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13 pages, 1588 KiB

Open AccessArticle

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

by Chanika Ouephanit, Nassapat Boonvitthya, Sophie Bozonnet and Warawut Chulalaksananukul

Molecules 2019, 24(19), 3515; https://doi.org/10.3390/molecules24193515 - 27 Sep 2019

Cited by 13 | Viewed by 2865

Abstract

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 [...] Read more.

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 (P_GAP) and alcohol oxidase 1 (P_AOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of P_GAP and P_AOX1 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 P_AOX1 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 K_m(app) of 2.8 mg/mL, and a k_cat 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. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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17 pages, 3836 KiB

Open AccessArticle

Insight into Improved Thermostability of Cold-Adapted Staphylococcal Lipase by Glycine to Cysteine Mutation

by Jiivittha Veno, Raja Noor Zaliha Raja Abd Rahman, Malihe Masomian, Mohd Shukuri Mohamad Ali and Nor Hafizah Ahmad Kamarudin

Molecules 2019, 24(17), 3169; https://doi.org/10.3390/molecules24173169 - 30 Aug 2019

Cited by 16 | Viewed by 2828

Abstract

Thermostability remains one of the most desirable traits in many lipases. Numerous studies have revealed promising strategies to improve thermostability and random mutagenesis often leads to unexpected yet interesting findings in engineering stability. Previously, the thermostability of C-terminal truncated cold-adapted lipase from Staphylococcus [...] Read more.

Thermostability remains one of the most desirable traits in many lipases. Numerous studies have revealed promising strategies to improve thermostability and random mutagenesis often leads to unexpected yet interesting findings in engineering stability. Previously, the thermostability of C-terminal truncated cold-adapted lipase from Staphylococcus epidermidis AT2 (rT-M386) was markedly enhanced by directed evolution. The newly evolved mutant, G210C, demonstrated an optimal temperature shift from 25 to 45 °C and stability up to 50 °C. Interestingly, a cysteine residue was randomly introduced on the loop connecting the two lids and accounted for the only cysteine found in the lipase. We further investigated the structural and mechanistic insights that could possibly cause the significant temperature shift. Both rT-M386 and G210C were modeled and simulated at 25 °C and 50 °C. The results clearly portrayed the effect of cysteine substitution primarily on the lid stability. Comparative molecular dynamics simulation analysis revealed that G210C exhibited greater stability than the wild-type at high temperature simulation. The compactness of the G210C lipase structure increased at 50 °C and resulted in enhanced rigidity hence stability. This observation is supported by the improved and stronger non-covalent interactions formed in the protein structure. Our findings suggest that the introduction of a single cysteine residue at the lid region of cold-adapted lipase may result in unexpected increased in thermostability, thus this approach could serve as one of the thermostabilization strategies in engineering lipase stability. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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17 pages, 2229 KiB

Open AccessArticle

Molecular Characterization and Heterologous Production of the Bacteriocin Peocin, a DNA Starvation/Stationary Phase Protection Protein, from Paenibacillus ehimensis NPUST1

by Chung-Chih Tseng, Lini Murni, Tai-Wei Han, Diana Arfiati, Hui-Tsu Shih and Shao-Yang Hu

Molecules 2019, 24(13), 2516; https://doi.org/10.3390/molecules24132516 - 9 Jul 2019

Cited by 15 | Viewed by 3674

Abstract

The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic Paenibacillus ehimensis NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in Escherichia coli BL21(DE3). Peocin [...] Read more.

The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic Paenibacillus ehimensis NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in Escherichia coli BL21(DE3). Peocin was identified as a DNA starvation/stationary phase protection protein, also called DNA-binding protein from starved cells (Dps), by gel overlay and LC-MS/MS analysis. For mass production of peocin, fed-batch cultivation of E. coli was performed using a pH-stat control system. Purification by simple nickel affinity chromatography and dialysis yielded 45.3 mg of purified peocin from a 20-mL fed-batch culture (49.3% recovery). The biological activity of the purified peocin was confirmed by determination of the MIC and MBC against diverse pathogens. Purified peocin exhibited antimicrobial activity against aquatic, food spoilage, clinical and antibiotic-resistant pathogens. In an in vivo challenge test, zebrafish treated with purified peocin exhibited significantly increased survival rates after A. hydrophila challenge. The present study is the first to show the antimicrobial activity of Dps and provides an efficient strategy for production of bioactive peocin, which will aid the development of peocin as a novel antimicrobial agent with potential applications in diverse industries. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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12 pages, 4435 KiB

Open AccessArticle

A Novel Cold-Adapted and Salt-Tolerant RNase R from Antarctic Sea-Ice Bacterium Psychrobacter sp. ANT206

by Yatong Wang, Yanhua Hou, Ping Nie, Yifan Wang, Xiulian Ren, Qifeng Wei and Quanfu Wang

Molecules 2019, 24(12), 2229; https://doi.org/10.3390/molecules24122229 - 14 Jun 2019

Cited by 17 | Viewed by 2871

Abstract

A novel RNase R, psrnr, was cloned from the Antarctic bacterium Psychrobacter sp. ANT206 and expressed in Escherichia coli (E. coli). A bioinformatics analysis of the psrnr gene revealed that it contained an open reading frame of 2313 bp and [...] Read more.

A novel RNase R, psrnr, was cloned from the Antarctic bacterium Psychrobacter sp. ANT206 and expressed in Escherichia coli (E. coli). A bioinformatics analysis of the psrnr gene revealed that it contained an open reading frame of 2313 bp and encoded a protein (PsRNR) of 770 amino acids. Homology modeling indicated that PsRNR had reduced hydrogen bonds and salt bridges, which might be the main reason for the catalytic efficiency at low temperatures. A site directed mutation exhibited that His 667 in the active site was absolutely crucial for the enzyme catalysis. The recombinant PsRNR (rPsRNR) showed maximum activity at 30 °C and had thermal instability, suggesting that rPsRNR was a cold-adapted enzyme. Interestingly, rPsRNR displayed remarkable salt tolerance, remaining stable at 0.5–3.0 M NaCl. Furthermore, rPsRNR had a higher k_cat value, contributing to its efficient catalytic activity at a low temperature. Overall, cold-adapted RNase R in this study was an excellent candidate for antimicrobial treatment. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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9 pages, 878 KiB

Open AccessArticle

Efficient Conversion of Cane Molasses Towards High-Purity Isomaltulose and Cellular Lipid Using an Engineered Yarrowia lipolytica Strain in Fed-Batch Fermentation

by Zhi-Peng Wang, Qin-Qing Wang, Song Liu, Xiao-Fang Liu, Xin-Jun Yu and Yun-Lin Jiang

Molecules 2019, 24(7), 1228; https://doi.org/10.3390/molecules24071228 - 28 Mar 2019

Cited by 28 | Viewed by 4696

Abstract

Cane molasses is one of the main by-products of sugar refineries, which is rich in sucrose. In this work, low-cost cane molasses was introduced as an alternative substrate for isomaltulose production. Using the engineered Yarrowia lipolytica, the isomaltulose production reached the highest [...] Read more.

Cane molasses is one of the main by-products of sugar refineries, which is rich in sucrose. In this work, low-cost cane molasses was introduced as an alternative substrate for isomaltulose production. Using the engineered Yarrowia lipolytica, the isomaltulose production reached the highest (102.6 g L⁻¹) at flask level with pretreated cane molasses of 350 g L⁻¹ and corn steep liquor of 1.0 g L⁻¹. During fed-batch fermentation, the maximal isomaltulose concentration (161.2 g L⁻¹) was achieved with 0.96 g g⁻¹ yield within 80 h. Simultaneously, monosaccharides were completely depleted, harvesting the high isomaltulose purity (97.4%) and high lipid level (12.2 g L⁻¹). Additionally, the lipids comprised of 94.29% C₁₆ and C₁₈ fatty acids, were proved suitable for biodiesel production. Therefore, the bioprocess employed using cane molasses in this study was low-cost and eco-friendly for high-purity isomaltulose production, coupling with valuable lipids. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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14 pages, 1393 KiB

Open AccessArticle

Amylase and Xylanase from Edible Fungus Neurospora intermedia: Production and Characterization

by Zohre Shahryari, Mohammad H. Fazaelipoor, Younes Ghasemi, Patrik R. Lennartsson and Mohammad J. Taherzadeh

Molecules 2019, 24(4), 721; https://doi.org/10.3390/molecules24040721 - 17 Feb 2019

Cited by 13 | Viewed by 4455

Abstract

Integrated enzyme production in the biorefinery can significantly reduce the cost of the entire process. The purpose of the present study is to evaluate the production of two hydrolyzing enzymes (amylase and xylanase) by an edible fungus used in the biorefinery, Neurospora intermedia [...] Read more.

Integrated enzyme production in the biorefinery can significantly reduce the cost of the entire process. The purpose of the present study is to evaluate the production of two hydrolyzing enzymes (amylase and xylanase) by an edible fungus used in the biorefinery, Neurospora intermedia. The enzyme production was explored through submerged fermentation of synthetic media and a wheat-based waste stream (thin stillage and wheat bran). The influence of a nitrogen source on N. intermedia was investigated and a combination of NaNO₃ and yeast extract has been identified as the best nitrogen source for extracellular enzyme production. N. intermedia enzymes showed maximum activity at 65 °C and pH around 5. Under these conditions, the maximum velocity of amylase and xylanase for starch and xylan hydrolysis was found to be 3.25 U mL⁻¹ and 14.77 U mL⁻¹, respectively. Cultivation of N. intermedia in thin stillage and wheat bran medium resulted in relatively high amylase (8.86 ± 0.41 U mL⁻¹, 4.68 ± 0.23) and xylanase (5.48 ± 0.21, 2.58 ± 0.07 U mL⁻¹) production, respectively, which makes this fungus promising for enzyme production through a wheat-based biorefinery. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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13 pages, 1646 KiB

Open AccessArticle

Anti-α-Glucosidase Activity by a Protease from Bacillus licheniformis

by Chien Thang Doan, Thi Ngoc Tran, Minh Trung Nguyen, Van Bon Nguyen, Anh Dzung Nguyen and San-Lang Wang

Molecules 2019, 24(4), 691; https://doi.org/10.3390/molecules24040691 - 15 Feb 2019

Cited by 20 | Viewed by 3817

Abstract

Anti-α-glucosidase (AAG) compounds have received great attention due to their potential use in treating diabetes. In this study, Bacillus licheniformis TKU004, an isolated bacterial strain from Taiwanese soil, produced AAG activity in the culture supernatant when squid pens were used as the sole [...] Read more.

Anti-α-glucosidase (AAG) compounds have received great attention due to their potential use in treating diabetes. In this study, Bacillus licheniformis TKU004, an isolated bacterial strain from Taiwanese soil, produced AAG activity in the culture supernatant when squid pens were used as the sole carbon/nitrogen (C/N) source. The protein TKU004P, which was isolated from B. licheniformis TKU004, showed stronger AAG activity than acarbose, a commercial anti-diabetic drug (IC₅₀ = 0.1 mg/mL and 2.02 mg/mL, respectively). The molecular weight of TKU004P, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 29 kDa. High-performance liquid chromatography (HPLC) analysis showed that TKU004P may be a protease that demonstrates AAG activity by degrading yeast α-glucosidase. Among the four chitinous sources of C/N, TKU004P produced the highest AAG activity in the culture supernatant when shrimp head powder was used as the sole source (470.66 U/mL). For comparison, 16 proteases, were investigated for AAG activity but TKU004P produced the highest levels. Overall, the findings suggest that TKU004P could have applications in the biochemical and medicinal fields thanks to its ability to control the activity of α-glucosidase. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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10 pages, 2078 KiB

Open AccessArticle

Purification and Characterization of A New Cold-Adapted and Thermo-Tolerant Chitosanase from Marine Bacterium Pseudoalteromonas sp. SY39

by Yu Zhou, Xuehong Chen, Xiao Li, Yantao Han, Yanan Wang, Ruyong Yao and Shangyong Li

Molecules 2019, 24(1), 183; https://doi.org/10.3390/molecules24010183 - 6 Jan 2019

Cited by 34 | Viewed by 3716

Abstract

Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new chitosanase, CsnM, with an apparent [...] Read more.

Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides. Full article

(This article belongs to the Special Issue Biotechnology of Microbial Enzymes)

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