Elsevier

Process Biochemistry

Volume 39, Issue 12, 29 October 2004, Pages 2115-2122
Process Biochemistry

Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14

https://doi.org/10.1016/j.procbio.2003.11.002Get rights and content

Abstract

Statistical optimization of culture conditions for production of a widely suited detergent protease from Bacillus sp. RGR-14 was carried out using a two-step approach. A quick identification of the important factors with simple screening experiment was followed by application of complex response surface design for further optimization. The production of extracellular alkaline protease by Bacillus sp. was favored in the presence of complex carbon and nitrogen sources, viz. starch, casamino acid and soybean meal. A reduced quadratic model was found to fit the alkaline protease production. Response surface analysis revealed the significant role of phosphate ions in determining alkaline protease production. A steep, stretched out response surface showed direct relation between the level of protease production and casamino acid and starch concentration in the medium. A 12.85 fold increase in protease production could be obtained within the design space. Protease production was found to be repressed in the presence of high concentrations of casamino acid. The model could be validated in up to 2 l shake flasks (3914 U ml−1). The same statistical design could explain economic protease production in cost-effective medium as well.

Introduction

Recent years have witnessed a phenomenal increase in the use of enzymes as industrial catalysts. The estimated value of the world enzyme market is about US $1.3×109 and it has been forecasted to reach US $2×109 by 2005. Detergent enzymes comprise 37% of global enzyme market. Alkaline proteases enjoy a big share of the enzyme market primarily as detergent additives. Microbial alkaline proteases are also of immense utility in other industrial sectors, viz. leather, food, textile, organic synthesis, waste water treatment [1], [2], [3], [4], [5].

Thermostable alkaline proteases from Bacillus spp. (viz. alcalase, savinase, esperase, maxatase, maxacal, opticlean, optimase, proleather) constitute 20% of the world enzyme market. With the growing awareness of energy conservation and acceptance of synthetic fabrics, which cannot tolerate high temperature of the conventional hot water washings, there is a constant thrust to discover and design novel detergent alkaline proteases active at low temperatures. The search for proteases capable of functioning at low temperatures has made psychrophiles an attractive research target [6], [7], [8]. There are several reports on improved protease yields/properties by strain improvement using rDNA technology/protein engineering, respectively [1], [9]. We have reported an inherently SDS-stable alkaline protease from Bacillus sp. showing good wash performance when used in conjunction with detergent over a broad range of temperature (25–60 °C) [10]. This enzyme is a promising candidate for application in both high and ambient temperature compacts.

Considering the commercial importance of the SDS-stable alkaline protease we have attempted to study and maximize detergent protease production and economize it. In view of the available literature [1], [2], [5], [11], [12], production optimization was carried out using a two-step strategy. In the first phase, screening of carbon and nitrogen sources and statistical screening of primary physiological and nutritional determinants of alkaline protease production from Bacillus sp. was done. There are several reports on the use of Plackett–Burman design for screening purposes [13], [14], [15]. This was followed by use of a multi-factorial response surface approach as a quick and effective tool to study the effect of both the primary factors and their mutual interactions on extracellular alkaline protease production from Bacillus sp. for production optimization.

The cost of enzyme production is a major obstacle in its successful industrial application. In view of the promising applicability of the alkaline protease as a builder for detergents, it should be produced in high yields in a low-cost medium. Thus, we have also attempted to bring down the detergent protease production costs by using low-cost fermentation medium [11]. Here, we report statistical optimization of culture conditions for enhanced production of a widely suited detergent protease from Bacillus sp. RGR-14.

Section snippets

Chemicals and experimental statistics

Casein for protease assay was purchased from Sigma (St. Louis, USA) and bovine serum albumin (BSA) was purchased from ICN Biomedicals Inc., Ohio. Soy flour/defatted soy flour were agrowastes obtained from soybean processing plant. All other chemicals used were of analytical grade commercially available in India. All the experiments were carried out independently in triplicates and repeated twice. The standard deviation in results was within 10%.

Microbial strain

Bacillus sp. RGR-14, known to produce SDS-stable

Selection of most suitable carbon and nitrogen sources by one-variable-at-a-time approach

The Bacillus sp. produced maximum alkaline protease in the presence of starch (1265 U ml−1) followed by mannitol (1113 U ml−1), maltose (1086 U ml−1) and glycerol (970 U ml−1) as shown in Table 3. The readily assailable simple carbon sources, viz. fructose, glucose, mannose, sucrose, lactose supported poor alkaline protease production. Similarly, complex organic nitrogen sources, viz. soybean meal (1480 U ml−1), casamino acid (1466 U ml−1) and peptone (1265 U ml−1) were observed to induce high protease

Discussion

The production of extracellular alkaline protease by Bacillus sp. is 102 fold higher in the presence of complex carbon and nitrogen sources, viz. starch, casamino acid and soybean meal as compared to simple sugars, viz. glucose, mannose, fructose, sucrose, or inorganic nitrogen sources, viz. potassium nitrate, ammonium sulfate, etc. There are similar reports of enhanced alkaline protease production in the presence of complex carbon [11], [17], [18], [19] and organic nitrogen sources [1], [5],

Acknowledgements

Ms. Bhavna Chauhan acknowledges the JRF grant from CSIR, Government of India (sanction no. 9/45/282/2001 EMR-I). The authors are grateful to the Department of Microbiology, UDSC for providing the necessary infrastructure.

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