Improved phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation due to statistical optimization

doi:10.1016/j.biortech.2007.01.007

Bioresource Technology

Volume 99, Issue 4, March 2008, Pages 824-830

https://doi.org/10.1016/j.biortech.2007.01.007 Get rights and content

Abstract

Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation were optimized. Soluble starch, peptone, Tween-80 and sodium phytate were identified by Plackett–Burman design as the most significant factors to affect phytase production. The 2⁴ full factorial central composite design of response surface methodology was applied for optimizing the concentrations of the significant variables and to delineate their interactions. Starch, Tween-80, peptone and sodium phytate at 0.4%, 1.0%, 0.3% and 0.3% supported maximum enzyme titres, respectively. An overall 3.73-fold improvement in phytase production was achieved due to optimization. When sodium phytate was substituted with wheat bran (3%), the phytase titre in the former was comparable with that in the latter.

Introduction

The screening and evaluation of nutritional and other culture variables of a microorganism is an important step in the development of a bioprocess. Since microbial fermentations are affected by cultural variables, a suitable approach has to be applied for their optimization. The conventional methods used in the optimization of the cultural variables are time consuming, tedious and expensive (Vohra and Satyanarayana, 2002, Bogar et al., 2003a, Bogar et al., 2003b). These methods also tend to overlook the effects of interactions among them. Optimization of all the variables by statistical experimental designs can eliminate the limitations of ‘one variable at a time’ approach (Stanbury et al., 1997). Several research groups have applied this approach for screening and optimization of the parameters affecting phytase production (Sunitha et al., 1999, Vohra and Satyanarayana, 2002, Kaur and Satyanarayana, 2005). The use of statistical approaches helped in enhancing yield of yeast phytase that led to the reduction in the cost of production, and therefore, making the fermentation process economical and cost effective (Vohra and Satyanarayana, 2002, Kaur and Satyanarayana, 2005).

Phytases (myo-inositol hexaphosphate phosphohydrolase EC 3.1.3.8) are histidine acid phosphatases, a subclass of acid phosphatases which catalyze the hydrolysis of phosphate moieties from phytic acid (myo-inositol hexakis dihydrogen phosphate), thereby mitigating its anti-nutritional properties (Vohra and Satyanarayana, 2003, Greiner and Konietzny, 2006). The market volume of phytases is in the range of ∈ 150 milllion which is still rising further (Greiner and Konietzny, 2006). The supplementation of animal feed with phytases enhances the bioavailability of minerals, protein and phosphorus in monogastric animals, besides reducing the phosphorus pollution in the areas of intensive livestock production. In grains, roughly 60–80% of phosphorus is tied up in phytin, which is not digestible by monogastric animals due to the lack of adequate levels of phytase in their digestive tracts. The phytin thus excreted in the manure reaches the soil, where it is degraded by microflora that leads to the eutrophication of water bodies and thus environmental pollution (Wodzinski and Ullah, 1996). The pollution can be tackled by supplementing animal feeds with phytase.

In view of ever increasing demand for phytase, it is essential to produce phytase in a cost-effective manner. In this investigation, an attempt has been made to improve phytase production by a thermophilic mould Sporotrichum thermophile by statistical approaches using Plackett–Burman design and response surface methodology (RSM) in submerged fermentation.

Section snippets

Source of the strain and inoculum preparation

The thermophilic mould, S. thermophile BJTLR50 was isolated from a soil sample collected from Rohtak, Haryana (India), and routinely grown on Emerson’s YpSs (Emerson, 1941) agar medium (Singh and Satyanarayana, 2006). The conidiospores from 6 day-old fully sporulated solid media slopes were harvested by flooding with normal saline containing 0.1% (v/v) Tween-80 and the spore suspension thus prepared was adjusted to 1 × 10⁷ CFU/ml for inoculating the medium.

Phytase assay

Phytase was assayed by measuring the

The optimization of medium components for phytase production by S. thermophile

Plackett–Burman design is a widely used statistical design for the screening of the medium components (Plackett and Burman, 1946). The design screens important variables that affect enzyme production as well as their significant levels, but does not consider the interaction effects among the variables as in RSM. In RSM, each selected variable is studied at five different levels along with other variables, and therefore, the interactions among the variables at their different levels could be

Conclusions

Plackett–Burman design made possible to consider a large number of variables and to identify the most important variables that affect phytase production. Plackett–Burman and RSM designs have been proved to be effective in optimizing phytase production by S. thermophile in submerged fermentation, which resulted in a 3.73-fold enhancement in phytase production. Sodium phytate could be substituted with wheat bran that resulted in sustained secretion of phytase, and thus making the fermentation

Acknowledgements

BS gratefully acknowledges the financial assistance from the Council of Scientific and Industrial Research (CSIR, New Delhi, India) by awarding Junior/Senior Research Fellowship during the course of this investigation.

References (24)

P. Kaur et al.
Production of cell-bound phytase by Pichia anomala in an economical cane molasses medium: optimization using statistical tools
Process Biochem.
(2005)
O.H. Lowry et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem.
(1951)
A. Pandey et al.
Production, purification and properties of microbial phytases
Biores. Technol.
(2001)
M. Papagianni et al.
Production of phytase by Aspergillus niger in submerged and solid state fermentation
Process Biochem.
(1999)
P. Vats et al.
Production studies and catalytic properties of phytases (myo-inositolhexakisphophate phosphohydrolases): an overview
Enzym. Microb. Technol.
(2004)
A. Vohra et al.
Statistical optimization of the media components by response surface methodology to enhance phytase production by Pichia anomala
Process Biochem.
(2002)
B. Bogar et al.
Optimization of phytase production by solid substrate fermentation
J. Ind. Microbiol. Biotechnol.
(2003)
B. Bogar et al.
Production of phytase by Mucor racemosus in solid-state fermentation
Biotechnol. Progr.
(2003)
B.S. Chadha et al.
Phytase production by the thermophilic fungus Rhizomucor pusillus
World J. Microbiol. Biotechnol.
(2004)
R. Emerson
An experimental study of life cycle and taxonomies of Allomyces
Lloydia
(1941)

C.H. Fiske et al.

The colorimetric determination of phosphorus

J. Biol. Chem.

(1925)

R. Greiner et al.

Phytase for food application

Food Technol. Biotechnol.

(2006)

Cited by (51)

Microbial phytase: Their sources, production, and role in the enhancement of nutritional aspects of food and feed additives
2023, Journal of Agriculture and Food Research
Phosphorous is one of the important elements for life but its surplus amount is not good. It can be retrieved for being reused but can not be produced. It's a good thing that phosphorus can be retrieved and used again since it cannot be produced. Reducing, Reusing, and Recycling is the only method to prevent a supply shortage. Phytic acid is a common putative nutritional supplement found in many plant-based diets that functions as the primary phosphorus storage form. However, the animals are unable to effectively use it, resulting in two major issues: the requirement of inorganic phosphorus supplementation in their diets and the excretion of a considerable amount of phosphorus in manure. Another nutritional problem is that phytate has been proven to form complexes with metal ions, rendering them inaccessible to the body through the diet. As a result, interest has grown in the finding of natural enzymes that are produced as a byproduct or consequence of continual metabolic activity within living organisms. In the next coming years, phytase is likely to play a key role in the dephosphorylation of phytate, a phosphorus-locking molecule that is antinutritional and indigestible, into digestible phosphorus, calcium, and other mineral elements. Because the existing technique is costly and time-consuming, it is necessary to make efforts to produce cost-effective phytase with quick upstream and economical downstream processing. The current review highlights the sources of phytase, its production strategies, and its application in various sectors.
Contribution of microbial phytases to the improvement of plant growth and nutrition: A review
2020, Pedosphere
Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in plant cells. Phytase has been used globally to diminish phosphorus pollution and to enhance nutrition in monogastrics. In this review, the classification, sources, and diversity of microbial phytases, and their practical applications, as well as supplementation of the soil with transgenic and wild types of microbial strains, which can release phytase to enhance phosphorus availability for plant uptake and reduce the need for fertilizers, are discussed. The overexpressed microbial phytases in transgenic plants enhance the growth capacity of co-cultivated plants and can therefore be employed in agricultural and biotechnological practices, such as intercropping. The introduction of phytases into the soil for improved plant growth and enhanced crop yield can be accomplished without extra cost. A diverse group of photoautotrophic microalgae can synthesize phytase and will likely be useful in many human food and animal industries.
Bioprospects of extremophilic fungus Myceliopthrora thermophila: Insights from genomic analysis and recent developments
2020, Fungi Bio-prospects in Sustainable Agriculture, Environment and Nano-technology: Volume 2: Extremophilic Fungi and Myco-mediated Environmental Management
Filamentous fungi are the predominant microorganisms used to produce various industrially important products including enzymes, organic acids, etc. Thermophilic fungi have the to thrive between 45 and 55 °C and are particularly advantageous for the large-scale production of several compounds by fermentation. One such well-studied thermophilic fungi is Myceliophthora thermophila, which has strong ability to degrade biomass during its optimal growth at 45–55 °C. It produces a repertoire of biomass hydrolyzing enzymes which find many applications in biotechnology due to their higher stability and better catalytic rates at elevated temperatures during the process. In recent times, this fungus has been improved through metabolic engineering and other strain improvement strategies. The genome sequence data of M. thermophila has further helped in better understanding of its physiology for industrial exploitation. This chapter provides insights on bioprospects of M. thermophila supported by recent developments.
Molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould Sporotrichum thermophile reveals insights into molecular catalysis and biochemical properties
2018, International Journal of Biological Macromolecules
Citation Excerpt :
Moreover, this is the first report showing molecular docking with different substrates and inhibitors revealing reasons for its broad substrate specificity and role of important residues in catalysis. Sporotrichum thermophile BJTLR50 has been reported to secrete phytase in submerged [9] and solid state fermentations [10,11]. Pichia pastoris X-33 (Invitrogen, USA) was used as the host to express recombinant phytase (rSt-Phy) of S. thermophile BJTLR50 [12].
A thermostable and protease-resistant HAP-phytase of Sporotrichum thermophile was over-expressed in Pichia pastoris X-33. Purified recombinant phytase displayed all its biochemical properties similar to wild type. Molecular modeling and docking of phytase with various substrates showed differential binding patterns with GoldScore values ranging from 40.61 to 79.78. Docking with different substrates revealed strong binding affinity with ATP and phytic acid, while the lowest with AMP and phosphoenol pyruvate. This was further confirmed using biochemical assays, as the recombinant enzyme displayed broad substrate specificity. Docking with inhibitors also showed differential binding with GoldScore values ranging from 22.94 (2,3-butanedione) to 85.72 (myo-inositol hexasulphate). Validation using biochemical analysis revealed that both 2,3-butanedione and phenyl glyoxal inhibited the phytase activity significantly. Furthermore, presence of inorganic phosphate in the reaction mixture also inhibited the phytase activity, as there was no activity at and beyond 0.8 mM. Docking of phytase with metavanadate showed binding at the same atom in the active-site where the substrate i.e. phytic acid binds. Vanadium incorporation resulted in the catalytic conversion of phytase into a peroxidase with concomitant inhibition of phytase activity. Peroxidase activity was high in acidic range and the product formation showed correlation with reaction time. Furthermore, molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould S. thermophile reveals insights into molecular catalysis that is validated by the biochemical properties.
Multi-objective Based Superimposed Optimization Method for Enhancement of L-Glutaminase Production by Bacillus subtilis RSP-GLU
2018, Karbala International Journal of Modern Science
l-Glutaminase (l-glutamine amidohydrolase, EC 3.5.1.2) is a deamiding enzyme that has current focus on a therapeutic potentials and flavor enhancement agent in food industry. It was noticed that the amidase production was depended on the bacterial growth. However, the enhancement of bacterial growth by other substrates suppress the secretion of l-glutaminase. Governing the growth as well as amidase production is challenging task. In this study a multi-objective based superimposed optimization method was employed to control the biomass as well as to enhance the production of l-glutaminase. Various parameters such as volume of medium, pH, incubation temperature, inoculum level, l-glutamine concentration and rpm were optimized. The interactive influence of these parameters with their effect on enzyme and biomass production was analyzed using fractional factorial central composite design (FFCCD). The coefficient of determination (R²) was calculated as 0.9862 and 0.9513 for l-glutaminase and biomass production respectively. Overlay graphs were plotted by superimposing the contours for the various response surfaces of biomass and enzyme production. Based on superimposing method the cultural conditions obtained was volume of media 85 mL, temperature 37 °C, pH 7.24, inoculum concentration 2.47 mL, l-glutaminase concentration 1.01 g/L and agitation speed of 109 rpm. At these conditions 225 U/mL l-glutaminase and 0.79 g/L biomass were obtained. The current study provides a good description for balanced optimization of fermentation process of multi response or objective systems by using FFCCD method.
Evaluation of Candida tropicalis (NCIM 3321) extracellular phytase having plant growth promoting potential and process development
2018, Biocatalysis and Agricultural Biotechnology
Phytase is known to provide a solution for depletion of phosphorus (P). It helps it by hydrolyzing the insoluble P source in soil which is phytate. In this study, provides insight on yeast Candida tropicalis (NCIM 3321) which produces cell bound and extracellular thermostable phytase. The media components were optimized to enhance the enzyme production and checked for plant growth promoting activity. On optimization the isolate exhibited enhanced cell bound and extracellular phytase activity by four folds (from 236 to 1024 IU DCG⁻¹) and by five folds (from 0.46 to 1.95 IU ml⁻¹) respectively in 36 h. The production time decreased to 24 h compare to shake flask on Up-scaling the production process upto 10 L scale, thus increasing the productivity of cell bound (1810 IU DCG⁻¹day⁻¹) and extracellular phytase (6.08 IU ml⁻¹ day⁻¹). The crude phytase (12 IU) from NCIM 3321 strain was studied for plant growth promotion activity in lab scale and field level experiments with maize crop. Findings of the study revealed that the extracellular phytase derived from non pathogenic C. tropicalis (NCIM 3321) was found to be plant growth stimulating by increasing the available P in soil. Our findings of phytase isolated from non-pathogenic yeast C. tropicalis NCIM 3321 exhibited dephytinization potential. Therefore, current study may have profound application in sustainable agriculture.

View all citing articles on Scopus

View full text

Improved phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation due to statistical optimization

Abstract

Introduction

Section snippets

Source of the strain and inoculum preparation

Phytase assay

The optimization of medium components for phytase production by S. thermophile

Conclusions

Acknowledgements

Process Biochem.

J. Biol. Chem.

Biores. Technol.

Process Biochem.

Enzym. Microb. Technol.

Process Biochem.

Optimization of phytase production by solid substrate fermentation

J. Ind. Microbiol. Biotechnol.

Production of phytase by Mucor racemosus in solid-state fermentation

Biotechnol. Progr.

Phytase production by the thermophilic fungus Rhizomucor pusillus

World J. Microbiol. Biotechnol.

An experimental study of life cycle and taxonomies of Allomyces

Lloydia

The colorimetric determination of phosphorus

J. Biol. Chem.

Phytase for food application

Food Technol. Biotechnol.