You-Jung Lee

Statistical Optimization for Production of Carboxymethylcellulase of Bacillus amyloliquefaciens DL-3 by a Recombinant Escherichia coli JM109/DL-3 from Rice Bran Using Response Surface Method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.

Optimization of salts in medium for production of carboxymethylcellulase by a psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using two statistical methods

The concentration of four mineral salts in the medium for the production of carboxymethylcellulase (CMCase) by Psychrobacter aquimaris LBH-10 were optimized using orthogonal array method (OAM) and response surface method (RSM) and their results from two statistical methods were compared. The analysis of variance (ANOVA) of data from central composite design (CCD) based on OAM indicated that potassium phosphate gave the highest percentage participation for cell growth as well as production of CMCase. However, their relative participations of four salts for cell growth were different from those for production of CMCase. The ANOVA of results from RSM indicated that highly significant factors (“probe>F” less than 0.0001) for cell growth were K2HPO4 and (NH4)2SO4, whereas those for production of CMCase were K2HPO4, NaCl, and MgSO4·7H2O. The optimal concentration of K2HPO4, NaCl, MgSO4·7H2O, and (NH4)2SO4 for cell growth extracted by Design Expert Software based on RSM were 7.10, 0.84, 0.24, and 0.33 g/L, respectively, whereas those for production of CMCase were 3.00, 0.52, 0.34, and 0.45 g/L. The optimal concentrations of salts for cell growth and production of CMCase using RSM basically coincided with those using OAM as well as those from ‘one-factor-at-a-time’ method. The production of CMCase by P. aquimaris LBH-10 with optimized concentrations of salts was 273.0 U/mL, which was enhanced by 1.27 times higher than the previous report.

Characterization of Acidic Carboxymethylcellulase Produced by a Marine Microorganism, Psychrobacter equimeris LBH-10

A microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Psychrobacter aquimaris by analysis of 16S rDNA sequences, and named P. aquimari LBH-10. This strain produced an acidic carboxymethylcellulase (CMCase), which hydrolyzed carboxymethylcellulose (CMC), cellobiose, curdlan, filter paper, p-nitrophenyl-β-D-glucopyranoside (pNPG), pullulan, and xylan, but there was no detectable activity on avicel and cellulose. The optimal temperature for CMCase produced by P. aquimari LBH-10 was 50℃ and more than 90% of its original activity was maintained at broad temperatures ranging from 20 to 50℃ after 24 hr. The optimal pH of the CMCase was 3.5, and more than 70% of its original activity was maintained under acidic conditions between pH 2.5 and 7.0 at 50℃ after 24 hr. The optimal pH of CMCase produced by P. aquimaris LBH-10 seems to be lower than those produced by any other bacterial and fungal strain. CoCl₂, EDTA, and PbCl₂ at a concentration of 0.1 M enhanced CMCase-produced P. aquimaris LBH-10, whereas HgCl₂, KCl, MnCl₂, NiCl₂, and SrCl₂ inhibited it.