Industrial enzymes have been widely preferred in various industries such as chemical production, food & beverage, pharmaceutical, textile, cosmetics, etc. due to the advancements in recent years. ...They are considered more economic than using whole cells and more environmental-friendly than chemical alternatives. Since the demand for industrial enzymes has been rising, the development of production strategies has been gathered speed. In this respect, the efficiency of
Pichia pastoris
(
P. pastoris
) as a host for heterologous protein expression has proved and gained attention due to its great potential for large-scale studies. Especially high-cell density fermentation of
P. pastoris
is a well-studied and efficient method. Moreover, the improvements in the state of art gene-editing tools have broadened the possibilities of strain improvement for
P. pastoris
. This review summarized the role of
P. pastoris
as a cell factory by accentuating the accomplishments in biocatalyst production. Moreover, the benefits and challenges of the most relevant expression systems named
Escherichia coli
(
E. coli
),
Saccharomyces cerevisiae
(
S. cerevisiae
),
P. pastoris
and recent evolvements and future directions were revealed in detail. Subsequently, offers for prospects and the latest evolvements to enhance the recombinant protein production were discussed.
Valorization of waste materials has been an attractive approach due to ensuring circular bioeconomy model in bioprocesses depending on annual billion metric tons production of agro-industrial ...by-products and wastes. With being an influential platform of Pichia pastoris fermentation to produce value-added bioproducts, especially for recombinant proteins, the studies have been gaining speed to apply the circular bioeconomy model to these processes by enhancing production efficiency. In this study, solid and liquid dextrose from a local corn processing industry and watermelon peel from a local market were evaluated for production of Madurella mycetomatis laccase from P. pastoris X33 in a bioreactor. When compared to fermentation including alternative sources and glycerol/methanol which was performed as a control study, almost 2-fold higher laccase activity with 41 g DCW/L and 1.30-fold increase in laccase activity with 50 g DCW/L were determined, respectively for fermentation strategies including solid dextrose and liquid dextrose. Maximum laccase activity was obtained from watermelon peel hydrolysate utilization although lowest cell density was obtained in the growth stage. One-way ANOVA analysis confirmed the effect of carbon sources encouraging heterologous protein production in P. pastoris expression system. These results proved suitability of bioprocesses which were designed for the valorization of solid/liquid dextrose and watermelon peel hydrolysate as alternative carbon sources in P. pastoris fermentation for future scale-up.
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•Utilization of agro-industrial waste and by-products for P. pastoris fermentation was employed.•Usability potential of alternative carbon sources in recombinant protein production from P. pastoris was determined.•Laccase activity was enhanced by the valorization of by-products and waste as a carbon source in a complex medium.•ANOVA analysis was assigned effect of agro-industrial wastes on recombinant protein production in P. pastoris.
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•M. mycetomatis laccase was heterologously produced in P. pastoris and purified.•The laccase immobilised in a MOF followed by silica encapsulation.•The immobilization of laccase was ...confirmed by FT-IR, SEM and SEM-EDS techniques.•The immobilized laccases showed a better performance than free MmLac in bleaching.
In this study, a laccase from Madurella mycetomatis (MmLac) was produced heterologously in Pichia pastoris; the initial immobilization in a metal-organic framework (MOF) (MmLac/ZIF-8) was achieved using zinc nitrate and 2-methylimidazole. Due to the instability of MmLac/ZIF-8 in an acidic medium, a silica layer was created on the surface of MmLac/MOF-8. The immobilized laccase composite (silica@MmLac/ZIF-8) obtained was further treated with glutaraldehyde (silica@Glu-MmLac/ZIF-8) to increase stability of composite. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques were used to confirm the immobilization of MmLac and to investigate the morphology of the immobilized laccase samples. The MmLac samples were also characterised in terms of optimum pH, temperature and thermal stability. The optimum pH of all the MmLac samples was determined to be 4.0. The free MmLac showed maximum activity at 55 °C, whereas both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were maximumly active at 65 °C. The silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were 9.3- and 11.8-fold higher in stability, respectively, than the free MmLac at 65 °C. Furthermore, both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 showed a higher bleaching performance than free MmLac on cotton woven fabric. According to these results, silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 may be promising candidates for biocatalysts in laccase-based biotechnological applications.
In recent years, CO
2
reduction and utilization have been proposed as an innovative solution for global warming and the ever-growing energy and raw material demands. In contrast to various classical ...methods, including chemical, electrochemical, and photochemical methods, enzymatic methods offer a green and sustainable option for CO
2
conversion. In addition, enzymatic hydrogenation of CO
2
into platform chemicals could be used to produce economically useful hydrogen storage materials, making it a win-win strategy. The thermodynamic and kinetic stability of the CO
2
molecule makes its utilization a challenging task. However, Nicotine adenine dinucleotide (NAD
+
)-dependent formate dehydrogenases (FDHs), which have high selectivity and specificity, are attractive catalysts to overcome this issue and convert CO
2
into fuels and renewable chemicals. It is necessary to improve the stability, cofactor necessity, and CO
2
conversion efficiency of these enzymes, such as by combining them with appropriate hybrid systems. However, metal-independent, NAD
+
-dependent FDHs, and their CO
2
reduction activity have received limited attention to date. This review outlines the CO
2
reduction ability of these enzymes as well as their properties, reaction mechanisms, immobilization strategies, and integration with electrochemical and photochemical systems for the production of formic acid or formate. The biotechnological applications of FDH, future perspectives, barriers to CO
2
reduction with FDH, and aspects that must be further developed are briefly summarized. We propose that constructing hybrid systems that include NAD
+
-dependent FDHs is a promising approach to convert CO
2
and strengthen the sustainable carbon bio-economy.
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•A critical Asp188 at the subunit interface was discovered by molecular dynamics.•Integrating mutation-immobilization provides ∼25-fold increase in carbonate activity.•Formic acid ...production was increased ∼2-fold with the immobilized mutant enzyme.•An enhanced enzymatic method for hydrocarbons production from renewable sources.
Formate dehydrogenase (NAD+-dependent FDH) is an enzyme that catalyzes the reversible oxidation of formate to CO2 while reducing NAD+ to NADH. The enzyme has been used in industrial and chemical applications for NADH regeneration for a long time. However, discovering the unique ability of FDHs, which is to reduce CO2 and produce formic acid, leads studies focusing on discovering or redesigning FDHs. Despite using various protein engineering techniques, these studies mostly target the same catalytic site amino acids of FDHs. Here, for the first time, the effect of an Asp188 mutation on a potential allosteric site in NAD+-dependent CtFDH around its subunit-subunit interface was studied by molecular modelling and simulation in the presence of bicarbonate and formate. Biochemical and kinetic characterization of this Asp188Arg mutant and wild type CtFDH enzymes were performed in detail. Both enzymes were also immobilized on newly synthesized MWCNT-Ni-O-Si/Ald and MWCNT-Ni-O-Si/Glu supports designed to overcome well-known CtFDH stability problems including thermostability and reuse resistance. Integrating mutation and immobilization provided about a 25-fold increase in catalytic efficiency for carbonate activity. The one-way ANOVA analysis also ensured significant effect of the mutation and immobilization on kinetic constants. After characterizing the immobilization of highly purified wild type and mutant enzyme with instrumental analysis techniques, the thermal stability of MWCNT-Ni-Si@wt-CtFDH and MWCNT-Ni-Si@mt-CtFDH was found to increase about 11- and 18-fold, respectively, compared to their free counterparts at 50 °C. The mutant CtFDH and its immobilized counterpart produced around 2-fold more formic acid than those of wild type CtFDH and its immobilized counterpart under the same conditions. MWCNT-Ni-Si@wt-CtFDH and MWCNT-Ni-Si@mt-CtFDH remained around 82 % and 86 % of their initial activities respectively after lots of recycling. Integration of subunit interface amino acid position of NAD+ dependent FDHs engineering and immobilization provides a new insight can be scientifically and rationally employed for this current application FDHs as a solution to produce formic acids from renewable sources.
The biochemical conversion route in the utilization of biomass has required intensive development of processing methods for reducing recalcitrance of cellulose to enzymatic hydrolysis. Since ...discovering hydrophilic ionic liquids (ILs) are efficient lignocellulose solvents, considerable efforts have been made to demonstrate their potential applications in cellulose hydrolysis. However, most of the commercial lignocellulose hydrolyzing enzymes are largely deactivated or inhibited in the presence of even low concentrations of ILs. The link found between enzyme thermostability and IL tolerance leads to the consideration that hyperthermostable enzymes are good candidates to be used together with ILs. Hyperthermostable cellulases and xylanases that are highly tolerant to aqueous solutions of ILs show resistance to competitive inhibition by IL cations and have lower amounts of structural motifs (loop and helix structures) that are prone to denaturation by IL anions. In addition, they have a net negative protein surface charge that contributes to repulsion of anions. This review provides recent developments in understanding the behaviour of hyperthermostable cellulases and xylanases in ILs by elucidating protein-IL interactions. It also outlines the current research demonstrating the potential of rational enzyme engineering to improve enzyme tolerance to ILs based on comprehension of IL-induced deactivation mechanisms.
In this study, an epoxide hydrolase gene (hdeh) from extremophile Hypsibius dujardini was expressed heterologously in Pichia pastoris under the optimized conditions. The purified HdEH was immobilized ...in a metal-organic framework (HdEH@ZIF-8) using zinc nitrate and 2-methylimidazole. The HdEH@ZIF-8 was further treated with different concentration of glutaraldehyde (HdEH@Glu/ZIF-8) to investigate the effect of glutaraldehyde concentration on the activity and stability. The cross-linking of HdEH@ZIF-8 with glutaraldehyde resulted in decreasing the activity, while increasing the thermal stability. The optimum pH of free and immobilized HdEH samples was all determined as 8.0. The free and both immobilized HdEHs exhibit its optimal activity at 20 °C and 30 °C respectively. The HdEH@ZIF-8 and HdEH@Glu/ZIF-8 showed around 16- and 18-fold higher stability than free HdEH at 30 °C. All the HdEHs were used in the asymmetric hydrolysis of racemic styrene oxide (R/S-SO) and the results showed that R-enantiomer of epoxide was preferentially hydrolyzed by free HdEH, HdEH@ZIF-8 and HdEH@Glu/ZIF-8. After 360 min reaction time, (S)-styrene oxide was obtained with 99% enantiopurity for the all the HdEH preparations. HdEH@ZIF-8 and HdEH@Glu/ZIF-8 showed 1.9- and 1.4-fold higher enantioselectivity than that of the free HdEH. HdEH@ZIF-8 and HdEH@Glu/ZIF-8 were remained 91% and 93% of its initial activity after 5 reuses. The results show that HdEH@ZIF-8 and HdEH@Glu/ZIF-8 are a promising stable and enantioselective EH preparations for the asymmetric hydrolysis of racemic styrene oxide.
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•The novel epoxide hydrolase was produced heterologously from Hypsibius dujardini.•HdEH was immobilized in ZIF-8 using zinc nitrate and 2-methylimidazole.•The immobilization of HdEH was approved by instrumental techniques.•Thermal stability of HdEH increased up to 18 folds after immobilization at 30 °C.•The enantioselectivity of HdEH increased at least 1.4 folds after immobilization.
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