Effect of aeration and agitation on yeast inulinase production: a biocalorimetric investigation

Santharam, Leelaram; Easwaran, Sivanesh Nanjan; Mohanakrishnan, Anusha Subramanian; Surianarayanan, M.

doi:10.1007/s00449-019-02101-0

Cited by 10 publications

(3 citation statements)

References 30 publications

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“…Considering that K. marxianus is more strictly Crabtree negative than the model organism species Kluyveromyces lactis and other known Crabtree-negative yeasts, it cannot grow under strictly anaerobic conditions and its ethanol fermentation exclusively relies on oxygen limitation [ 16 ]. As described previously, dissolved oxygen tension is a key factor for the production of inulinase in K. marxianus [ 54 , 55 ]. In this study, expressions of the hemicellulolytic enzymes in the IMPαXPαR strain were samely driven by the inulinase promoter.…”

Section: Resultsmentioning

confidence: 99%

Coordinately express hemicellulolytic enzymes in Kluyveromyces marxianus to improve the saccharification and ethanol production from corncobs

Lan

Duan

et al. 2021

Biotechnol Biofuels

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Background Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K.marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. Results Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K.marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. Conclusions The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

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Section: Resultsmentioning

confidence: 99%

Coordinately express hemicellulolytic enzymes in Kluyveromyces marxianus to improve the saccharification and ethanol production from corncobs

Lan

Duan

et al. 2021

Biotechnol Biofuels

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“…Low agitation speed fails to generate sufficient turbulence and hold up air bubbles and consequently influences volumetric mass transfer due to high medium [8,15]. Low oxygen solution rate subsequently results in stunted bacterial growth and low metabolite production [24]. Similarly, lower keratinase production due to poor mixing as well as lower dissolved oxygen has been reported from Bacillus sp.…”

Section: Discussionmentioning

confidence: 99%

Optimization and validation of keratinase production by Bacillus aerius NSMk2 in a stirred tank reactor using response surface methodology

2021

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Keratinase is a robust enzyme that is produced in the presence of keratin substrates. This enzyme has been recognized for its applications in waste management, leather and detergent industries. Our group has isolated a potential keratinase producing strain of Bacillus aerius NSMk2 from poultry dump soil, and its hide dehairing and stain removal applications have been studied. Considering commercial applicability of keratinase, the present study reports the keratinase production in a stirred tank reactor (5 l). Central composite rotatable design of response surface methodology (RSM) was employed to study the effect of most influencing process variables, i.e., aeration (0.5–1.5 vvm), agitation (150–350 rpm) and incubation period (24–48 h) on keratinase production. The quadratic model predicted 15 experimental runs, and the influence of independent variables and their interaction on keratinase production were interpreted using analysis of variance (ANOVA) and t-test statistics. Coefficient of determination (R2) value close to 1 and Fisher F-value of 3743.77 showed good fit of experimental data to second-order polynomial equation. A reasonable agreement between experimental and predicted values showed the accuracy of deduced model. Applying the desirability function, aeration rate of 1.0 vvm, agitation rate of 276.88 rpm and incubation period of 33.68 h supported maximum keratinase production (318.38 U/ml). Confirmatory experiments were performed to evaluate the accuracy of desirability function. Maximum keratinase activity of 318.11 U/ml close to predicted value (318.38 U/ml) validates the model. The present study provides useful guidelines for large-scale production of keratinase that can be used for various commercial applications. Article highlights Keratinase production was optimized in a stirred tank reactor by RSM. The influence of aeration, agitation and incubation period on keratinase production was studied. A significant increase in keratinase production was observed at 1.0 vvm aeration and 276.88 rpm agitation after 33.68 h.

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“…Considering that, K. marxianus is more strictly Crabtree-negative than the model organism species Kluyveromyces lactis and other known Crabtree-negative yeasts that it cannot grow under strictly anaerobic conditions and its ethanol fermentation exclusively relies on oxygen limitation [58][59][60]. Meanwhile, dissolved oxygen tension is a key factor for the production of inulinase in K. marxianus [61,62]. Since expressions of hemicellulolytic enzymes were drived by the inulinase promoter in the IMPαXPαR strain, to ensure the hemicellulolytic enzymes were highly expressed and su cient for HSFs, HSFs starters were prepared by high-cell-density fed-batch cultures in 5L fermenters under aeration and agitation.…”

Section: Hsfs Of Ethanol From Pretreated Corncobsmentioning

confidence: 99%

Coordinately Express Hemicellulolytic Enzymes in Kluyveromyces Marxianus to Improve the Saccharification and Ethanol Production from Corncobs

Lan

Duan

et al. 2021

Preprint

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BackgroundHemicelluloses act as one factor contributing to the recalcitrance of lignocelluloses that prevent cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianu is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K. marxianus through a 2A-mediated ribosomes skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs.ResultsTwo polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from Foot and Mouth Disease virus (FMDV) in K. marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn, while in the IMPαX gene there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of IMPX and IMPαX strains were 21.34 and 15.50 U/mL repectively. By contrast, the IMPαX strain secreted 136.17 U/mLof the β-xylanase, which was much higher than that of IMPX strain 42.07 U/mL. Based on these, two recombinant strains, the IXαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes a β-D-xylosidase RuXyn1 and Xyn-CDBFV, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentations, extracellular activities of β-xylanase and β-xylosidase in the IMPαX strain were 1664.2 and 0.90 U/mL, while productions of secretory β-mannanase, β-xylanase, and β-xylosidase in the IMPαXPαR strain were 159.8, 2210.5, and 1.25 U/ml of respectively. Hemicellulolytic enzymes of these two strains enhanced the yields of both glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yields by 8.7% at 144 h. When using aqueous ammonia pretreated (AAP) corncobs as HSF feedstocks, the IMPαXPαR strain increased both ethanol and xylose yields, which were about 12.7% and 18.2% more than that of the control at 120 h. Our results indicated that coordinately expression of hemicellulolytic enzymes in K. marxianus could promote the saccharification and ethanol production from corncobs.ConclusionsThe FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus, and could be used for secretory expression of multiple enzymes in present of their own signal sequences. The IMPαXPαR strain that coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

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Effect of aeration and agitation on yeast inulinase production: a biocalorimetric investigation

Cited by 10 publications

References 30 publications

Coordinately express hemicellulolytic enzymes in Kluyveromyces marxianus to improve the saccharification and ethanol production from corncobs

Coordinately express hemicellulolytic enzymes in Kluyveromyces marxianus to improve the saccharification and ethanol production from corncobs

Optimization and validation of keratinase production by Bacillus aerius NSMk2 in a stirred tank reactor using response surface methodology

Coordinately Express Hemicellulolytic Enzymes in Kluyveromyces Marxianus to Improve the Saccharification and Ethanol Production from Corncobs

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