Characterization of Crude Cellulases From a Bacillus Sp. Isolated From Lake Bogoria, Kenya

doi:10.21203/rs.3.rs-41635/v1

Download PDF

Short report

Characterization of Crude Cellulases From a Bacillus Sp. Isolated From Lake Bogoria, Kenya

https://doi.org/10.21203/rs.3.rs-41635/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

The demand for a carbohydrate driven economy coupled with the abundant cellulosic biomass has driven the search for cellulases that can be applied for the utilization of the cellulosic biomass.

Context of the study

Of special focus for our study is cotton, which is the preferred textile fiber given its favorable high quality. In addition, sugar, which is an important feedstock for several chemical industries, can be readily produced from cellulosic biomass.

Purpose of the study

We therefore sought to characterize cellulases from favorable ecosystems in order to gauge the validity of their inclusion in cellulosic biomass industrial utilization.

Conclusion

We have shown the production of saccharides from cellulosic biomass (cotton and filter paper). Further, we established the stability of the cellulases in 40% (v/v) organic solvents; propanol, methanol, ethanol. In addition, the enzymes showed tolerance to the accumulation of sugars. Moreover, the cellulase enzymes were most stable at pH 6 and demonstrated a wide activity temperature range of 20°-80°C. Optimal cellulase activity was recorded at 60°C confirming the thermophilic nature of the enzymes.

Brief Summary

This validates and shows the potential of these enzymes in co-expression bio-factories and applications requiring high sugar yields

Potential Implications

This work adds to the study on the activity of the pure extracted cellulases from this microorganism under review at the journal of applied Biochemistry and Biotechnology. These findings substantiate the potential inclusion of either this microorganism or its isolated/extracted enzymes in the industrial processing of cellulosic biomass including but not limited to cotton and paper.

General Biochemistry

Cellulase

Bacillus

cellulose hydrolysis

Cotton

Filter paper

Previously, chemical methods of cotton treatment were employed in the textile industry. However, these ancient methods have presented with problems of environmental pollution, high temperature requirements, high alkaline pH and excessive energy costs involved in the maintenance of the high temperature and in the removal of the vast residual wastes during wastewater treatment (Verenium-cottonase ® and Novozymes).

This escalated production cost due to huge alkali chemical requirements, water requirements and energy costs as well as taxing decontamination processes and the poor fabric quality obtained at the end of the process, have driven the need for alternative processes that can alleviate the environmental exertions, reduce processing costs and minimize chemical requirements hence making the processes more sustainable.

Biological solutions in the form of enzymes have been found to alleviate the inconveniences associated with chemical treatments as they work optimally under mild conditions with minimal residual wastes. Moreover, the fabric obtained at the end of the process, is of good quality.

In nature, multiple enzymes are employed by microorganisms to efficiently degrade plant cell wall cellulosic polysaccharide material. In fact, microorganisms employ three principal systems for enzymatic breakdown of cellulosic plant cell wall. These work either in combination or in separation and include; free enzymes, multifunctional enzymes and multi enzyme complexes for example, cellulosome (Mcclendon et al., 2012).

Applications of cellulases have increased considerably especially in the textile industry during the last two decades. These include bio processing of natural fibers, bio polishing of cotton fabrics in order to enhance the softness and feel, appearance and treatment of recycled fibres to restore fibre texture and flexibility lost during operations (Karmakar & Ray, 2011).

It is therefore vital to bioprospect for and characterize novel cellulases as well as novel cellulase producers. This study aimed at characterizing new cellulase enzymes from the thermophilic, alkalophilic and saline Lake Bogoria found in the Rift valley region of Kenya.

Chemicals

G-75 sephadex (1.7 X 30cm, Pharmacia Fine chemicals, Sweden), salts used in making of microbial media were of analytical grade, TLC plates, TLC sprayer (CAMAG), sugars used here were of analytical grade, cotton, and p-anisaldehyde for TLC visualization.

Strains and media

Luria Bertani (LB) medium was used for Bacillus sp. LAF-A8 (gb|MT682523), cell growth , Mineral salt Medium (MSM) supplemented with 0.5% sodium carboxymethylcellulose (Na-CMC) was used for production of cellulase enzymes.

Enrichment medium

Bacillus sp. LAF-A8 were grown in Luria bertani (LB) nutrient medium (10g/l tryptone, 5g/l yeast, 10g/l NaCl). This was done in order to enrich the culture before sub-culturing in supplemented MSM with Na-CMC as the sole carbon source.

Enzyme Production

LB overnight culture (1ml) was inoculated into MSM made of tryptone 0.5%, yeast extract 0.1% , Na-CMC 0.5% , K₂HPO₄0.08% , KH₂PO₄0.06% , (NH)₂SO₄0.1%, MgSO4.7H₂O 0.02% , CaCl₂.H₂O 0.005% , NaCl 0.3% , FeCl₃0.00001% and supplemented with 0.5% Na-CMC to induce the expression of cellulase enzyme genes. The medium was incubated for 5 days at 37°C, 180rpm (Anish, Rahman & Rao , 2007).

Enzyme Purification

Concentration of the protein

Following growth, the supplemented MSM culture medium was centrifuged at 10,000rpm, 4°C for 20mins. The clarified supernatant containing the secreted cellulase enzyme proteins was then concentrated by precipitation in 50% ice-cold acetone overnight at -20°C. The precipitate was thereafter pelleted by centrifugation at 15,000xg, 4°C for 15mins. The pellet was subsequently resuspended in 20mM Tris-HCl pH8.8.

Gel filtration

A sephadex column (G-75, 1.7cm X 30cm, Pharmacia fine chemicals, Sweden) was used to fractionate the concentrated crude cellulase protein based on molecular weight. The sample buffer used was phosphate buffer with 0.15M NaCl, whereas the eluent buffer was phosphate buffer with 0.5M NaCl. Thirty fractions of 1.5ml each were collected and monitored at 280nm (UV-mini 1240, UV-Vis spectrophotometer, SHIMADZU). The fractions were tested for protein content using biuret assay.

Fractions that peaked at 280nm were then assayed for cellulase activity. Those showing the highest activity corresponded to elution fractions 8 to 16( LB pH7, chromatogram ) and elution fractions 40 and 45 (Na-CMC, MSM pH10, chromatogram).

Cellulase enzyme assays:

Cotton assay

A preliminary continuous assay was conducted using sodium acetate buffer (50mM, pH5), cotton (0.1%w/v) and crude enzyme concentrate. This cellulase activity assay using cotton was done over a 10-day incubation period. Aliquots of 1ml each were collected in time constant time intervals, and tested for enzymatic activity by checking for reducing sugars using DNS assay (Fig.6).

Cellulase activity assays

Cellulolytic activity was quantified using DNS assay (Miller, 1959; Dashtban et al. 2010). Due to the analytical complexities presented with pure crystalline cellulose substrates, we used Na-CMC; a cellulose derivative with a higher degree of polymerization and with better solubility for these analysis

Dinitrosalicylic acid (DNS) assay

Gel filtration fractions, were tested for cellulase activity using the 3,5 dinitrosalicylic acid method (Miller 1959). DNS reagent (750µl ), was added to a 1ml cellulase reaction test tube containing 100µl of the gel purification fractions. 40% sodium potassium tartrate (250µl) was added to the mixture and heated at 100°C for 5minutes. Optical density (O.D) was then recorded at 550nm using a spectrophotometer (UV, SHIMADZU). Using a predetermined glucose standard curve, the glucose concentrations of the samples were obtained and used to determine enzymatic activity.

Thin Layer Chromatography (TLC) assays

TLC was used to follow the cotton and filter paper cellulase hydrolysis. The spotted plate, were developed in acetonitrile: water (v/v) . The plates were then air dried and sprayed ( TLC sprayer, MERCK) with visualization solution (1ml P-anisaldehyde, 1ml 97%H₂SO₄ in 18ml ethanol) .This was followed by heating at 110°C for 30 minutes for staining.

Optimization of cellulase activity

Cellulase enzymatic activity parameters (temperature, pH and time), were determined.

Temperature Optimum

The temperature profile was determined by recording the cellulase activity between 20°C -100°C.

pH Optimum

The optimum pH was also determined by recording the cellulase activity between pH 2-14. Different solutions with potent buffering capacities at various points were used; Glycine-HCl buffer, pH2-3 , sodium acetate buffer pH 4-5, phosphate buffer pH 6-7, Tris-HCl buffer pH 8-9, Sodium bicarbonate –NaOH buffer pH 10 and Glycine –HCl buffer pH 11-14.

Reaction times

Optimum reaction/ incubation times was determined by recording the cellulase activity in 1-hour intervals over an 8-hour period. This was preceded by an initial 30minutes interval reading.

Enzyme Stability assays

pH and temperature stability was done by determining the residual enzymatic activity following pre-incubation at pH 2-14 and 20°-100°C.

Effect of different compounds on cellulase activity

The effect of various sugars (monosaccharides, disaccharides, polysaccharides), alcohols, chemical reagents (ions, metal chelators surfactants and detergents) on cellulase activity was also determined.

Cellulase substrate specificity assays

Cellulase substrate specificity was also determined on a number of soluble and insoluble substrates (Avicel, Na-CMC, cellobiose and cotton).

RESULTS AND DISCUSSION

Production and Purification of cellulase enzyme proteins

The microorganism was inoculated into LB medium and minimal salt medium supplemented with 0.5% sodium carboxymethylcellulose (Na-CMC) as the main carbon source. The role of Na-CMC was to induce the expression of the cellulase gene and cellulase production (Sang-Mok &Koo, 2001; Kubicek, 1993). The secreted cellulase enzymes were harvested by centrifugation at 4°C, 10,000rpm for 20 mins (Schallmey et al. 2004). The harvested proteins were purified on a shorter column (Fig. 2) (production in a LB medium), then a final run (production in MSM) as shown in Fig. 1. The fractions corresponding to C10 and C16 (Fig.2), Fraction 41, and Fraction 45 that showed the highest reading using UV were further analysed for enzyme activity using pNp-β-cellobioside as a substrate. They recorded enzyme activity of 4.93X10^^-6U and 7.01X10^^-6U respectively.

Optimization of cellulase activity

Reaction temperature

Bacterial cellulases have been shown to have optimal activity in the temperature range (35°-50°C) (Aygan et al. 2011). For this study, there was a significant change in enzyme activity at the various temperatures within the tested temperature range of 20°-100°C (p<0.05) (ANOVA). Further, the enzyme showed a working temperature range between 20°-80°C. Moreover, the highest enzymatic activity was recorded at 60°C (Fig 9). This confirms and validates the thermophilic nature of the microorganism which was isolated from Lake Bogoria; a hot water lake in the Rift valley region of Kenya. This is of interest particularly for industry as these enzymes could find application in both ambient and temperature intensive applications. Further, they could be used to substitute the temperature intensive processes by conducting the processes at the ambient conditions; subject to process optimization and cost/ benefit analysis.

Further the recorded optimum temperature is similar to those reported by Lima et al. 2005 but higher than those reported by Balasubramanian & Simoes, 2013.

Optimum pH

There was a significant effect on enzyme activity with change in pH (p<0.05) (ANOVA). The crude enzymes showed dual peaks of activity between pH3-7 and pH8-11 (Fig.9). The highest activity was observed at pH5 and pH 10.

Optimum reaction time

There was a significant effect on the enzyme activity with different reaction times. (p<0.05) (ANOVA). Significant reaction products were observed after a reaction time of 4hrs, 6hrs and reaction seemed to reach completion at 8hrs. The highest enzyme activity was recorded following a 4hour reaction incubation period (Fig.11).

Cellulase stability assays

Temperature stability

The crude cellulase enzymes showed triple peaks of thermal stability (Fig. 12) with a sharp peak at , 40°-70°C and flanking peaks at 20°-40°C and 70°-90°C. Moreover, it would be interesting to further test temperature ranges lower than 20°C as well as higher temperature ranges greater than 100°C due to the observed continued peaking at these points (Fig12). The enzymes retained 9.50-99.99% of activity between 40°-60°C and 5.97-39.55% of activity between 70°-90°C. With the enzymes showing preference for the temperature range 40°-60°C, in which they retained the highest activity. This is an important characteristic of these enzymes, which would be of interest in synthetic biology/industry where high reaction temperatures (50°-60°C) for prolonged periods are compulsory (Lima et al.2005).

pH stability

The crude enzymes showed stability peak between pH4-8 and was most stable at pH 6 (Fig 12). This is consistent with the results on the pure glycosyl hydrolase9 (GH9) enzyme we extracted from this microorganism (Under review)

Cellulase stability in various compounds

The effect of various compounds (saccharides, alcohols and chemical reagents; detergents/surfactants, ions and inhibitors). This is important especially in co-fermentation in bio-factories where cellulases could be applied. In addition, it is important to study for these because the cellulosic biomass is normally not pure and in some cases, pre-processing is needed. And to determine reagents to use in enzyme preparations. There was a significant effect on enzyme activity with the different chemical reagents (p<0.05) (ANOVA) (Table1) (Fig. 13).

Stability in Chemical reagents

Native crude cellulase activity was greatly inhibited by 10mM EDTA, urea, β-mercaptoethanol and moderately by 10mM PMSF, triton 1% (v/v), SDS 1% (w/v) and 10mM CaCl_{2 .} Moreover, native crude cellulase activity was moderately enhanced by 10mM: CuSO_4, Imidazole and tween-20 1 %( v/v).native crude cellulase activity was greatly enhanced by 10mM: NaCl, MgS0₄_, MnCl_2,MnS0₄_,ZnS0₄_,CoCl₂_,KCl, DTT and FeSO₄. Similar effect on cellulase activity profiles have been previously reported (Balasubramanian & Simões, 2014).

Stability in Sugars

Effect of different saccharides on native crude cellulase activity was tested (Fig.14). There is a significant effect on enzyme activity with different saccharides (p<0.05) (ANOVA).Cellulase activity was enhanced by mannitol, starch, inositol, sucrose, sorbitol, D-xylose, raffinose, lactose, maltodextrin, trehalose, D-glucose and cellobiose. This is contrary to previous studies that showed significant inhibitory effects on cellulase activity during cellulose hydrolysis by sugars (Xiao et al. 2004). This profile is however slightly similar to those reported by (Nigam & Prabhu, 1991) who showed that glucose, xylose and sucrose enhanced cellulase activity while cellobiose had severe inhibitory effects. The stability and activity in the presence of these sugars could be attributed to the low levels of sugars utilized (0.2µg/µl). Further, the stability and activity observed can be attributed to the presence of cellulase cocktail in the crude enzyme extract thus enabling a high substrate consistency and minimizing the classical product (sugar) inhibition.

Stability in alcohols

Effect of alcohol on native crude cellulase enzyme was tested. There is a significant effect on enzyme activity with different alcohols (p<0.05). 40% (v/v) ethanol, 2-propanol and methanol enhanced native cellulase activity (Figure. 20)

The findings from this study show cellulase enzymes stable over a wide range of processing conditions and stability in organic solvents as well as a high tolerance for sugar accumulation. In addition, the cellulase activity on cotton with the release of shorter saccharides has been demonstrated. It would therefore be important to test for the quality (functional and physical properties) of the cotton fibre achieved after this processing.

TLC –Thin layer chromatography

LB-Luria Bertani

MSM-Minimal salt medium

Na-CMC –sodium carboxymethylcellulose

UV-ultraviolet

DNS-dinitrosalicylic acid

OD-optical density

Rpm-revolutions per minute

ANOVA- analysis of variance

GH9-glycosyl hydrolase 9

PMSF –phenylmethylsulfonyl fluoride

SDS-sodium dodecyl sulfate

DTT-dithiothreitol

Ethics approval and consent to participate-N/A

Consent for publication –N/A

Availability of data and materials-data in report

Competing interests-N/A,

Funding

Ogonda Lydia was a recipient of PhD scholarship from the French Government managed by Campus France and a one-year (2015) WFS scholarship managed by the International center for insect physiology and ecology –KENYA( ICIPE)

Author’s Contribution

LAO- conceived and designed research, conducted experiments and data analysis and interpretation and wrote initial draft. EKM –revised initial draft. MFJ, EKM, BMW and CT-Supervised the work. All authors read and approved the manuscript

Acknowledgements

N/A

Anish, R., Rahman, M. S., & Rao, M. (2007). Application of cellulases from an alkalothermophilic Thermomonospora sp. in biopolishing of denims. Biotechnology and Bioengineering, 96(1), 48–56. http://doi.org/10.1002/bit.21175

Aygan A, Karcioglu L, Arikan B. Alkaline thermostable and halophilic endoglucanase from Bacillus licheniformis Afr J Biotechnol 2011; 10(5): 78996.

Cavaco-Paulo, A. (1998). Processing Textile Fibers with Enzymes: An Overview. In: Enzyme Applications in Fiber Processing, Eriksson, K.E. and A. Cavaco-Paulo (Eds.). American Chemical Society Washington, DC. ISBN-13: 9780841235472, pp: 180-189.
Dashtban, Mehdi & Schraft, Heidi & Syed, Tarannum & Qin, Wensheng. (2010). Fungal biodegradation and enzymatic modification of lignin. International journal of biochemistry and molecular biology. 1. 36-50

Jatinder Kaur; Bhupinder, S. Chadha; Badhan A. Kumar; Ghatora S.Kaur ; Harvinder, S. Saini.(2007). Purification and characterization of β-glucosidase from Melanocarpus sp. MTCC 3922. Electronic journal of Biotechnology ISSN : 0717-3458

Karmakar, M and Ray, R.R. (2011). Current Trends in Research and Application of Microbial Cellulases. Research Journal of Microbiology, 6: 41-53.
Kubicek, C.P. (1992), Biochem. Eng., 45, 1-27. The cellulase proteins of T. reesei:
structure, multiplicity, mode of action and regulation of formation

Kubicek, C.P., Messner, IL, Gruber, F., Maeh, ILL. and Kubicek-Pranz, E.M. (1993),
Enzyme Microb. Technol., 15, 90-99. The Trichoderma reesei cellulase regulatory puzzle--
from the interior life of a secretory fungus.

Lima AOS, Quecine MC, Fungaro MHP, Andreote FD, Maccheroni W, Araújo WL, Silva-Filho MC, Pizzirani-Kleiner AA, Azevedo JL (2005) Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain. Appl Microbiol Biotechnol 68:57–65 https://doi.org/10.1007/s00253-004-1740-1
Lynd, L. R., van Zyl, W. H., McBride, J. E., & Laser, M. (2005). Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol, 16(5), 577-583.
Lynd, L. R., Weimer, P. J., van Zyl, W. H., & Pretorius, I. S. (2002). Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev, 66(3), 506-577, table of content
Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry, 31, 426-429.
Balasubramanian, Natesan & Simões, Nelson. (2014). Bacillus pumilus S124A carboxymethyl cellulase; a thermo stable enzyme with a wide substrate spectrum utility. International journal of biological macromolecules. 67. 10.1016/j.ijbiomac.2014.03.014.
Nigam, P., & Prabhu, K. A. (1991). Influence of sugars on the activity of cellulase system from two basidiomycetes cultures. Journal of Basic Microbiology, 31(4), 279–283. http://doi.org/10.1002/jobm.3620310408
Verenium-cottonase ®http://www.verenium.com/prod_cottonase.html

Xiao, Z., Zhang, X., Gregg, D. J., & Saddler, J. N. (2004). Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates. Applied Biochemistry and Biotechnology, 115(1-3), 1115–1126. http://doi.org/10.1385/ABAB:115:1-3:1115

Table 1: Effect of chemical reagents on crude cellulase enzymatic activity

Reagent	Concentration	Relative activity (%)
EDTA	10mM	-425.1396648
NaCl	10mM	334.6368715
MgS04	10mM	692.1787709
CuS04	10mM	44.13407821
MnCl2	10mM	831.8435754
Tween 20	1%	88.82681564
MnS04	10mM	513.4078212
SDS	1%	-67.59776536
Imidazole	10mM	60.89385475
Triton	1%	-62.01117318
CaCl₂	10mM	-84.3575419
β-mercaptoethanol	10mM	-291.0614525
ZnS0₄	10mM	206.1452514
CoCl₂	10mM	1329.050279
KCl	10mM	256.424581
Control (without chemical)		100
DTT	10mM	2485.47486
PMSF	10mM	-134.6368715
UREA	10mM	-386.0335196
FeSO₄	10mM	1083.240223

Table 3: Substrate specificity of crude native cellulase

Substrate	Cellulase activity	Concentration
Avicel	ND	1%
CMC	0.020284	1%
Cellobiose	0.439805	1%
Cotton	0.0069	1%

Download PDF

Version 1

posted

You are reading this latest preprint version

Characterization of Crude Cellulases From a Bacillus Sp. Isolated From Lake Bogoria, Kenya

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results And Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Status:

Version 1