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G. Singh et al.
low pH-xylanase, provided by M/s Biocon India Pvt. Ltd. Subsequently, several other trials were organized in di ff erent mills by using di ff erent types of raw materials and practicing di ff erent pulping and bleaching processes, using xylanase enzymes of di ff erent qualities (including al- kaline and thermo-tolerant) (Singh et al., 2016). According to the news bulletin on pulp and paper R&D, CPPRI Saharanpur (2011), evaluated the potential of bleaching enzymes at pilot and mill scale that was sponsored by the Department of Biotechnology, Govt. of India, New Delhi. Aim of the project was to evaluate prebleaching e ffi ciency of xylanase/laccase biocatalysts produced by Department of Micro- biology, South Campus, Delhi University and Kurukshetra University on hardwood pulps at both pilot and mill scale. The outcome of this trial was quite favorable to commercialize the biobleaching process (http:// www.cppri.org.in). Through the extensive literature search, there were several patents found on xylanases and laccases on the name of Indian laboratories and industries up to the extent for the production of bleaching enzymes but not yet evaluated for their biobleaching po- tential. Moreover Biocon India, Bangalore alone is, selling its com- mercial xyalnase under the name of Bleachzyme F for the deligni fi ca- tion of pulps (Table 3).
6. Heterologous expression of xylanase, laccase and protein engineering
Garg et al. (2012) reported the cloning and expression of Cyathus bulleri laccase in Pichia pastoris. In this study, complete cDNA encoding laccase (Lac) from white rot fungus C. bulleri was ampli fi ed by RACE- PCR, cloned and expressed in P. pastoris under the control of alcohol oxidase ( AOX ) 1 promoter. Later it was also observed, CuSO 4 increased the synthesis of laccase up to 12-fold when added in production media. Goswami et al. (2014) reported cloning and heterologous expression of cellulase free thermostable xylanase from Bacillus brevis. Xylanase gene was isolated and expressed in E. coli BL21. The recombinant xylanase was predominantly secreted to culture medium and showed mesophilic nature (optimum working was at 55 °C, pH 7.0). Both rational design and directed evolution has been widely applied for designing of pro- teins in technically advanced molecular biology laboratories world- wide. Generally most of the enzymes are produced by mesophilic or- ganisms, like fungi, molds, yeasts and several bacteria. Commonly enzymes produced by these types of organisms have less thermo-sta- bility/pH stability and least consistency in the presence of salts and also less speci fi city of enzymes towards their substrates. Therefore it is ne- cessary to bring an improvement in the catalytic performance of en- zymes by applying the protein engineering that is the vital tool of molecular biology. Verma et al. (2013) reported increased thermo- stability of xylanase (Mxyl) retrieved from a compost-soil-based meta- genomic library. After scrutinizing the structure of xylanase by mole- cular dynamics simulation exposed more structural fl uctuations in β - sheets. The surface of β -sheets was enriched with arginine residues by substituting serine/threonine by site-directed mutagenesis; the enzyme with four arginine substitutions (MxylM4) exhibited enhanced ther- mostability at 80 °C. The Half life (t 1/2 ) of MxylM4 at 80 °C, in the presence of birchwood xylan, increased from 130 to 150 min, without any alteration in optimum pH and temperature. The K m of MxylM4 was also, increased from 8.01 ± 0.56 of Mxyl to 12.5 ± 0.32 mg ml − 1 but reduced the a ffi nity as well as speci fi c enzyme activity. Both Mxyl and MxylM4 xylanases remained e ff ective for deligni fi cation of pulp. Lac- case enzyme is metallic biocatalyst unlikely xylanases, it needs med- iators (small molecules) for the deligni fi cation of pulps. Biobleaching of pulps by laccases in absence of mediator component is not feasible due to the less redox potential (E0) of laccases (470 to ~ 800 mV) than non phenolic structures of lignin (1400 mV) like veratryl alcohol (Camarero et al., 2007; Singh et al., 2015). In the case of laccases, protein en- gineering may not consider as worthy if there is only increase in en- zyme activity not E0. Kenzom et al. (2014) have performed the random mutagenesis to Cyathus bulleri lcc gene (WtLcc) by using an error prone
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