreover, antibodies to both PfEBA-175II F2 and PfCP-2.9, isolated from rabbits immunized with both constructs, inhibited parasite growth in vitro. The combination of high yield, functional folding, antibody inhibition, and lack of Ag competition provides support for inclusion of these merozoite proteins in a combination vaccine against infection with blood-stage parasites.

Cloning of a Cellobiohydrolase Gene (cbh1) from Aspergillus niger and Heterogenous Expression in Pichia pastoris

2011 ◽  
Vol 347-353 ◽  
pp. 2443-2447
Author(s):  
Guo Qing Li ◽  
Chang Sheng Chai ◽  
Song Fan ◽  
Lin Guo Zhao
Keyword(s):  
Amino Acid ◽  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Working Condition ◽  
Industrial Applications ◽  
Gene Encoding ◽  
Acid Protein ◽  
Wide Range ◽  
Ph Range ◽  
Amino Acid Protein

A gene encoding a cellobiohydrolase (CBH) was isolated from Aspergillus niger-NL-1 and designated as cbh1. The cbh1 gene contains 1,515 nucleotides with three introns and encodes a 452-amino acid protein with a molecular weight of approximately 60 kDa. The amino acid sequence encoded by cbh1 shows high homology with the sequence of glycoside hydrolase fimily 7. The cellobiohydrolase (cbh1) gene was succussfully expressed in Pichia pastoris KM71H. The recombinant CBHⅠshowed an optimal working condition at 60 °C, pH 4.0 with Kmand Vmaxtoward CMC-Na of 13.81 mM and 0.269 μmol/min, respectively. The enzyme retained more than 80 % of its initial activity after 2 h of incubation at 90 °C and was stable in pH range 1.0～10.0. Because of its moderately stable at high temperature and stability through wide range of pH, this enzyme has potential in various industrial applications.

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