Krishna Kant Sharma

Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498

Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol. In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pretreatment to produce more sugar, to be fermented to ethanol. Dilute H(2)SO(4) (3.0%,v/v) treatment resulted in hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byproducts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated substrate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%,w/v) and sodium chlorite (3.0%,w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M citrate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of beta-glucosidase per mL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%) was achieved after 28 h incubation at 50 degrees C. The fermentation of both acid and enzymatic hydrolysates, containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced 7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.

Laccase: enzyme revisited and function redefined

One enzyme, one physiological role, that’s how most scientists have traditionally looked at it but there is a growing appreciation that some enzymes “moonlight” i.e. in addition to their “primary” catalytic function, they carry other functions as well. Moonlighting refers to a protein that has multiple functions, which are not because of gene fusion; splice variants or multiple proteolytic fragments. Until recently laccases were reported from eukaryotes, e.g. fungi, plants, insect. However there is some evidence for its existence in prokaryotes, a protein with typical features of multi-copper oxidase enzyme family. The present available knowledge of its structure provides a glimpse of its plasticity, revealing a multitude of binding sites responsible for multifunctional activity. Laccase represents an example of a ‘moonlighting’ protein that overcomes the one gene-one structure-one function concept to follow the changes of the organism in its physiological and pathological conditions. It is wide spread in plants, where it is involved in biosynthesis of lignin; in fungi it is involved in lignin degradation, development associated pigmentation (melanin synthesis), detoxification and pathogenesis, and in bacteria, laccases are involved in the synthesis of endospore coat protein (cot A).

In vivo enzymatic digestion, in vitro xylanase digestion, metabolic analogues, surfactants and polyethylene glycol ameliorate laccase production from Ganoderma sp. kk-02

Aims:  The effect of in vivo enzymatic digestion (IVED), in vitro xylanase digestion (IVXD), metabolic analogues, surfactants and polyethylene glycol (PEG) on laccase production from Ganoderma sp. kk‐02 was studied.

Middle-redox potential laccase from Ganoderma sp.: its application in improvement of feed for monogastric animals

The variables influencing laccase production by white-rot fungus Ganoderma sp. rckk-02 were optimized employing response surface methodology. Malt extract (6.0% w/v), lignin (0.5% w/v) and pH (5.5) were found to be the most significant factors for enhanced laccase production by 7 fold (226.0 U/ml) as compared to unoptimized growth conditions (32.0 U/ml). The N-terminal sequence of laccase revealed its distinct amino acid profile (S- I- R- N- S- G), which suggested it as a novel enzyme. The Far-UV CD spectrum of the laccase showed single broad negative trough at around 213 nm, a typical signature of all β proteins. The laccase was found to fall in the range of middle redox potential laccases. Purified laccase at dosage of 2.5 Ug−1 body weight when supplemented with pelleted diet of rats, a significant improvement (p < 0.05) in nutrients digestibility without causing any elevation of blood stress enzymes was observed.

Laccase from an alkalitolerant basidiomycetes Crinipellis sp. RCK-1: production optimization by response surface methodology.

A newly isolated alkalitolerant basidiomycetous fungus, identified as Crinipellis sp. RCK‐1 was observed to produce laccase. The effect of different physicochemical factors on laccase production was studied. The identification of the important factors (initial pH of the medium, copper and tryptophan) with simple screening experiment involving optimization using single factor at a time strategy, was followed by application of complex response surface design for further maximizing the laccase production and was helpful in defining the effects and interactions of the physiological and nutritional factors. The statistical optimization by response surface methodology resulted in a 27.0‐fold (619.9 U ml–1) increase in the production of laccase from Crinipellis sp. RCK‐1 when compared to laccase production in unoptimized medium (23.0 U ml–1). The results from the response surface curve suggested that there was interaction between tryptophan and copper in a way that might resulted in positive effect on laccase production from Crinipellis sp. RCK‐1. The relatively higher laccase production by Crinipellis sp. RCK‐1 showed promise of offering great potential in various biotechnological applications. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Agrobacterium-mediated delivery of marker genes to Phanerochaete chrysosporium mycelial pellets: a model transformation system for white-rot fungi

The lack of an efficient transformation system in filamentous fungi is one of the major problems in carrying out cloning and expression of genes to produce a value‐added product at the commercial level. Therefore an attempt has been made to develop an efficient, convenient and expeditious genetic transformation system for successful transfer of transferred DNA, carrying the genes coding for GUS (β‐glucuronidase) (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of Phanerochaete chrysosporium, a widely studied lignin‐degrading white‐rot fungus. The transformation of the marker gene was confirmed through GUS, PCR and Southern‐blot hybridization. Transformation with acetosyringone, in the presence of light with partial bleaching for 1 h, were found to be a better combination for fungal transformation with 48% efficiency. The agro‐transformation method to transform the modified mycelial pellets offers a practical means for exploiting transgenic approaches in genetic manipulation and improvement of P. chrysosporium, a model white‐rot fungus. Moreover, it has the potential to overcome the technical hurdles in genetic manipulation to bio‐technologically exploit many other filamentous fungi of industrial importance.

An evidence of laccases in archaea

Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are a diverse group of multicopper oxidases that catalyze the oxidation of a variety of aromatic compounds. Here we present evidence for distribution of laccases among archaea and their probable functions. Putative laccase genes have been found in different archaeal groups that might have branched off early during evolution, e.g. Haloarcula marismortui ATCC 43049, Natronomonas pharaonis DSM2160, Pyrobaculum aerophilum IM2, Candidatus Nitrosopumilus maritimus SCM1, Halorubrum lacusprofundi ATCC 49239. Most of the archaeal multicopper oxidases reported here are of Type 1 and Type 2 whereas type 3 copper-binding domain could be found in Pyrobaculum aerophilum IM2 and Halorubrum lacusprofundi ATCC49239. An analysis of the genome sequence database revealed the presence of novel types of two-domain laccases in archaea. ed using this method. CyMVin the positive samples of Phalaenopsis sp. and Arachnis sp. was confirmed by DNA sequencing and cp gene homeology blast. The results showed that CyMV extracted from the leaves of orchid in Hangzhou, Zhejiang Province, China, could be derived from Kunming city (KM), Yunnan Province, China. This method characterized by high sensitivity, specificity, and precision is suitable for early diagnosis and quantitative detection of CyMV.

Microorganisms and Enzymes Involved in Lignin Degradation Vis-à-vis Production of Nutritionally Rich Animal Feed: An Overview

Lignocellulosics are the major structural component of woody and nonwoody plants and represent a major source of renewable organic matter. The plant cell wall consists of three major polymers: cellulose, hemicellulose, and lignin. Lignocellulose biomass, available in huge quantity, has attracted considerable attention as an alternate resource for pulp and paper, fuel alcohol, chemicals, and protein for food and feed using microbial bioconversion processes. The current industrial activity of lignocellulosic fermentation is limited because of the difficulty in economic bioconversion of these materials to value-added products. Lignin is degraded to different extents by variety of microorganisms including bacteria, actinomycetes, and fungi, of which wood-rotting fungi are the most effective, white-rot fungi in particular. White-rot fungi degrade wood by a simultaneous attack on the lignin, cellulose, and hemicellulose, but few of them are specific lignin degraders. The selective lignin degraders hold a potential role in economically bioconversion of plant residues into cellulose-rich materials for subsequent bioethanol and animal feed production. Different fungi adapt in accordance to conditions existing in the ecosystem and complete their task of carbon recycling of the lignified tissues, and some white-rot fungi have capability to completely mineralize it. It is known that white-rot fungi are able to perform lignin degradation by an array of extracellular oxidative enzymes, the best characterized of which are lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase. However, the regulation of the production of individual enzymes and lignin degradation is a complex phenomenon. Unfortunately, even selected white-rot fungi take long in delignifying the lignocellulosic substrates. Therefore, it is necessary to improve these fungi for their ability to degrade lignin through various conventional and modern approaches. A considerable progress has been made in this direction during the past two decades; LiP, MnP, and laccase genes have been cloned, and an efficient Agrobacterium-mediated transformation system has been developed, which will eventually help in successful expression of the desired protein. This chapter presents an overview of diversity of lignin-degrading microorganisms and their enzymes especially in developing animal feed. In addition to that, advances in molecular approaches to enhance the delignification capability of microorganisms are also discussed.

Plant-Microbe Interaction and Genome Sequencing: An Evolutionary Insight

Abstract The survival of human population depends on the balance between the demand and supply of plant resources for food, feed, and fuels. Interestingly, plants are in continuous interaction with both aerial and terrestrial microbes, which creates a challenge for the crop productivity and evolution of new varieties. The opportunistic and avirulent plant microbial symbiont after strong colonization releases a variety of compounds that causes nonreversible changes to the plant genome, proteome, and secretome. Furthermore, the advancement in genome- and transcriptome-based studies on epiphytic, endophytic, phyllospheric, and rhizospheric microorganisms have transformed our understanding of many phytopathogens and have significantly broadened our knowledge of plant-microbe interactions.