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


 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 studyWe therefore sought to characterize cellulases from favorable ecosystems in order to gauge the validity of their inclusion in cellulosic biomass industrial utilization. ConclusionWe 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 SummaryThis validates and shows the potential of these enzymes in co-expression bio-factories and applications requiring high sugar yields Potential ImplicationsThis 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.


Introduction
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 (Vereniumcottonase ® 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 e ciently 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 Applications of cellulases have increased considerably especially in the textile industry during the last two decades. These include bio processing of natural bers, bio polishing of cotton fabrics in order to enhance the softness and feel, appearance and treatment of recycled bres to restore bre texture and exibility 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.

Materials And Methods
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.

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 Puri cation
Concentration of the protein Following growth, the supplemented MSM culture medium was centrifuged at 10,000rpm, 4°C for 20mins. The clari ed 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 ltration
A sephadex column (G-75, 1.7cm X 30cm, Pharmacia ne 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 10day 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 quanti ed 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 ltration 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 puri cation 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 lter 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%H 2 SO 4 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 pro le was determined by recording the cellulase activity between 20°C -100°C.

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

RESULTS AND DISCUSSION
Production and Puri cation 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 puri ed on a shorter column (Fig. 2) (production in a LB medium), then a nal 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^-6 U and 7.01X10^-6 U 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 signi cant 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 con rms 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 nd 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/ bene t 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 signi cant 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 signi cant effect on the enzyme activity with different reaction times. (p<0.05) (ANOVA). Signi cant 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 anking 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 signi cant effect on enzyme activity with the different chemical reagents (p<0.05) (ANOVA) (Table1) (Fig. 13).

Stability in Sugars
Effect of different saccharides on native crude cellulase activity was tested (Fig.14). There is a signi cant effect on enzyme activity with different saccharides (p<0.05) (ANOVA).Cellulase activity was enhanced by mannitol, starch, inositol, sucrose, sorbitol, D-xylose, ra nose, lactose, maltodextrin, trehalose, Dglucose and cellobiose. This is contrary to previous studies that showed signi cant inhibitory effects on cellulase activity during cellulose hydrolysis by sugars (Xiao et al. 2004). This pro le 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 signi cant effect on enzyme activity with different alcohols (p<0.05). 40% (v/v) ethanol, 2-propanol and methanol enhanced native cellulase activity ( Figure. 20)

Conclusions
The ndings 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 bre achieved after this processing.    Figure 1