Che Nyonya Abdul Razak

Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co2+ and Hg2+, whereas Mg2+, Fe2+, Cu2+, Zn2+ and Sr2+ had little or no inhibitory effect. However, Mn2+ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca2+.

Isolation and screening of an extracellular organic solvent-tolerant protease producer

Abstract An extracellular organic solvent-tolerant protease producer has been successfully isolated out of 11 isolates of benzene–toluene–xylene–ethylbenzene (BTEX) tolerant bacteria. This organic solvent-tolerant microorganism was found to be a polycyclic-aromatic-hydrocarbons (PAHs) degrader and identified as Pseudomonas aeruginosa strain K. It was selected based on the stability of its proteolytic enzyme in the presence of various organic solvents. P. aeruginosa strain K protease tolerated up to at least 50% (v/v) of benzene, n -hexane, 1-decanol, isooctane and n -hexadecane and was also stable in the presence of 25% (v/v) n -decane and n -dodecane. This enzyme strain K was activated 2.5, 1.5 and 1.2 times by 75% (v/v) of 1-decanol, isooctane and n -dodecane, respectively. This organic solvent stable protease could be used as a biocatalyst for enzymatic synthesis in the presence of organic solvents.

Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).

Some characteristics of lipases from thermophilic fungi isolated from palm oil mill effluent

Rhizopus oryzae and Rhizopus rhizopodiformis are thermophilic fungi isolated from palm oil mill effluent (POME). They are able to grow up to 50 °C and produce remarkable amounts of extracellular lipases. The extracellular lipases from both fungi displayed quite similar characteristics. The optimum pH and temperature for both lipases were 6.0 and 45 °C, respectively. However, lipase from R. rhizopodiformis was slightly more thermostable than that of R. oryzae lipase but the latter was more stable over broader pH ranges compared to the former, specially at acidic pH. Both the enzyme showed rapid loss of activities at temperatures above 50 °C and pH above 7.0. Lipase from R. oryzae showed broader specificities to substrates than R. rhizopodiformis, but both displayed little reactivity for triacetin and polyoxysorbitan (Tween 20, 40, and 60). Both the lipases are 1,3 specific.

Lipase immobilized on poly(VP-co-HEMA) hydrogel for esterification reaction

Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate)(poly[VP-co-HEMA]) hydrogel, and divinylbenzene was the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases was also determined. Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90∶10 exhibited the highest activity. Lipase immobilized on VP(%):HEMA(%) 50∶50 showed the highest thermal, solvent, storage, and operational stability compared to lipase immobilized on other compositions of hydrogels as well as the native lipase.

Enzymatic synthesis of fatty esters by alkylated lipase

Lipase from Candida rugosa was chemically modified by reductive alkylation with aldehydes of various chain lengths. The derivatised lipases showed a higher esterification activity compared to native enzyme. The degree of activity enhancement depended on the type and molecular weight of the modifiers used and the degree of modification of the enzyme. They exhibited higher activities in non-polar than in polar solvents. The optimum esterification temperature and preference of fatty acids as acyl donors of the derivatised lipases were very similar to those of native enzyme. Lipase derivatised with dodecyldehyde was more thermostable than those modified with acetaldehyde. Alkylated lipases are relatively more stable in organic solvents than the native enzyme.