The Dual Carboxymethyl Cellulase and Gelatinase Activities of a Newly Isolated Protein from Brevibacillus agri ST15c10 Confer Reciprocal Regulations in Substrate Utilization

Abstract

A protein showing endoglucanase-peptidase activity was prepared from a newly isolated bacterium (ST15c10). We identified ST15c10 as Brevibacillus agri based on electron-microscopic images and its 16S-rDNA sequence (GenBank accession No. HM446043), which exhibits 98.9% sequence identity to B. agri (KZ17)/B. formosus (DSM-9885T)/B. brevis. The enzyme was purified to homogeneity and gave a single peak during high-performance liquid chromatography on a Seralose 6B-150 gel-matrix/C-18 column. MALDI-TOF mass-spectrometry and bioinformatics studies revealed significant similarity to M42-aminopeptidases/endoglucanases of the CelM family. These enzymes are found in all Brevibacillus strains for which the genome sequence is known. ST15c10 grows optimally on carboxymethyl cellulose (CMC)-gelatin (40°C/pH 8-9), and also shows strong growth/carboxymethyl cellulase (CMCase) activity in submerged bagasse fermentation. The purified enzyme also functions as endoglucanase with solid bagasse/rice straw. Its CMCase activity (optimal at pH 5.6 and 60°C/K_m = 35.5 µ<smlcap>M</smlcap>/V_max = 1,024U) was visualized by zymography on a CMC-polyacrylamide gel, which provided a strong band of approximately 70 kDa. The purified enzyme also showed strong peptidase (gelatinase) activity (pH 7.2/40°C during zymography on 6-12% gelatin/1% gelatin-PAGE (at approx. 70 kDa). The CMCase activity is inhibited by the metal ions Mn/Cu/Fe/Co (50%), Hg/KMnO₄ (100%), and by glucose or lactose (50-75%; all at 1 m<smlcap>M</smlcap>). The observed dose/time-dependent inhibition by Hg ions could be prevented with 2-mercaptoethanol. A comparison of the B. agri endoglucanase-aminopeptidase (ELK43520; 350 aa) with other members of the M42-family revealed the conservation of active-site residues Cys256/Cys260, which were previously identified as metal-binding sites. Regulation of the endoglucanase activity probably occurs via metal binding-triggered changes in the redox state of the enzyme. Studies on this type of enzyme are of high importance for basic scientific and industrial research.

Characterization of gelatinase produced by Antarctic Mrakia sp.

“…The highest activity of gelatinase of Mrakia sp. at 36°C and pH 7, which is in the range reported for gelatinases from other microorganisms, such as E. faecalis (30°C and pH 7.5–8.0) , Bacillus strains (50°C and pH 7) , Brevibacillus agri (40°C and pH 7.2) , and Salinicoccus roseus (37°C and pH 8) . As has been reported for gelatinases from Enterococcus , Bacillus strains , and Cyprinus carpio , the activity of the gelatinase from Mrakia sp.…”

“…Gelatinase has been described from Enterococcus faecalis [9], Bacillus spp. isolated from the sediment sample of Porto Novo Coastal sites [11], Brevibacillus agri ST15c10 [12], and some fungal species [13].…”

One-Pot Ionic Liquid-Mediated Bioprocess for Pretreatment and Enzymatic Hydrolysis of Rice Straw with Ionic Liquid-Tolerance Bacterial Cellulase

“…However, the size of CMCase produced from CBD3 was smaller than those reported in identified Brevibacillus sp. (~70–90 kDa [ 37 , 38 ]).…”

Endoglucanase gene of M42 aminopeptidase/endoglucanase family from thermophilic Bacillus sp. PW1 and PW2 isolated from Tattapani hot spring, Himachal Pradesh, India

“…These results raise the possibility that the putative ORFs encode for dual enzyme activities. Although there are not many such reports, Maiti et al [30] reported the dual activity of a protein isolated from Brevibacillus agri, which exhibits gelatinase as well as cellulase activities in vitro.…”