Aravind Madhavan

Strategies for design of improved biocatalysts for industrial applications

Biocatalysts are creating increased interest among researchers due to their unique properties. Several enzymes are efficiently produced by microorganisms. However, the use of natural enzymes as biocatalysts is hindered by low catalytic efficiency and stability during various industrial processes. Many advanced enzyme technologies have been developed to reshape the existing natural enzymes to reduce these limitations and prospecting of novel enzymes. Frequently used enzyme technologies include protein engineering by directed evolution, immobilisation techniques, metagenomics etc. This review summarizes recent and emerging advancements in the area of enzyme technologies for the development of novel biocatalysts and further discusses the future directions in this field.

Water hyacinth a potential source for value addition: An overview

Water hyacinth a fresh water aquatic plant is considered as a noxious weed in many parts of the world since it grows very fast and depletes nutrients and oxygen from water bodies adversely affecting the growth of both plants and animals. Hence conversion of this problematic weed to value added chemicals and fuels helps in the self-sustainability especially for developing countries. The present review discusses the various value added products and fuels which can be produced from water hyacinth, the recent research and developmental activities on the bioconversion of water hyacinth for the production of fuels and value added products as well as its possibilities and challenges in commercialization.

Recent advances in the production of value added chemicals and lipids utilizing biodiesel industry generated crude glycerol as a substrate – Metabolic aspects, challenges and possibilities: An overview

One of the major ecological concerns associated with biodiesel production is the generation of waste/crude glycerol during the trans-esterification process. Purification of this crude glycerol is not economically viable. In this context, the development of an efficient and economically viable strategy would be biotransformation reactions converting the biodiesel derived crude glycerol into value added chemicals. Hence the process ensures the sustainability and waste management in biodiesel industry, paving a path to integrated biorefineries. This review addresses a waste to wealth approach for utilization of crude glycerol in the production of value added chemicals, current trends, challenges, future perspectives, metabolic approaches and the genetic tools developed for the improved synthesis over wild type microorganisms were described.

Promoter and signal sequence from filamentous fungus can drive recombinant protein production in the yeast Kluyveromyces lactis.

Cross-recognition of promoters from filamentous fungi in yeast can have important consequences towards developing fungal expression systems, especially for the rapid evaluation of their efficacy. A truncated 510bp inducible Trichoderma reesei cellobiohydrolase I (cbh1) promoter was tested for the expression of green fluorescent protein (GFP) in Kluyveromyces lactis after disrupting its native β-galactosidase (lac4) promoter. The efficiency of the CBH1 secretion signal was also evaluated by fusing it to the lac4 promoter of the yeast, which significantly increased the secretion of recombinant protein in K. lactis compared to the native α-mating factor secretion signal. The fungal promoter is demonstrated to have potential to drive heterologous protein production in K. lactis; and the small sized T. reesei cbh1 secretion signal can mediate the protein secretion in K. lactis with high efficiency.

Recent developments in l-glutaminase production and applications – An overview

l-glutaminases is an important industrial enzyme which finds potential applications in different sectors ranging from therapeutic to food industry. It is widely distributed in bacteria, actinomycetes, yeast and fungi. l-Glutaminases are mostly produced by Bacillus and Pseudomonas sp. and few reports were available with fungal, actinomycete and yeast system. Modern biotechnological tools help in the improved production as well as with tailor made properties for specific applications. Most of the genetic engineering studies were carried out for the production of l-glutaminase with improved thermo-tolerance and salt tolerance. Considering the potential of in vitro applications of l-glutaminase, extracellular enzymes are important and most microbes produce this enzyme intracellularly. Several research and developmental activities are going on for the extracellular production of l-glutaminase. This review discusses recent trends and developments and applications of l-glutaminases.

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. One of the most promising alternatives to petroleum for the production of fuels is microbial production. Yeasts are highly efficient producer of bioethanol with several superior traits over bacterial counterparts. Tools of synthetic biology has revolutionised the field of microbial cell factories especially in the case of ethanol and fatty acid production. Era of yeast synthetic biology began with the well-studied industrial work horse Saccharomyces cerevisiae. Despite many highly beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, non-conventional yeast has for synthetic biology, it currently lags behind Sachharomyces cerevisiae in the number of synthetic networks that have been described. Currently synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris and Yarrowia lipolytica. Here we review basic synthetic biology tools that we can apply to non-conventional yeasts. Moreover we discuss how metabolic engineering and synthetic biology tools can be applied in nonconventional yeasts for improved biofuel production.

Molecular improvements in microbial α-amylases for enhanced stability and catalytic efficiency

α-Amylases is one of the most important industrial enzyme which contributes to 25% of the industrial enzyme market. Though it is produced by plant, animals and microbial source, those from microbial source seems to have potential applications due to their stability and economic viability. However a large number of α-amylases from different sources have been detailed in the literature, only few numbers of them could withstand the harsh industrial conditions. Thermo-stability, pH tolerance, calcium independency and oxidant stability and starch hydrolyzing efficiency are the crucial qualities for α-amylase in starch based industries. Microbes can be genetically modified and fine tuning can be done for the production of enzymes with desired characteristics for specific applications. This review focuses on the native and recombinant α-amylases from microorganisms, their heterologous production and the recent molecular strategies which help to improve the properties of this industrial enzyme.

Metagenome Analysis: a Powerful Tool for Enzyme Bioprospecting

Microorganisms are found throughout every corner of nature, and vast number of microorganisms is difficult to cultivate by classical microbiological techniques. The advent of metagenomics has revolutionized the field of microbial biotechnology. Metagenomics allow the recovery of genetic material directly from environmental niches without any cultivation techniques. Currently, metagenomic tools are widely employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the uncultivable component of microbial communities. The employment of next-generation sequencing techniques for metagenomics resulted in the generation of large sequence data sets derived from various environments, such as soil, the human body and ocean water. This review article describes the state-of-the-art techniques and tools in metagenomics and discusses the potential of metagenomic approaches for the bioprospecting of industrial enzymes from various environmental samples. We also describe the unusual novel enzymes discovered via metagenomic approaches and discuss the future prospects for metagenome technologies.

Secreted expression of an active human interferon‐beta (HuIFNβ) in Kluyveromyces lactis

Interferon‐beta (IFNβ) is a cytokine involved in the antiviral, anti‐proliferative and immunomodulatory responses of cells, and a potent drug for multiple sclerosis. Human interferon beta (HuIFNβ) gene fused with the glucoamylase signal sequence in the N‐terminus and 6 His Tag in the C‐terminus was cloned into pKlac1 vector and introduced in Kluyveromyces lactis to allow secreted expression and one‐step purification of the protein. Recombinant yeast transformant with the highest level of HuIFNβ production was identified, and this secreted up to 1 mg/L of the cytokine after 72 h of incubation. Glycosylated and non‐glycosylated forms of the cytokine were elaborated by the yeast, the latter in higher quantities. His tag of the protein allowed easy one‐step purification by nickel‐nitriloacetic acid affinity chromatography, yielding close to 100% purity. SDS‐PAGE, western blot and MALDI‐TOF‐TOF confirmed the identity of the protein. The biological activity of the recombinant HuIFNβ was confirmed by its anti cell proliferative activity on HeLa cells. Expression of HuIFNβ in K. lactis is advantageous since it is a very safe organism to produce proteins for therapeutic applications, allows glycosylation and offers a cost effective method for large scale production as it can be grown in cheap carbon sources.

Applications of Microbial Enzymes in Food Industry

The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed.