J. K. Gupta

Strain improvement for the production of a thermostable α-amylase

Abstract Bacillus sp. MK716, a thermophilic and amylolytic soil isolate, produced a thermostable α-amylase active even at 100°C. The enzyme yield was increased 40 times through mutagenesis with ethylmethane sulfonate. The mutated gene was cloned in Escherichia coli -pBR322 system, and the 4.8 kb cloned fragment was mapped with restriction enzymes. Subcloning in Bacillus subtilis of a 2.0 kb portion of the cloned fragment resulted in 107 times increased enzyme yield. The recombinant plasmid was completely stable in B. subtilis .

Purification and properties of an endoglucanase from a Bacillus isolate.

Abstract The thermophilic soil isolate, Bacillus sp. PDV, produced endoglucanase when grown on glucose and could not utilize carboxymethyl cellulose (CMC) or Avicel as growth substrates. The CMCase was purified on a DEAE-Sephacel ion exchange column followed by Biogel-A-0.5m and Superose-6 gel filtration columns. The purified CMCase was found to be homogeneous on polyacrylamide gel electrophoresis with silver staining and had a molecular weight of 33,000 (SDS-PAGE and Sephadex G-100 gel filtration). It showed activity towards crystalline forms of cellulose such as cotton and Avicel and was stable in the pH range of 4–10 and temperature up to 60°C. The enzyme hydrolysed CMC with pH and temperature optima of 5.0 and 60°C and a Km of 0.588 mg ml−1. The enzyme activity was completely inhibited by Hg2+, while other metalions, chelators, various oxidizing agents, and repeated freezing and thawing had no marked effect on activity. Reducing agents, in general, increased the activity.

Molecular cloning and expression in Escherichia coli of a thermophilic Bacillus sp. PDV endo-β-1,4-glucanase gene

Abstract The gene encoding endoglucanase in thermophilic Bacillus sp. PDV was cloned in Escherichia coli strain TB1 using pUC 8 as vector. The cloned 3.1 kb Pst I DNA fragment was found to express the endoglucanase activity in either orientation. The deletion analysis of pSD 81 suggested that the Bacillus endoglucanase gene expressed in E. coli under the control of its own natural promoter, contained putatively in the 0.2 kb Hind III fragment at the 5′ end of the insert. The relative level of endoglucanase expression in E. coli was about three times higher than that in parent Bacillus sp. PDV. The cloned organism secreted about 84% of the total synthesized CMCase into the culture medium. The CMCase was stable up to 60°C and in the pH range of 4–10 .

Occurrence of an organic inhibitor in cane molasses affecting citric acid production by Aspergillus niger

Abstract A highly potent inhibitor of citric acid fermentation by Aspergillus niger is shown to be present, in addition to minerals, in cane molasses. Sephadex G-10 fractionation of molasses shows the inhibitor to be present in the hexose fraction. This fraction, after concentration reduces the citric acid yield from a citric acid-producing system. The hexose fraction of the sulphitated syrup is much more inhibitory than the hexose fraction of unsulphitated syrup, indicating enhanced activity of the inhibitor at this step of cane-juice processing in sugar-mills .

Short communication: Thermostability of α-amylase produced by Bacillus sp. E2-a thermophilic mutant.

An α-amylase from a hyper-producing strain of Bacillus (sp. E2) was stable at 70°C for 30 min but was quickly inactivated at higher temperatures. In the presence of 10mm Ca2+ and starch (20% w/v), however, the enzyme was stable at 90°C for 10 min and after 30 min at 100°C still retained 26% of its initial activity.