Swati Joshi

Biotechnology of cold-active proteases.

The bulk of Earth’s biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

Characteristics and applications of a recombinant alkaline serine protease from a novel bacterium Bacillus lehensis.

A highly alkaline protease (BLAP) from a novel psychrotolerant and alkaliphilic bacterium, Bacillus lehensis was cloned and expressed in Escherichia coli. BLAP belongs to subtilase S8 family of proteases, comprising 27 aa secretion signal, 83 aa prosequence and 269 aa mature BLAP. The amino acids Asp 141, His 171 and Ser 324 form catalytic triad, while Ile 214, Leu 233 and Asn 267 are other active site moieties. Recombinant alkaline protease (rBLAP) is a monomeric protein of 39.0±1.0kDa, and it is active over broad pH (8-12) and temperature (30-60°C) ranges, with optima at pH 12.8 and 50°C. rBLAP is stimulated by SDS, Co(2+), Ca(2+), β-ME, and inhibited by Hg(2+) and PMSF. The rBLAP is compatible with commercial detergents, useful in silk degumming and silver recovery from the used photographic films and a potent biocontrol agent for arresting the development of eggs of the nematode Meloidogyne incognita.

In vitro engineering of microbial enzymes with multifarious applications: prospects and perspectives.

The discovery of a novel enzyme from a microbial source takes anywhere between months to years, and therefore, there has been an immense interest in modifying the existing microbial enzymes to suit the present day needs of the industry. The redesigning of industrially useful enzymes for improving their performance has become a challenge because bioinformatics databases have been revealing new facts on a day-to-day basis. Modification of the existing enzymes has become a trend for fine tuning of biocatalysts in the biotech industry. Hydrolases are employed in pharmaceutical, biofuel, detergent, food and feed industries that significantly contribute to the global annual revenue, and therefore, the emphasis has been on engineering them. Although a large data is accumulating on making alterations in microbial enzymes, there is a lack of definite information on redesigning industrial enzymes. This review focuses on the recent developments in improving the characteristics of various biotechnologically important enzymes.

Biotechnological Applications of Biocatalysts from the Firmicutes Bacillus and Geobacillus Species

Bacillus and Geobacillus species are the dominant workhorses in industrial biotechnology. The ability of these bacteria to produce a variety of extracellular enzymes, such as amylases, xylanases, proteases, phytases, carbonic anhydrases, catalases, pectinases and others, has ranked them among the most important enzyme producers. These bacteria produce enzymes that are active in broad pH and temperature ranges, and thus, have led to the development of processes for large scale production of a variety of enzymes with suitable properties for industrial applications. The industrial enzymes are produced by submerged as well as solid state fermentations. Classical mutation and selection techniques, and protein engineering strategies have been used for developing enzymes with the desirable characteristics. The enzymes find applications in starch, paper, food, feed, textile and several other industries.

Recombinant thermo-alkali-stable endoglucanase of Myceliopthora thermophila BJA (rMt-egl): Biochemical characteristics and applicability in enzymatic saccharification of agro-residues

Codon adaptation index (CAI) of a 1263bp long endoglucanase encoding gene from the thermophilic mould Myceliopthora thermophile BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (Mt-egl) has been constitutively expressed in Pichia pastoris under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of ∼47kDa. rMt-egl is optimally active at pH 10 and 50°C, displaying stability in broad pH and temperature ranges, with a t1/2 of 60 and 15min at 90 and 100°C, respectively. This retained ∼70% of activity after 3h incubation at pH 5-12. The Km, Vmax, kcat and kcat/Km of rMt-egl were 5mgmL-1, 20μmolesmin-1mg-1, 1.02×103s-1 and 204s-1mg-1mL-1, respectively. Homology modeling and bioinformatics analysis confirmed catalytically important role of glutamate 234 and 344. rMt-egl released high amounts of reducing sugars from wheat bran and corn cobs (421 and 382mgg-1), thus making it a useful biocatalyst for producing bioethanol and fine chemicals from agro-residues.

Bioprocess for efficient production of recombinant Pichia anomala phytase and its applicability in dephytinizing chick feed and whole wheat flat Indian breads

Abstract The phytase of the yeast Pichia anomala (PPHY) is a suitable biocatalyst as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. The cell-bound nature and low phytase titres are the main bottlenecks for its utility in food and feed industries. In this investigation, we have overcome the problems by constitutive secretory expression of PPHY under glyceraldehyde phosphate dehydrogenase (GAP) promoter. A ~44-fold increase in rPPHY titre has been achieved after optimization of cultural variables by one-variable-at-a-time approach and two factorial statistical design. The use of GAP promoter makes the cultivation of the recombinant P. pastoris straight forward and eliminates the requirement of methanol for induction and hazards associated with its storage. Among metal-phytate complexes, Ca2+ phytate is hydrolyzed more efficiently by rPPHY than Co2+, Mn2+, Mg2+, Fe3+ and Zn2+ phytates. The enzyme is effective in dephytinizing whole wheat unleavened flat Indian breads (naan and tandoori) and different broiler feeds, thus mitigating anti-nutritional effects of phytates.

Atmospheric Carbon Dioxide Levels in Garhwal Himalaya, India

Measurements of atmospheric were made in the mountainous region of Srinagar-Garhwal, India (January to December 2006). Concentrations of averaged ppm in 2006. Daily variations of values showed minimum during the daytime (376.2 ppm) and peaked in the morning/evening (410.1 ppm). At monthly intervals, the values varied from (May) to ppm (March). If divided on a seasonal basis, the values declined to minimum amounts in post-monsoon ( ppm) and reached maximums during winter ( ppm). Although phenology is significant in controlling levels, short-term changes cannot be explained without the anthropogenic perturbations (e.g., vehicular pollution and forest fires). The concentrations in Srinagar-Garhwal (393.4 ppm) were generally higher than those of other major monitoring locations around the world.

Recombinant exochitinase of the thermophilic mould Myceliopthora thermophila BJA: Characteristics and utility in generating N‐acetyl glucosamine and in biocontrol of phytopathogenic fungi

Chitinase from the thermophilic mould Myceliopthora thermophila BJA (MtChit) is an acid tolerant, thermostable and organic solvent stable biocatalyst which does not require any metal ions for its activity. To produce high enzyme titres, reduce fermentation time and overcome the need for induction, this enzyme has been heterologously expressed under GAP promoter in the GRAS yeast, Pichia pastoris. The production medium supplemented with the permeabilizing agent Tween‐20 supported two‐fold higher rMtChit production (5.5 × 103 U L−1). The consensus sequences S(132)xG(133)G(134) and D(168)xxD(171)xD(173)xE(175) in the enzyme have been found to represent the substrate binding and catalytic sites, respectively. The rMtChit, purified to homogeneity by a two‐step purification strategy, is a monomeric glycoprotein of ∼48 kDa, which is optimally active at 55°C and pH 5.0. The enzyme is thermostable with t1/2 values of 113 and 48 min at 65 and 75°C, respectively. Kinetic parameters Km, Vmax, kcat, and kcat/Km of the enzyme are 4.655 mg mL−1, 34.246 nmol mg−1 s−1, 3.425 × 106 min−1, and 1.36 × 10−6 mg mL−1 min−1, respectively. rMtChit is an unique exochitinase, since its action on chitin liberates N‐acetylglucosamine NAG. The enzyme inhibits the growth of phytopathogenic fungi like Fusarium oxysporum and Curvularia lunata, therefore, this finds application as biofungicide at high temperatures during summer in tropics.