INTRODUCTION:

Establishment and development of environmental friendly technologies are highly challenging processes in the current scenario. Furthermore, the usage of conventional and physico-chemical process for bioremediation is not applicable for large scale processes. Hence, to curtail these challenges and limitations the biocatalysts are utilized as a promising robust tool for the degradation of intoxicating micropollutants.

Oxygen is indispensable for all the living organisms but generation of dioxygen compounds such as Reactive Oxygen Species (ROS) can be toxic. In order to destroy these free radicals, aerobes recruit the large panel of proteins, including the heme peroxidases.

Peroxidases (EC 1.11.1.7) are oxidoreductases, capable of catalysing the oxidation of wide range of organic and inorganic substrates, with the reduction of H₂O₂.

These enzymes can also utilize lipid peroxides as electron donors or acceptors¹. The Horse Radish peroxidase was the first promising candidate, known for more than 20 decades and it has been the vital topic of studies till now for several peroxidase mediated applications². Later, these enzymes were found to be distributed diversely in nature, their vast presence in bacteria, fungi, algae, actinobacteria and plants systems have been documented³.

Peroxidases are broadly classified as heme and non-heme peroxidases. The heme peroxidases are composed of a protoporphyrin IX (heme), a prosthetic agent, contradictorily, the prosthetic group are truantry in non-heme peroxidases. The heme peroxidases are phylogenetically classified into two major superfamilies (peroxidase-cyclooxygenase and peroxidase-catalase superfamilies) and three other families such as dyP-type peroxidases (DyPPrx), haloperoxidases (HalPrx) and di-heme peroxidases⁴.

The Peroxidase-cyclooxygenase superfamily is made up of members from all domains of life, whose classification was formerly restricted to only animal peroxidases. These multifarious enzymes are dominant to catalyze oxidation of halides⁵. The peroxidase-catalase superfamily can be additionally categorized into three vital classes. Class I incorporates intracellular peroxidases like yeast cytochrome c peroxidase (CcP) which acts as scavenger of reactive oxygen species (ROS) and significant degrader of hydrogen peroxide during the electron transport chain.⁶ This periplasmic enzyme can also function as a signalling protein and mitochondrial peroxide sensor in Saccharomyces cerevisiae⁷.

Ascorbate peroxidase (APx) comes next, is affiliated with the removal of hydrogen peroxide in the chloroplast of higher eukaryotes like plants⁸ and ultimately, the catalase-peroxidases (KatG) exhibiting the hybrid catalytic activities of both peroxidase and catalase, can palliate the oxidative stress exhibited by the organism due to the formation of free radicals or ROS⁹.

Class II is recognized as peroxidase-catalase superfamily, in which the ligninolytic extracellular enzymes generated by fungus such as lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) are incorporated. Whereas Class III family includes the plant secretory peroxidases such as horseradish peroxidase (HRP), soya bean peroxidase (SBP),switch grass peroxidase (SGP), Neem peroxidase, tree legume peroxidases, papaya peroxidases etc¹⁰-¹⁴.

REASONS FOR INTEREST IN RECOMBINANT PEROXIDASE:

The global market for industrial enzymes was estimated about $4.2 billion in 2014 and expected to develop at a compound annual growth rate (CAGR) of approximately 7% over the period from 2015 to 2020 to reach nearly $6.2 billion¹⁵. Figure 1 enlists the top global producers of horse radish peroxidase. The production of peroxidases from their native source, leads to inadequate outturns and isoenzymes mixtures differing in glycosylation which impedes the subsequent enzyme applications. Thus, the heterologous production of these enzymes in suitable host is a reasonable alternative¹⁶.

With the advent of recombinant DNA technology, a microbe can be genetically manipulated and cultured in large quantities to meet increased demand¹⁷. Recombinant enzymes can also be characterized due to their auxiliary advantages such as quality by design guidelines, easier isolation, purification, well-defined enzyme preparations and export outside the cell. Additionally, the aptitude to control the recombinant protein or enzyme production by regulatory expression vectors is also an undeniable and worthwhile advantage^16,18.

Figure 1: Overture of global countries, dwelling as commercial producers of Horse radish peroxidase (HRP) with high market values (USD Million) from 2012-2017 (Source:Absolute reports market research department)

In this consistent prospect, peroxidases are crucial component for widespread applications like dye degradation¹⁹, delignification²⁰,biosensor development²¹, biodegradation oftoxic nitroaromatic compounds²², immunoassay kits²³, effluent treatments²⁴ and melanin reduction²⁵. With these uses, recombinant peroxidases have acquired the potential to become the largest group of commercially used biocatalysts worldwide. Due to their multifarious targeted applications, heterologous expression of peroxidases has been efficiently carried out in wide panel of cell factories such as bacteria²⁶, yeast¹⁹, plant²⁷, and fungus²⁸.

So far, reviews on only recombinant production of horse radish peroxidase have been reported. Thus, this comprehensive review for the first time, aims to focus on recombinant peroxidases from distinct sources and their expression systems, engineering and biotechnological applications.

BACTERIAL CELL FACTORIES:

Peroxidases are often expressed in E.coli via recombinant gene expression, as other expression host like yeast has the undesirable tendency of hyper-glycosylating the heterologously expressed glycoproteins, which restrains the subsequent downstream processing and resulting in the hindrance for enzymatic applications. Furthermore, up to 20-fold higher outturns can be accomplished in E. coli than yeast P. pastoris¹⁶. In accordance to these adorable traits, the heterologous expression of BsDyp from Bacillus subtilis in E.coli, has facilitated the structure solving and deep characterization of recombinant enzyme; higher recombinant peroxidase yield of 100mg/l is another benchmark triumph reported using E.coli cell factory^26,29.

Despite these motivating strategies of protein expression in E.coli, the formation of inclusion bodies (IB) in the cytoplasm, making the protein refolding yield meagre is an abrogating scenario for higher enzyme outturns (Figure 2)¹⁶. However, besides, this limitation the synthesis of target protein as IBs withholds several advantages such as i) IBs are comprised of 95% recombinant enzyme ii) the expressed recombinant protein cannot be degraded easily iii) IBs can be separated easily from cellular debris because of their density difference^30-33. Thus supporting this strategy, the final yield of refoldedand purified tobacco peroxidase was productive, about 60 mg per litre, quantified in E. coli culture³⁴. The peroxidase from phytopathogenic rice blast fungus, Magnaporthegrisea has also been expressed successfully in E.coli.A collection of references on heterologous expression of peroxidases in E.coli is listed out in Table 1.

Figure 2: A pictorial representation of Escherichia coli cell presenting the expression and localization choice of recombinantly expressed peroxidases.Recombinant peroxidases are expressed in E.coli host systemoften as a misfolded protein, which accumulates as inclusion body(IB).These IBs are either stored in the cytoplasm(A) or can be targeted to the periplasm(B) of E.colicellthrough specific protein transporters (such asTat- and Sec-pathways).Energy-driven and active processes may confer their functional role for the deposition of misfolded protein in IBs.

Table 1: List of various recombinantly expressed peroxidases inE.coli cell factory systems.

Gene	Source	Host	Reported yields	Remarks	References
Imnp	Irpexlacteus F17	E.coli strain Rosetta (DE3)	76mg/l	Inclusion bodies; enzyme refolded to be active.	[36]
Ec-apx1	Eleusinecoracana	E. coli BL-21	n/a	Fusion protein; intracellular.	[37]
TPx1	Branchiostomabelcheritsingtaunese	E. coli strain BL21	n/a	Fusion protein; intracellular.	[38]
katG1	Magnaporthe Grisea	Escherichia coli BL21 DE3	30mg/l	Constitutively expressed, intracellular.	[35]
LmTRYP6	Leishmania Major	E. coli BL21, E. Coli XL1-Blue	n/a	Detection of full tryparedoxin peroxidase gene sequence.	[39]
GPx	Pseudoalteromonas sp. ANT506	E. coli BL21	18.68%	Intracellular, active.	[40]
Hrp	Horse radish	E. coli BL21(DE3)	48mg/l	Soluble; targeted to periplasm.	[26]
KatG	Mycobacterium sp. Strain PYR-1	Escherichia coli	n/a	Intracellular, active.	[41]
n/a	Lepidiumdraba	E. coli BL21	n/a	Insoluble inclusion bodies.	[42]
LDP	Lepidiumdraba	Escherchia coli BL21	16mg/l	Refolded from inclusion bodies	[43]
Hrp	Horse radish	E.coliBL21(DE3)	100mg/l	Refolded from inclusion bodies	[26]
TOP	Nicotianatabacum	E.coli BL21(DE3)	2.55mg/l	Refolded from inclusion bodies	[44]
MnP3	Phlebiaradiate	E. coli W3110	6% - 7%	Refolded from inclusion bodies	[45]
Pbapx	Pimpinellabrachycarpa	E. coli	n/a	Recombinant expressionresulted in a 1.8 fold increase in Pbapxactivity after treatment with IPTG for 1h	[46]
hrp	Horse radish	Escherichia coli BL21	24%	Refolded from inclusion bodies.	[47]
HRP C	Horse radish	Escherichia coli	8–10 mg/L, 0.5 mg/L	Targeted to periplasm; active; refolded from inclusion bodies.	[48]
HRP	Horse radish	E. coli strainBL21	110 µg/L	14-fold higher HRP activity of mutant gene than wild-type	[49]
TXNPx	Crithidiafasciculate	E. coli BL21(DE3)	2.51U/mg	Intracellular	[50]
HRP	Horse radish	E. coli KIT strain	3–4%	Refolded from inclusion bodies	[51]
PodC	Brassica napus	Escherichia coli Rosetta 2	36 mg/l	Lactose induced; Intracellular; refolded	[52]
BnAPX	Brassica napus	E. coli BL21 (DE3)	n/a	Fusion protein; Targeted to chloroplast.	[53]
Mlip	Trametescervina	E. coli BL21(DE3)	0.6%	Refolded from inclusion bodies	[54]
sscp	Glycine max	E. coli BL21(DE3	11.23 U/ml	Active protein yield without invitro folding.	[55]

n/a-no data available.

Peroxidases are expressed on the ER; the expressed proteins travel to golgi apparatus through secretory vesicles (SV) for post-translational modification. The modified proteins are carried by the secretory vesicles either to the hyphal tip for the extracellular apical secretion or to the septa for in an alternative secretory pathway.

PEROXIDASE ENGINEERING:

Protein engineering makes the enzyme active, stable and specific towards the substrate thus recombinant proteins can be efficiently produced in relevance to the requirement of the users and the process. Furthermore, the enzyme productivity can be augmented by strong promoters, multiple gene copies, and efficient signal sequences, suitably designed to target proteins into the extracellular medium, thus making the downstream processes easy. Several invitro molecular biology techniques such as superior site-directed or random mutagenesis can offer proficient yields, where a single variation in aminoacidic sequences confers improvement in physiochemical properties, such as stability to acidic pH and radical attacks. For instance, introducing a tryptophan residue in the catalytic site, enhanced the acidic stability and Mn²⁺oxidative activity of the engineered peroxidase, with consequent manifestation of oxidative activities towards veratyl alcohol and synthetic dye, Reactive Black 5⁹². Highlighting the importance of peroxidase engineering, mutation of four phenylalanine residues with alanine residues resulted in marginal improvement of radical stability HRP C⁹³.

Also, engineering peroxidases by incorporating the diverse prominent techniques like MolCraft ‘in vitro protein evolution systems, Cell sorting, Computational protein design, directed evolution, DNA shuffling, Peptidomimetics, mRNA display, De novo enzyme engineering, Cell-free translation systems, Stimulus responsive peptide systems and Designed divergent evolution can establish a remarkable improvement in protein in conjunction with the higher outturns in shorter duration. Thus, for future investigations, these networks of engineering techniques can be highly encouraged to meet the high global market demand of peroxidases.

APPLICATIONS OF PEROXIDASES:

Peroxidases have shown a great aptitude within a variety of industrial applications, where they represent a vital route for “greening” chemical processes. Owing to the heterogeneous properties observed among peroxidases from various sources, an ever-increasing suite of native peroxidases has been applied to different biotechnological processes, with the aim to find the most suitable enzyme for a specific application. However, only few examples of industrial uses of peroxidases currently exist. Most of biotechnological applications with recombinant peroxidases are based on the same commercial preparation. Although, despite these factors, recombinant peroxidases dwell as unbeatable options, for variegated biotechnological applications(Figure 5)

Figure 5: An schematic representation of commercial, industrial and biotechnological applications of peroxidases

Recombinant peroxidase based electronic tongue (biosensor):

Biological electron transfer (ET) reactions are responsible for regulating many biological processes, due to their potentialistic high selectivity, proficiency of the photosynthetic and respiratory ET chains, pH and ionic gradients and ultimately for metabolic transformations^94,95. Under conditions when an electrode replaces natural redox partners of ET proteins or redox enzymes, electrochemistry enables the characterisation of the enzyme/protein function, quantitative estimations of ET rates at electrodes and revealing the mechanisms of biological ET reaction. At this point, there emerges a particular interest in scrutinizing the direct ET reactions between the electrode and the protein where, the electrode can accept or donate a electrons for the protein^96-105.

Recombinant horseradish peroxidase (rHRP) structurally a, dimer are used for biosensing applications. For example, three forms of (rHRP), such as wild type recombinant HRP, and two recombinant forms with six-His tag at the C and the N-terminus were adsorbed with gold electrode with high and stable responses to hydrogen peroxide due to the bioelectrocatalytic reduction based on electron transfer between HRP and gold. The rHRP modified gold electrode based biosensor was able to detect a lower detection limit of 10mM of H2O2¹⁰⁶. In a similar study, Andrey and co-workers, used gold electrode with wild-type rHRP and recombinant forms with cysteine and histidine tags at Asn57, Gln1, Asn189, or Ser309 residues of the protein with long range electron transfer based on the Chemisorption of the various rHRPs onto the gold electrodes through the tags introduced in different positions of the protein surface offering anisotropic orientations of the rHRPs on the gold surface, which permitted a restricted ‘‘rotation’’ of the rHRP molecules on the electrodes¹⁰⁷.

The genetically manipulated recombinant tobacco peroxidases (rTOP), are another promising commercial candidates, in addition to HRP. The rTOP outranks as a more active enzyme than rHRP in the reaction of direct electron transfer from the enzyme into the gold surface.In accordance to this statement, rHRP has been recounted to exhibit 63±7 % of direct electron transfer¹⁰⁸while that of rTOP was 68±3 %¹⁰⁹. The rTOP can also exist as a benchmark in advanced biodetection system such as amperometric flow immunosensors that are based on a peroxidase chip (P-chip) principle. In these sensors, recombinant peroxidase and a monoclonal antibody are immobilized to a particular analyte like Simazin (herbicide) on a gold electrode. The concentration of Simazin is determined by the virtue of its competition with simazin conjugate and glucose oxidase for binding with antibodies at the electrode¹¹⁰.

Recombinant Peroxidase for dye degradation:

Recombinant peroxidases are prolific option for dye biotransformation. For instance, polyacrylonitrile based entrapped horse radish peroxidase beads manifested a potential use of acid orange 20 degradation¹¹¹. A similar strategy has been explored with extracellular fluid entrapped using chitosan-Fe2O3 nanoparticles with peroxidase activity¹¹². Recombinant peroxidases also represent an attractive route for degradation of phenol and synthetic dyes such asDrimaren Blue CL-BR, Drimaren Yellow X-8GN, Drimaren Red K-4Bl and Disperse Navy Blue HGL¹⁹. Out of ordinary, to other recombinant peroxidases, the heterologous enzyme expressed in Trichodermareesei from Pleurotussapidus displayed an advanced bleaching feature of β-carotene and annatto in addition to the anthraquinonic dye Reactive Blue5¹¹³. These eminent catalyst also dwell as oxidizer of inducers like veratryl alcohol and synthetic dyes, such as bromophenol blue and 2,6-dimethoxyphenol dyes¹¹⁴. Hence, in future, the improvement of textile effluent and waste remediation can be highly influenced by molecular docking and dynamic simulation studies.

Recombinant Peroxidase for analytical applications:

The enzymatic immunoassays are based on coupling of antigens or antibodies with marker enzymes such as horseradish peroxidase (HRP, EC 1.11.1.7), for the quantitative analysis and detection of wide panel of substances. However, all the eminent analytical approaches used for the chemical conjugation of haptens and proteins result in the partial inactivation of the conjugate and enzyme heterogeneity, which influences the specificity and sensitivity of the ELISA. Genetic engineering is highly useful for obtaining the recombinant protein conjugates with antigens or antibodies. Such conjugates offers a series of advantages; firstly, they have 1: 1 stoichiometry; secondly, they possess a homogenous composition; thirdly they exhibit the functional activities of both the antigen/antibody and the marker protein, in addition to the reproducibility and simplicity of their production. Recombinant conjugates of antibodies with luciferase¹¹⁵, Arthromycesramosus peroxidase¹¹⁶, alkaline phosphatase¹¹⁷-¹¹⁹, were recounted previously. Recombinant fusion antigens can also be obtained with the bacterial enzymes like β-galactosidase^120,121.

The main problem concerned with alkaline phosphatase and β-galactosidase conjugates is the oligomeric structure of these two biocatalysts (dimeric and tetrameric, respectively) and enhancing the conjugates affinity compared to the free antigen, significantly. This is an undesirable strategy, when competitive schemes of enzyme immunoassay are used. On the other hand, HRP that is used extensively for preparation of the conjugates⁷⁷. can be expressed in E.colicells as inclusion bodies and as well as periplasmic enzyme; however, resulted outturn are similar with low quantity²⁶. Besides, there is a successful evidence of heterologous expression of the HRP gene in E. coli cells aided with the reactivation of the recombinant HRP containing an oligohistidine sequence on the C terminus¹²², favouring the production of peroxidase as the reporter enzyme with recombinant fusion proteins and their application in the enzymatic immunodetective assay. Supporting this view, the recombinant conjugate of HRP with fatty-acid-binding protein (FABP)¹²³, expressed in E. Coli system has been recounted to be successfully used as an immunotracer while performing the enzymatic immunoassay for early diagnosis of myocardial infarction.

CONCLUSION AND FUTURE PROSPECTIVES:

Bio-economic strategy makes a vision, as a significant platform for bioremediation. The contribution of peroxidases as eminent green biocatalysts is highly multifaceted withholding a promising benefits and scope. Besides these prospective, the first and foremost trait required is comprehending and designing of bio-based processes around what happens in nature and mechanisms involved in it; that is, understanding the synergy between all the biocatalysts involved in the degradation of noxious aromatic pollutants, and customization of this knowledge for designing the novel bio-based processes will cogently contribute towards more productive processes. In a similar vein, exploring the active site topology of peroxidase is highly indispensable for acquitance of the oxidative capacity of the enzyme. In the consistent notion, mediators are also playing significant role in determining the enzymatic catalysis. Although, the contribution of synthetic mediators systems tend to enhance the peroxidase activity and their production, some other synthetic mediators like tannic acid might result in meagre secretion of peroxidases in peculiar cases. However, this is not consistent with the other synthetic mediators like gallic acid, guaiacol and veratryl alcohol as they are addressed to offer a higher peroxidase outturn.

On the other hand, natural mediators such as lignin, lignosulphonic acid could also equally commit their role. The empowerment of engineering in mining enzymes from microbial communities has become more feasible due to advances in tagging, sequencer technology, CRISPER (Clustered Regularly Interspaced Short Palindromic Repeat)/cas9 mediated genome editing, and oligonucleotide directed mutagenesis and artificial gene synthesis. These techniques pose a focussed channel for the discovery of novel peroxidases. The establishment of new bioinformatics tools such as the fungal peroxidase Database (fpoxDB) has been a cumulative support for researchers to design prominent peroxidases. Such technologies can be adapted in the search of better commercial enzyme systems which can be encouraged for remediation and industrial applications.

Peroxidases have been exploited widely in textile industry for textile bleaching, in the food industry as an index of blanching vegetables and fruits; crosslinking of proteins and carbohydrates to impart novel properties, in the pulp and paper industry for pulp bleaching and for delignification of lignocelluloses. They also play a conversant role in bioremediation, biosensing, effluent treatment, nanobiotechnology and cosmetic industry. Despite of all the adroit applications of peroxidases, there is a need for this biocatalyst to be explored in the biofuel production, synthetic application and pharmaceutical compounds removing systems as they are considered as the major micropollutants exhibiting toxic impacts on marine and aquatic creatures such as fishes. The pharmaceutical drugs such as diphenhydramine, albuterol, testosterone, sertraline and estrogens exhibited structural variation of the endocrine glands of fishes, molluscs, frogs. These endocrine disruptors are mainly plastic additives, pesticides and phthalates. The exposure of these disruptors to the fishes accounts to ultimate consequence of ‘intersex fish’ (male fishes with female characteristics) and death. Henceforth, these undesirable effects of drugs on the environment, necessitates to attest clone and produce proficient recombinant and engineered peroxidases to wield them as a robustsustainable and environment friendly tool.

ACKNOWLEDGEMENTS:

The authors thank VIT University, Vellore, Tamil Nadu, India for providing necessary facilities to carry out this work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest. The authors themselves are responsible for the content and writing of the paper.

ABBREVIATIONS USED:

ROS:Reactive oxygen species;

DyPPrx:dyP-type peroxidases;

HalPrx: Haloperoxidases;

CcP:Cytochrome c peroxidase;

APx: Ascorbate peroxidase;

KatG:Catalase-peroxidase;

LiP: Lignin peroxidase;

MnP:Manganese peroxidase;

VP:Versatile peroxidase;

SBP: soya bean peroxidase;

SGP: switch grass peroxidase;

CAGR: Compound annual growth rate;

IB: Inclusion Bodies;

GRAS:Generally regarded as safe;

MIA: methanol-inducible AOX1;

ELIZA: Enzyme-linkedimmunosorbent assay;

SV:Secretory vesicles;

ER: Endoplasmic reticulum;

ET: Biological electron transfer;

rHRP: recombinant horse radish peroxidase;

rTOP: recombinant tobacco peroxidase;

FABP: Fatty-acid-binding protein;

CRISPER:Clustered Regularly Interspaced Short Palindromic Repeat;

fpoxDB:fungal peroxidase Database

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Received on 06.03.2018 Modified on 29.04.2018

Research J. Pharm. and Tech 2018; 11(7): 3186-3196.

DOI: 10.5958/0974-360X.2018.00586.3

Gene	Source	Host	Reported yields	Remarks	References
GluMnP1	Ganodermalucidum	Pichiapastoris	126 mg/L	Extracellular; methanol induced expression	[19]
mnp1	Phanerochaetechrysosporium	Pichiapastoris	40mg/L	Intra(with) and extracellular(without) expression using native and α-factor secretion signal.	[79]
lipH2	Phanerochaetechrysosporium BKM-F-1767	PichiapastorisX-33	15U/L	Extracellular; produced only by multicopyrecombinantsyeast cells.	[80]
HRP	Horse radish	Pichiapastoris	113 mg/L	Chemical conjugation of Horse radish peroxidase with fusion protein, streptococcal protein G.	[74]
HRP	Horse radish	Saccharomyces cerevisiae	260 U/L/OD600	Extracellular; active	[72]
HRP	Horse radish	Pichiapastoris	600 U/L/OD600 15 U/mL	methanol induced expression with synthetic AOX1 promoter	[81]
HRP	Horse radish	Pichiapastoris ∆och1	101mg/L	Extracellular; recombinant peroxidase production highly influenced by hemin.	[75]
LP1	Pycnoporuscinnabarinus	Kluyveromyceslactis	n/a	Enzyme accumulation within the cell, revealing the retainment of protein in pellet.	[78]
LP2	Pycnoporuscinnabarinus	Kluyveromyceslactis	n/a	Extracellular; secreted efficiently.	[78]
LP3	Pycnoporuscinnabarinus	Kluyveromyceslactis	n/a	Enzyme accumulation within the cell, revealing the retainment of protein in pellet.	[78]
n/a	S. viridosporus	Pichiapastoris	2.47g/L	Extracellular; methanol induced expression with AOX1promoter.	[76]
HRP	Horse radish	Pichiapastoris	3-10mg/L	Expression of peroxidase with Fab fragments of antibodies by recombinant conjugation.	[77]

Gene	Source	Host	Reported yields	Remarks	References
ylpA	Phanerochaetesordida YK-624.	Phanerochaetesordida YK-624 mutant,UV-64	n/a	Extracellular; active	[28]
CPO	Caldariomycesfumago	Aspergillusniger	10 mg/litre	Extracellular; active.	[88]
mnp3	n/a	PleurotusostreatusTMG1	0.195 U/flask	Extracellular	[89]
		Pleurotusostreatus TMG5	0.975 U/flask	Extracellular	[89]
		Pleurotusostreatus TMG7	Nd		[89]
		Pleurotusostreatus TMG8	Nd		[89]
		Pleurotusostreatus TMG9	1.560 U/flask	Extracellular	[89]
		PleurotusostreatusTMC1	0.195 U/flask	Extracellular	[89]
		PleurotusostreatusTMC3	0.390 U/flask	Extracellular	[89]
		PleurotusostreatusTMC5	Nd		[89]
		P. ostreatusstrain #261	Nd		[89]
lipA	Phanerochaetechrysosporium	Aspergillusniger	n/a	Expression in a protease-deficient A. nigerstrain using different expression cassettes; extracellular; non active due to incorrect processing of the recombinant enzyme.	[86]
mnp1	Phanerochaetechrysosporium	Aspergillusniger	9.6 ± 1.2 mg/L(Aspergillus minimum growth medium-maltodextrin) 10.7±1.4 mg/L(Aspergillus minimum growth medium-maltodextrin+ 0.02 g/litre of FeSO4) 66.9 ± 1.2 mg/L (Aspergillus minimum growth medium-maltodextrin +500 mg/litre of hemin) 31.5 ± 0.9 mg/L (Aspergillus minimum growth medium-maltodextrin + 5 g/litre of apohemoglobin) 105.9±19.4 mg/L (Aspergillus minimum growth medium-maltodextrin+ 5 g/litre of haemoglobin)	Expression in a protease-deficient A. nigerstrain using different expression cassettes; extracellular; active.	[86]
Mnpl	P. chrysosporium	P. chrysosporium	5–10 mg/L	Extracellular.	[90]
mnp1	P. chrysosporium	A. oryzae	5 mg/L	Extracellular.	[91]
ylpB	Phanerochaetesordida YK-624.	Phanerochaetesordida YK-624 mutant,UV-64	n/a	Extracellular; active	[28]