Rice bran as a substrate for proteolytic enzyme production

FOOD SCIENCE AND TECHNOLOGY

Rice bran as a substrate for proteolytic enzyme production

Alagarsamy Sumantha^I; Paul Deepa^I; Chandran Sandhya^I; George Szakacs^II; Carlos Ricardo Soccol^III; Ashok PandeyI, ^*

^IBiotechnology Division; Regional Research Laboratory (CSIR); Trivandrum - India

^IIDepartment of Agricultural Chemical Technology; Technical University of Budapest; Budapest - Hungary

^IIILaboratório de Processos Biotecnológicos; Universidade Federal do Paraná; Curitiba - PR - Brasil

ABSTRACT

Rice bran was used as the substrate for screening nine strains of Rhizopus sp. for neutral protease production by solid-state fermentation. The best producer, Rhizopus microsporus NRRL 3671, was used for optimizing the process parameters for enzyme production. Fermentation carried out with 44.44 % initial moisture content at a temperature of 30 C for 72 h was found to be the optimum for enzyme secretion by the fermenting organism. While most of the carbon supplements favored enzyme production, addition of casein resulted in a marginal increase in protease yield. Fermentation was then carried out under optimized conditions to obtain the crude extract of the enzyme, which was partially purified by precipitation and dialysis. A 3-fold increase in the enzyme purity was achieved in this manner. The enzyme was found to be a metalloprotease, being activated by Mn²⁺, with maximal activity at a temperature of 60 C and pH 7.0.

Key words: Solid-state fermentation, Rice bran, Rhizopus, neutral protease, agro-industrial residues

RESUMO

Farelo de arroz foi utilizado como substrato para seleção de nove linhagens de Rhizopus sp. com vistas a produção de protease neutra. A linhagem que apresentou maior produtividade da enzima foi Rhizopus microsporus NRRL 3671, sendo utilizada na otimização dos parâmetros do processos e produção da enzima. As condições otimizadas para produção da enzima foram 44% de umidade inicial, temperatura de 30ºC e 72h de fermentação.A suplementação do farelo de arroz com uma fonte de carbono favoreceu a produção da enzima, porém a adição de caseína resultou em um aumento marginal do rendimento em protease. Condições otimizadas foram utilizadas para obtenção do extrato cru da enzima que foi parcialmente purificado por precipitação e diálise. A enzima purificada teve sua atividade incrementada 3 vezes. A enzima foi classificada como metalo-protease, sendo ativada pelo Mn²⁺ , sendo que sua atividade máxima foi obtida a temperatura de 60ºC e a pH 7.0.

INTRODUCTION

Rice bran is a by-product of the rice milling industry. In India, nearly one million tons of bran is produced every year. It has a high nutritive value and serves as a valuable feed for cattle, poultry, and pigs. Rice bran supplies almost the same amount of protein (10-15 %) as wheat and oats and its protein is of considerably better quality than maize (The Wealth of India, 2001). Rice bran has been used as a fermentation substrate for the production of enzymes such as lipase by Candida sp. (Rao et al., 1993a; b), in combination with cassava starch and rice hulls for the production of glucoamylase by Aspergillus sp. (Tani et al., 1986) and also with wheat bran for the production of alkaline protease by Trichoderma koningii (Manonmani and Joseph, 1993).

Proteases are proteolytic (protein-digesting) enzymes that are mainly classified on the basis of their pH optimum as acidic, neutral, and alkaline proteases. These biocatalysts find wide applications in many industries such as textile, laundry, healthcare etc. Neutral proteases are mainly used in food processing such as baking, brewing, and also in the healthcare sector. One of the more recent applications of these proteases exploit their eco-friendly nature and hence their suitability to act as food-processing aids, wherein these enzymes can be used for the extraction of plant oils thus largely replacing hazardous organic solvents such as hexane which has been traditionally used for such processes.

Neutral protease production has been carried out under both submerged (SmF) and solid-state fermentation (SSF) using substrates such as wheat bran (Fernandez-Lahore et al., 1998; Couri et al., 2000; Sandhya et al., 2005), steamed rice (Chou and Rwan, 1995), mango peel and banana peel (Couri et al., 2000), etc. Fermentation of rice bran by Rhizopus sp. has been reported, but only for the production of acid protease (Ikasari and Mitchell, 1996; 1998). There are no reports on the production of neutral protease by Rhizopus sp. Hence, the potential of rice bran to be used as a substrate for the production of neutral protease by Rhizopus sp. was investigated.

MATERIALS AND METHODS

Substrate

The substrate used in this study, rice bran, was obtained from a local market in Trivandrum.

Microorganism and maintenance of culture

The Rhizopus strains, R. oligosporus NRRL 2710, 5905, R. microsporus NRRL 3671, R. oryzae NRRL 1526, 1891, 6431, 395, 3562, 1472 were obtained from Northern Regional Research Laboratory, USA and grown on Potato Dextrose Agar (PDA) slants at 30 °C with fortnightly transfer to fresh medium.

Inoculum preparation

The inoculum was prepared by dispersing the spores from a week-old fungal slant culture in 0.1 % Tween-80 solution with a sterile inoculation loop.

Solid-state fermentation

Five grams of rice bran was taken in a 250 ml Erlenmeyer flask, moistened with salt solution [composition (% w/v): ammonium nitrate 0.5, potassium dihydrogen orthophosphate 0.2, sodium chloride 0.1, and magnesium sulphate 0.1] to achieve the desired moisture content, sterilized at 121.5 °C for 15 min, cooled, inoculated with 1 ml of fungal spore suspension (10⁶ spores/ml) and incubated at 30 °C for 72 h, unless otherwise mentioned. All experiments were carried out in two sets. The results shown are average values ± SD.

Extraction of crude enzyme

A solution of Tween-80 (0.1 %) in distilled water was added to the fermented substrate and the substrate was homogenized on a rotary shaker at 180 rpm for 1 h. The solids were removed by centrifuging the homogenate at 8000 x g at 4 °C for 15 min and the resultant clear supernatant was used for analytical studies.

Analytical methods

Assay for neutral protease

To 200 µl of crude enzyme extract, 500 µl of casein (1 %) and 300 µl of 0.2 mol/l phosphate buffer (pH 7.0) were added. The reaction mixture was incubated at 60 °C for 10 min and arrested by the addition of 1 ml of 10 % trichloroacetic acid (Keay and Wildi, 1970). The reaction mixture was centrifuged at 8000 x g for 15 min and to the supernatant, 5 ml of 0.4 mol/l Na₂CO₃ and 1 ml of 3-fold diluted Folin and Ciocalteau's phenol reagent, were added. The resulting solution was incubated at room temperature for 30 min and the absorbance of the blue color developed was read at 660 nm using a tyrosine standard (Lowry et al., 1951). One unit of enzyme activity was defined as the amount of enzyme that liberated 1 µg of tyrosine from substrate (casein) per minute under assay conditions and reported in terms of protease activity per gram dry fermented substrate.

Estimation of total soluble protein

Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard and was expressed as milligram protein per gram dry fermented substrate.

Screening of fungal neutral protease producers

The nine different Rhizopus strains were screened for neutral protease production by performing SSF using rice bran as substrate.

Optimization of process parameters for neutral protease production

The protocol adopted for the optimization of process parameters was to evaluate the effect of an individual parameter at a time and to incorporate it at the standard level before optimizing the next parameter.

Optimization of incubation period

The production profile of neutral protease was studied by conducting fermentation for different time intervals (0, 24, 48, 72, 96, 120, 144, and 168 h).

Optimization of incubation temperature

The inoculated substrates were incubated at different temperatures to determine the optimum fermentation temperature for neutral protease production (10, 25, 30, 37, and 44 °C).

Optimization of initial moisture content

Optimum initial moisture content for neutral protease production was determined by adjusting the initial moisture content of the fermentation substrate to varying levels (37.5, 44.4, 50, 54.5, and 58.3 %).

Effect of nutrient supplementation

Effect of inorganic nitrogen supplements

Different sources of inorganic nitrogen (NH₄HCO₃, NH₄H₂PO₄, (NH₄)₂HPO₄, NH₄NO₃, NaNO₃, KNO₃, (NH₄)₂SO₄, and NH₄Cl) at 1 % (w/w) were added to the fermentation medium to study its effect on enzyme production.

Effect of organic nitrogen supplements

Various organic nitrogen supplements (beef extract, casein, corn steep liquor, corn steep solids, malt extract, peptone, tryptone, and yeast extract) at a concentration of 1 % (w/w) were added to the fermentation media to study its effect on enzyme production.

Effect of carbon supplements

Influence of various carbon supplements on enzyme production was studied by adding different sugars (dextrose, maltose, sucrose, mannitol, sorbitol, xylose, lactose, and galactose) at 1 % (w/w) to the fermentation media.

Solid-state fermentation under optimized conditions

Solid-state fermentation of rice bran by the selected strain of Rhizopus was carried out under the optimized conditions of time, temperature, initial moisture content, and nutrient supplements.

Partial purification of the enzyme

The crude enzyme sample was separated into three fractions, based on the percentage saturation of ammonium sulphate, at 4 °C under constant stirring. The precipitated proteins were pelletized by centrifugation at 10000 x g at 4 °C for 15 min. These proteins were dissolved in 0.2 mol/l phosphate buffer (pH 7.0) and stored at 4 °C. The precipitate was dialyzed against the same buffer at 4 °C for 24 h and the buffer was changed at regular intervals.

Characterization of the partially purified enzyme

The partially purified fraction showing highest specific activity was characterized by varying the parameters that influence enzyme activity.

Substrate concentration

The effect of assay substrate concentration on the activity of neutral protease was studied by using different concentrations of casein (5, 10, 15, 20, 25, and 30 mg/ml).

pH optimum

The pH optimum of the neutral protease enzyme was determined by using buffer solutions of different pH (phosphate buffer 6.0, 6.5, 7.0, 7.5, 8.0, and tris-glycine buffer 8.5, 9.0) for enzyme assay. The buffers used were of the concentration 0.2 mol/l.

Temperature optimum

The influence of temperature on the activity of neutral protease was studied by incubating the assay reaction mixture at different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 °C).

Effect of enzyme modulators

Different enzyme modulators (PMSF, EDTA, EGTA, DTT, CuSO₄, SDS, CaCl₂, MgSO₄, MnSO₄, FeSO₄, ZnCl₂) of concentration 0.1 mol/l, was added to the assay mixture to study their effect on enzyme activity.

RESULTS AND DISCUSSION

The process parameters for the production of neutral protease by the highest protease-producing Rhizopus strain grown on rice bran substrate were done by the single-parameter mode. Fermentation was done under optimized conditions. The enzyme was partially purified and characterized. The results are presented and discussed below.

Screening of microorganisms

R. microsporus NRRL 3671 proved to be the best strain for neutral protease production on rice bran substrate giving 129 U/gds of enzyme activity (Table 1). This Rhizopus strain was selected to optimize the process parameters for enzyme production by the SSF of rice bran.

Fermentation time

Maximum enzyme production was observed after 72 h of fermentation (Fig. 1). A gradual decrease in enzyme units was observed with increasing incubation time clearly suggesting the enzyme's role as a primary metabolite, being produced in the log phase of the growth of the fungus for utilization of nutrients (proteins) present in the solid substrate. The subsequent decrease in the enzyme units could probably be due to inactivation of the enzyme by other constituent proteases.

Initial moisture content

Initial moisture content is a crucial factor affecting the formation of products through solid-state fermentation. A moisture level of 44.4 % was found to be optimum for neutral protease production (Fig. 2). Moisture content of 35-40 % facilitated neutral protease production by Aspergillus oryzae NRRL 2160 on a combined substrate of rice hulls and rice bran (Battaglino et al., 1991).

Fermentation temperature

R. microsporus NRRL 3671, being a mesophilic culture was found to be highly sensitive to temperature changes below and above its optimum for both enzyme production and growth (data not shown). Fermentation carried out at 30 °C was best suited for enzyme production (Fig. 2).

Supplementation of nutrients

Supplementation of the fermentation medium with NH₄HCO₃ was found to enhance enzyme production (Table 2). Since no other inorganic nitrogen compounds, e.g. NH₄NO₃ which has a higher molar concentration of nitrogen than NH₄HCO₃, enhanced protease production, the enhancing effect of NH₄HCO₃ can not be attributed to the nitrogen present in it, but could rather be due to the carbonate group. Carbonate as a constituent of the extraction buffer enhanced protease recovery from rice bran fermented by Aspergillus niger (Anupama and Ravindra, 2001).

None of the organic nitrogen supplements enhanced protease production significantly, though casein showed a slightly promoting effect. Casein was found to be an inducer for protease synthesis by Bacillus licheniformis MIR 29 (Ferrero et al., 1996).

Almost all of the carbon supplements, especially sucrose, enhanced enzyme production. Sucrose probably provides the much required carbon in the carbohydrate-deficient rice bran substrate, which contains only about 1.3 % of reducing sugars (The Wealth of India, 2001).

Partial purification

A better understanding of the function of enzyme could be determined by purification of enzyme (Sandhya et al., 2004). Partial purification of the enzyme by ammonium sulphate precipitation, followed by dialysis resulted in nearly a 3-fold increase in the specific activity of the enzyme (Table 3).

Enzyme characterization

Substrate concentration

The assay substrate, casein, when used at increasing concentrations resulted in the saturation of the enzyme (Fig. 3). From Lineweaver-Burk plot, the K_m and V_max of the reaction was found to be 2.6 and 19.9 mg/min, respectively (data not shown).

pH

A gradual increase in specific activity to reach a peak at pH 7.0 was then followed by a sharp decline as shown in fig. 4 indicating the enzyme's instability at any pH other than its optimum, viz. 7.0. A neutral metalloprotease from Staphylococcus epidermidis also has pH optima in the range 5.0-7.0 (Teufel and Gotz, 1993).

Temperature

The partially purified enzyme was stable at a temperature range of 50-60 °C. The enzyme activity gradually increased with increasing temperature, followed by a steep decrease at temperatures above 60 °C (Fig. 5). A neutral metalloprotease exhibiting maximal activity at a temperature of 60 °C was purified from Aspergillus fumigatus (Markaryan et al., 1994).

Effect of activators/inhibitors

The effect of various activators and inhibitors at 0.1 mol/l concentration showed that the neutral protease is a metalloenzyme requiring Mn²⁺ for its activity (Fig. 6). The inhibition of enzyme activity by EGTA, EDTA also proved this. The enzyme was comparatively resistant to PMSF and SDS, but highly sensitive to DTT. Cu²⁺ slightly enhanced enzyme activity, but addition of other metal ions such as Ca²⁺, Mg²⁺, Fe²⁺, and Zn²⁺ did not improve enzyme activity. A neutral protease from Bacillus subtilis that was activated by Mn²⁺ and a few other metal ions and susceptible to metal chelators such as EDTA was described by Yang et al. (2000), while the production of a neutral metalloprotease by Bacillus thuringiensis var. kurstaki was dependent upon the presence of Mn²⁺ in the fermentation medium (Li and Yousten, 1975).

ACKNOWLEDGEMENTS

The authors thank CSIR, India for providing financial support under the networked Task Force Project (CMM 0006). A. Sumantha gratefully acknowledges a Junior Research Fellowship from CSIR, India.

Received: March 04, 2005;

Revised: December 12, 2005;

Accepted: July 12, 2006.

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Author for correspondence

Publication Dates

• Publication in this collection
  23 Feb 2007
• Date of issue
  Sept 2006

History

• Accepted
  12 July 2006
• Reviewed
  12 Dec 2005
• Received
  04 Mar 2005