Statističko optimiranje proizvodnje proteaza otpornih na povećani salinitet iz umjereno halofilne bakterije Virgibacillus sp. SK37

The objectives of this study are to optimize the conditions for providing high yield of NaCl-tolerant extracellular protease from Virgibacillus sp. SK37 based on a fi sh-based medium and to investigate the eff ects of the key factors (mass per volume ratios of dried anchovy, yeast extract and NaCl, and initial pH of the medium) on the secretion patt ern of proteases. Based on the predicted response model, the optimized medium contained 1.81 % of dried anchovy, 0.33 % of yeast extract and 1.25 % of NaCl at pH=7.8. Under these conditions, a 5.3-fold increase in protease production was achieved, compared with the broth containing only 1.2 % of dried anchovy (5 % of NaCl at pH=7). The cubic regression adequately described the protease production. Protease activity was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the synthetic substrate (Suc-Ala-Ala-Pro-Phe-AMC). Proteases of molecular masses of 19, 34, 35 and 44 kDa were secreted in the presence of NaCl, whereas those of 22 and 42 kDa were the main proteases detected in the absence of NaCl. In addition, no secreted proteases were detected when initial pH of the medium was pH=6. The peptide mass fi ngerprint of the medium cultured with 10 % NaCl showed a higher abundance of peptides with lower mass of 500– 1000 m/z compared with the medium containing 0 % NaCl, indicating the higher proteolytic activity of the high-salt medium. The Virgibacillus sp. SK37 proteases showed a marked preference towards Lys, Arg and Tyr in the presence of NaCl and towards Lys and Arg in the absence of NaCl.Svrha je ovog rada bila optimirati uvjete proizvodnje izvanstaničnih proteaza otpornih na povećani salinitet s pomoću bakterije Virgibacillus sp. SK37, te ispitati utjecaj glavnih čimbenika (masa sušenih inćuna, ekstrakta kvasca i soli, te početna pH-vrijednost podloge) na proizvodnju proteaza. Metodom odzivnih površina utvrđeno je da optimalna podloga sadržava 1,81 % sušenih inćuna, 0,33 % ekstrakta kvasca i 1,25 % soli, te da je optimalni pH=7,8. Pri tim je uvjetima proizvodnja proteaze porasla 5,3 puta, u usporedbi s podlogom koja je sadržavala samo 1,2 % sušenih inćuna (5 % soli pri pH=7). Proizvodnja proteaze dobro je opisana kubnim modelom. Aktivnost je proteaze određena na sintetičkoj podlozi (Suc-Ala-Ala-Pro-Phe-AMC) pomoću SDS-PAGE metode. U podlozi sa soli izlučene su proteaze molekularne mase od 19, 34, 35 i 44 kDa, dok su u podlozi bez soli dominirale proteaze molekularne mase od 22 i 42 kDa. Osim toga, ustanovljeno je da se proteaze nisu izlučivale pri početnoj pH-vrijednosti podloge od 6. Identifikacijom peptida utvrđeno je da je u podlozi s 10 % soli bilo više peptida manje molekularne mase (500-100 m/z), u usporedbi s podlogom bez soli, iz čega se može zaključiti da je u podlozi s većim salinitetom aktivnost proteaza bila veća. Proteaze iz Virgibacillus sp. SK37 koje su se lučile u slanoj podlozi hidrolizirale su lizin, arginin i tirozin, a one u podlozi bez soli lizin i arginin

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillus sp. SK37

<p>Abstract</p> <p>Background</p> <p>Microbial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the <it>Bacillus </it>species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications.</p> <p>Results</p> <p>A gene encoding a novel member of the subtilase superfamily was isolated from <it>Virgibacillus </it>sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the <it>aprX </it>gene from <it>Bacillus </it>species. This gene, designated <it>aprX-sk37 </it>was cloned into pET21d(+) and over-expressed in <it>E. coli </it>BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl₂. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl₂at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H₂O₂. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX).</p> <p>Conclusions</p> <p>The stability towards H₂O₂and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.</p

Silage production and silage lactobacillus survival in the digestive tract of dairy cows.

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Potential microorganism for the direct production of L-Lactic acid from cassava starch without carbon dioxide production.

โครงการหนึ่งอาจารย์หนึ่งผลงา

Bacterial stains for the direct prodution of L-lactic acid from cassava and sago starch.

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Edible mushrooms in dry dipterocerp forest of Tup Lan National Park in Thailand

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Scanning electron micrscope and nucleic acid technique aid the identification and diversity study of Thai rice-field crab.

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Study of lectins from wild mushrooms

ผลงานวิชาการคณาจารย์มหาวิทยาลัยเทคโนโลยีสุรนาร