Production of Hydrophobins from fungi

Accepted Manuscript Title: Production of Hydrophobins from fungi Authors: Shraddha Kulkarni, Sanjay Nene, Kalpana Joshi PII: DOI: Reference:

S1359-5113(16)31175-8 http://dx.doi.org/doi:10.1016/j.procbio.2017.06.012 PRBI 11071

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

4-1-2017 9-6-2017 13-6-2017

Please cite this article as: Kulkarni Shraddha, Nene Sanjay, Joshi Kalpana.Production of Hydrophobins from fungi.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Production of Hydrophobins from fungi 1

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Shraddha Kulkarni, 2Sanjay Nene, 1*Kalpana Joshi

Department of Biotechnology, Sinhgad College of Engineering

Affiliated to Savitribai Phule Pune University, Pune-411041, India E-mail: [email protected] 2

Innovation Biologicals Private Limited 100 NCL Innovation Park Dr. Homi Bhabha Road, Pashan, Pune- 411 008, India E-mail: [email protected] Address for correspondence: Dr. Kalpana Joshi 1*

Department of Biotechnology, Sinhgad College of Engineering

Affiliated to Savitribai Phule Pune University, Pune-411041, India E-mail: [email protected]

2 Highlights: 

Focuses on different sources of hydrophobins and diverse attempts made for production

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Gives overview of different traditional production methods used such as use of wild type fungi, solid state fermentation etc.

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Recombinant production methods for Hydrophobins are discussed in details

Abstract

Hydrophobins (HPs) are industrially important surface active, amphipathic proteins produced by fungi. There are many applications reported for HPs in the literature notably as, agents for enhancing bioavailability of water insoluble drugs, food stabilizers, antifouling agents for biomedical devices like catheters, fusion partner for recombinant proteins for purification, low friction coatings on biomaterials, immobilizing enzymes in biosensors, etc. However, there are limitations for industrial scale production of HPs. Various methods have been reported for their production e.g. use of wild fungi from natural hydrophobic environments, use of modified bioreactors for submerged and solid state fermentation and recombinant homologous as well as heterologous microbes. Knowing the industrial importance of HPs many reviews have been published focusing on technical and medical applications of these proteins; however there is no comprehensive overview of HP production in the literature. This review summarizes the efforts made to improve yields of HPs by various bioprocesses and also highlights the strategies designed to overcome problems of low yield of HPs.

Key Words: Hydrophobins; ascomycetes; basidiomycetes; fermentation

3 1. Introduction: Hydrophobins (HPs) are surface active proteins found only in fungi. Their surface activity is on par with some classes of commercial surfactants. These are small proteins comprising of 100-150 amino acids. HPs found in different fungal species perform different roles, including breaching the water layer by decreasing the surface tension for growth of hyphae, attaching with hydrophobic substrates by making lining of spores and fruiting bodies hydrophobic, invading insects by eliciting secretion of enzymes like chitinase, helping to hide antigens from immune system of mammals, endophytic association with plants etc. [1]. These roles are attributed to HPs due to their amphipathic nature. Hydrophobic and hydrophilic combinations of amino acids enable them to work at surfaces and also at water-air, air- oil, and water-oil interfaces. Each HP contains 8 cysteine molecules beside their different amino acid sequence. Amino acid sequence homology studies have shown that HPs performing similar roles show homology with each other across the species. The amphipathic nature of HPs is central to many industrial applications. Applications like bioavailability of drugs, surface coatings, dispersal of hydrophobic materials in aqueous solutions, stabilized foam in food products, biosensors, effective downstream processing of recombinant proteins, and antifouling of biomaterials can be listed as most recent and advanced applications of HPs. Considering their industrial importance attempts have been made to improve yield of HPs and large scale process development. The present review focuses on current information on major HP sources from ascomycetes and basidiomycetes and attempts made for their production. 2. Sources of HPs: The Interpro database (http://www.ebi.ac.uk/interpro/) currently lists 1678 separate entries of HPs with 428 from ascomycetes and 1250 from basidiomycetes. Some evidence suggests that HPs are also present in zygomycetes [2]. Depending upon their self-assembling patterns HPs have been divided into two classes I and II. Their hydropathic patterns are different, so also their purification requirements. Class I HPs require harsh treatment like use of trifluroacetic acid (TFA) to dissolve their protein rodlet layer.

4 Class II HPs do not have an interfacial rodlet layer, consequently they can be easily dissolved with the help of ethanol and sodium dodecyl sulphate (SDS). Species from ascomycetes express HPs from both classes I and II. Basidiomycetes express only class I HPs. Recent research findings suggest that an intermediate class of HPs as well as a unique class I HP is also present. (Table 1) 2.1 HPs from species of Ascomycetes: Many species of genera Aspergillus, Trichoderma, Fusarium, Penicillium, and Neurospora are already known for industrial production of acids, enzymes, proteins, specialty products etc. Some of the species listed above also produce one or more HPs. A bioinformatics approach was applied to study class of HPs from Aspergillus species. Around 74 putative HPs were identified in eight species of Aspergillus. Most of the HPs were from class I category except A.terreus which produced Class II HPs [3] and A.clavitus which produced a HP in a category intermediate to class I and II HPs. HPs have been successfully isolated and characterized from A. fumigatus, A.oryzae and A.nidulans. HPs from Trichoderma genus have in general, a high gene copy number of HPs as exemplified by T.reesei (6 genes) T. virens (9 genes) and T.atroviridis (10 genes) [4]. HPs HFB (hydrophobin) I and II from Trichoderma reesei have been successfully isolated, characterized and produced for many industrial applications e.g. food foams, surface modification, immobilization of molecules, antifouling of biomaterials etc. Isolation and characterization of HPs from other ascomycete genera like Cladosporium, Fusarium, and Neurospora is an on-going process for the past two decades. Information about different sources of HPs has been summarized in Table 1. 2.2 HPs from species of Basidiomycetes:

HPs were first isolated from a basidiomycete, Schizophyllum commune; found in the lining of fruiting bodies of spores. Successive findings provided the information about HPs like Sc1 (Schizophyllum

5 commune hydrophobin 1), Sc4 and Sc6 [45] [46]. Each of the three HPs have a different role to play e.g. Sc3 is expressed both in monokaryons and dikaryons but Sc4 is only expressed in dikaryons. Sc3 has got a role in breaching the water layer for growth of hyphae and attachment with substrate [46]. Sc4 prevents gas channels of fruiting bodies to be filled with water in wet conditions. The roles of Sc1 and Sc6 are unknown.

In the past, many basidiomycetes Agaricus bisporus, Pleurotus ostreatus, Dictoynema

glabratum, etc. have been studied for presence of HPs and their specific roles. All these fungi carry multiple genes encoding for HPs. Many HPs have been isolated and characterized from these species. HPs from basidiomycetes have great Scope as most of these genera are GRAS (Generally Recognized As Safe) cleared. Table 2 gives major species of basidiomycetes which have been investigated for their expression and role of HPs in growth and survival of fungi.

3. Production of HPs: The wide spectrum of applications of HPs underlines the need for further research for successful production and purification of these proteins [68]. However, till date production of these proteins has not progressed beyond the laboratory, which has adversely affected industrial applications [68] [69]. From the outset there appears to be a conflict between HP production using wild strains and recombinant strains. Some wild strains do not secrete HPs in submerged culture, which makes their large scale production difficult [70]. Except for one or two reports (production of HFB I from T.reesei (600mg/L) and production of fusion hydrophobin by BASF (Kilogram scale)) which are the genetically modified strains most of the references report a low intrinsic ability of the fungi to produce hydrophobins [98] [129].This is a major lacuna in large scale production of HP. It would need to be addressed by improved production using genetically modified strains. Understanding of mechanism by which hydrophobins accomplish their multiple roles in the fungal life cycles would also help in improving production of HP [70]. We realize this lacuna and at this point of time are unable to provide a realistic solution. Selection of genetically modified strains would also have an impact on their capability to properly fold the produced hydrophobin. In case of Aspergillus niger due to lack of specific foldases and or /chaperons recombinant production of Sc3 hydrophobin was not possible [71]. Sc3 mRNA was accumulated at relatively high levels but Sc3

6 levels in culture medium were negligible [71]. Major strategies used for improving yield of HPs are 1) Use of fungi from natural suitable habitat 2) Recombinant DNA Technology for intracellular expression of HPs. Surface hydrophobicity of fungi can be increased by growing them in a suitable environment. Fusarium solani grown on solid media with hexane showed more hydrophobicity in comparison to the same fungus grown in liquid culture containing less hydrophobic compounds [72]. This indicates that surface hydrophobicity of fungi is directly dependent upon the environment from which it is isolated or adapted for its end use. Another study suggested that surface hydrophobicity of fungi is related with surface expression of HPs. Fungus Paecilomyces lilacinus was grown in a biofilter used for biodegradation of toluene gas. Biodegradation of toluene took around 28 days and the degradation profile was analyzed using gas chromatography. HPs were extracted from biomass using a procedure described by Ying S. and Feng M. [74]. Class I category HP was identified with molecular weight of 11 kDa named as PLHYD. The process yield was 1.1 mg PLHYD /g of biomass [75]. Fungi secreting HPs have been selected for effective insertion of multiple copy number of HP genes and attempts were made to increase their yield. HP production is species specific hence recombinant processes for production of HP in industrial practice has many limitations as compared to production of other homologous and heterologous proteins. In the following sections different production practices and trends are discussed in details. 3.1Production from wild type fungi: 3.1.1Traditional Production Approaches: The first attempt made for production of HP cerato-ulmin (CU) was from wild type fungi Ceratocystsis ulmi. During this work CU was not recognized as a HP; instead it was studied as a toxin responsible for white elm disease, similar to Dutch elm disease. A process was developed to improve production of this toxin by media optimization for cultivation of C.ulmi. Different carbon source, nitrogen source and complex media, were used for studying their effect on production of CU along with monitoring of pH change. HP CU production in culture medium was increased from 25 to 140 mg/l [76] [77].

7 Production of SC3 was the second attempt made to produce HP from monokaryotic wild strain of S.commune [46]. The fungal strain was grown on minimal medium. Production levels of Sc3 in liquid shaken culture of S. commune reached up to 60 mg/l .This form of Sc3 was a monomeric secretary form of the HP [46]. Surface activity of Sc3 was also examined and it was found to decrease 72 mJm-2 culture medium surface tension to 30 mJm-2. Production of HP ABH3 (Agaricus bisporus hydrophobin 3) from Agaricus bisporus was developed along with Sc3. This basidiomycete fungus showed presence of three HP genes viz. ABH1, ABH2 and ABH3. Among these three HPs only ABH 3 was secreted into the culture medium. It had a performance role similar to what Sc3 had in S.commune [46]. A.bisporus was grown for 10 days in static condition on the same minimal medium used for growing Schizophyllum commune. A thin film was detected on surface of the mycelium. It was collected by bubbling air and examined by performing SDS-PAGE. A single band of 19 kDa was observed on the gel. The yield of this process was negligible. To improve HP yield gas bubbles were generated by electrolysis of the medium and a yield of 2 mg/l was obtained [78]. A novel type of class II HP CFTH1 (Claviceps fusiformis tri-partite hydrophobin 1) from Claviceps fusiformis was also separated from shake-flask culture medium after 8 days of fermentation. CFTH1 is a unique HP consisting of hydrophobic leader sequence and three class II HP domains typically called as tripartite HP. It remained attached to submerged hyphae in shake flask culture of C.fusiformis and started accumulating in the medium from day 3 of the 8 day incubation. After day 4, a fraction of CFTH1 was secreted in to the medium. CFTH1 continued to increase in concentration till the day 8 after which concentration was depleted along with glucose depletion. CFTH1 (including mycelial bound and secreted fractions) was harvested from culture medium by treatment with 60% ethanol. Purification yielded 3.5 mg/l of CFTH1 [79]. Production of HPs from wild type fungi has failed on account of low yield. Irrespective of HP type and producing strain all the examples of HP production mentioned above suffered from very poor yield. In an attempt to improve yield of the process by using effective purification technique HFB II (class II HP) was produced from wild type Trichoderma reesei QM 9414. The strain QM 9414 was grown on a medium

8 containing whey and a complex nitrogen source in a 15 L bioreactor for 4 days. Initial production yield of the process was 0.1-0.3 g/L. Purification of HFB II was followed by aqueous two phase extraction of the culture fluid using a surfactant, Berol 532 (C11EO2). HFB II partitioned in surfactant phase due to its amphipathic nature and was extracted back from surfactant phase using isobutanol. Practical purification yield of the process was 74% with concentration of 71 mg/L [80]. A counterpart of the same study will be explored in next section of recombinant production of HP for HFB I and purification using overproducing strain of T.reesei VTT-D- 98692. This method of aqueous two phase extraction was advantageous especially with surfactant C11EO2; as after interaction with hydrophobins it formed the most concentrated surfactant phase and at the same time the partitioning of hydrophobins was effective. Use of isobutanol has shown additional benefits that it did not denature the proteins [80]. Grifola frondosa, an edible mushroom took 9 days to grow for expression of HPs and was prone to contamination. HPs were isolated from cell wall using trifluroacetic acid and SDS. Purification was followed using RP-HPLC and analysis performed using SDS-PAGE. The HP was named as HGF I (Class I hydrophobin of Grifola frondosa ). Yield of HP was near to 300 mg/L [81]. In the same work surface modification properties of HPs were studied in detail. It showed surface activity of 45mN/m, in comparison with SC3 (24 mN/m) [46] this activity was less with same concentration of 50ug/ml [81]. Fermentative production of HPs from wild type fungi remains difficult due to long run times and frequent contamination [81]. 3.1.2 Solid state Fermentation for production of HPs: Bearing in mind the large requirements of HPs very few novel approaches have been developed till date to overcome problems of low productivity. Solid state fermentation (SSF) has been used for production of HPs from wild fungal strains, as SSF mimics living conditions of fungi in their terrestrial habitats. The low production levels of HPs in submerged culture, using wild fungal strains can be improved by using SSF. The probable reason for the improved production levels could be lower water activity, resistance to drying and higher germination rates leading to more hydrophobic conidia [93]. Further improvement in production of HPs from wild strains could be expected by use of hydrophobic substrate for solid state

9 fermentation [82]. Peñas et al. observed that both HP expression and metabolism in SSF differs with respect to submerged culture [82]. In another study, improvement in HP yield was achieved using hydrophobic substrates like n-hexadecane. The fungus P.lilacinus was grown in submerged liquid fermentation (SmF) and solid state fermentation (SSF) using an inert support, perlite [83]. In both the processes n-hexadecane was used as an elicitor of HP production. The findings suggested that PLHYD (Paecilomyces lilacinus hydrophobin) was found on the walls of fungi growing in SSF but not in mycelium of fungi growing in SmF. An HP yield of 1.3mg PLHYD/g of total protein was obtained in SSF. However, increased hydrophobicity of culture medium by addition of n-hexadecane did not increase total yield of the process. In the same work surface activity of PLHYD was studied. It minimized the surface tension of water from 72 mN/m to 36.7mN/m with critical micelle concentration of 0.45mg/mL of HP. The studies concluded that HP expression by fungi is different in SSF and SmF. It was additionally supported by expression of HP Rod A in Penicillium camemberti during SSF [84]. Production of HPs from other species of genus Paecilomyces was explored. Class I HP PfaH1 (Paecilomyces farinosus hydrophobin-1)was isolated, studied and produced in SmF process from P.farinosus [85]. This fungus was grown in two types of media, minimal and standard nutrient liquid (SNL) medium. Submerged fermentation was conducted in an orbital shaker and also under static conditions. After 8-10 days supernatant and mycelium was separated from each other and analyzed for presence of HP PfaH1. Maximum biomass yield of the process was 2.9 mg/g moist biomass when P.farinosus grown at static conditions on minimal media. PfaH1 concentration was negligible in supernatants of culture followed using different media and rotation conditions. In successive work second HP named PfaH2 was isolated and studied from same species in static and submerged liquid fermentation [86]. Entamopathogenic fungi act on chitin present in cuticles of insect and shells of crustaceans by secreting chitinases [87]. Chitinases are extracellular cuticle degrading enzymes responsible for degradation of chitin and penetration of fungus into insects and shells of crustaceans [88]. The process of chitin

10 degradation begins with attachment of hyphae and spores on to hydrophobic surface like insect cuticles. HPs help in this attachment process [89] [90]. Antagonistic relationship between secretion of chitinases and HPs is important concept to be explored for effective HP production [87][88][1]. The same concept was studied in case of entomopathogenic fungi Lecanicillium lecanii. It was grown in submerged liquid and solid state fermentation process [83]. Colloidal chitin was added in culture medium as an inducer of HP production and substitute for carbon source. Solid state fermentation was carried out with polyurethane foam (PUF) as an inert support. Production of HPs and chitinolytic activity was more in SSF compared to SmF [91]. Yields of the HP production were 627.3 mg/L and 57.4 mg/L for SSF and SmF respectively [91]. Requirement of HPs seems to be more for attachment of fungus to solid support in SSF. Application of solid state fermentation for production of HPs will be an effective alternative to submerged processes; when fungi typically grow well in attachment with increased hydrophobicity. This effect could be supported by study done on effect of environmental conditions on production of class II HPs from Rhinocladiella similis [92]. The same study was extended to check effect of different inert substrate on production of HP in solid state fermentation. In this work two inert substrates have been used namely Perlite (P) and Polyurethane (PUF) as they do not contribute nutritionally to growth of fungi. It improved extraction of product without contamination from the support. Simultaneously effect of quality of chitin was also tested for yield of HPs produced. L. lecanii produces class I and Class II HPs. Pure chitin produced 14 % more class I HP like protein (HfbL) than produced by impure chitin with high degree of acetylation. The comparison of production of class I HP like protein using PUF and P showed that Polyurethane (PUF) supported 3-fold more production of HP like proteins than Perlite. This difference in production levels is because of the different nature of both supports (polyurethane is hydrophobic in nature whilst perlite, being of mineral origin is hydrophilic in nature). There was a significant difference in production of Class I HfbL using Polyurethane and perlite. Production of class I HfbL was much higher using polyurethane compared to production using perlite. There was no significant difference in production of class II HfbL using either of the support substrate. The overall result indicated that Class I HfbL was produced for anchoring of fungal

11 cells on polyurethane support and Class II HfbL might be produced for protection against desiccation [93]. 3.1.3 Novel bioreactors in SmF for production of HPs: Nowadays development in bioreactor designs has widened the scope and effectiveness of bio-process. In case of large scale production of HPs these types of applications are rare. In one of the exceptional work, a fungal bioreactor was designed and evaluated as a new cultivation platform for production of HFB II by T.reesei [94]. In this study along with optimization of production and its scale up the effect of carbon source on production of HFB II was evaluated. Effect of glucose, galactose and lactose was studied on production of HFB II. Galactose was unsuitable for production of HFB II because of an extended lag phase displayed by T.reesei [95] [96]. Glucose and lactose were the only carbon sources that resulted in HFB II production [94]. A comparison of production of HP HFB II in submerged liquid fermenter and a fungal biofilm reactor showed that a specially designed biofilm reactor was very effective in increasing productivity of the overall process [94]. Production was increased from 29.6±1.6 mg/L to 48.6±6.2 mg/L after only 48 h. The same system was scaled up to a 10 L fermenter but production of HFB II decreased as evinced by 23% decline in HFB II production [94]. A scaled up operation was performed with intermittent feeding, that yielded thrice the efficiency. Production increased slightly in second cycle but then decreased in the third cycle [94]. Production levels are not at par with over expression in T.reesei but this work provides a platform for development of new bioreactor designs to enhance the HP production using wild type fungi. A summary of HP production using wild types moulds have been presented in table 3. This summary includes range of yields of different processes from 1.1-300 mg/L. Variations in yields can be explained by different roles played by hydrophobins even in same species. Taking the example of T.reesei and three hydrophobins HFB I, HFB II, HFB III produced by species, HFB II is found on the spore walls and is secreted in large amount in culture medium. In contrast HFB I and III remain on the mycelium and secretion is negligible. Hence yield of HFBII was always found to be more than HFB I and III [1]. Sometimes hydrophobins play similar roles but their characteristics are different so that they cannot be produced at the same concentrations. For example Sc3 and Sc4 play similar role in

12 aerial structure formation of S.commune but their characteristics are different so Sc3 is usually found in secreted with good yield while Sc4 remains attached to fruiting bodies [1]. 3.2 Production of fungal HPs by recombinant strains: In comparison with production of HPs by wild types, methods using recombinant strains are more attractive. Several attempts have been made to achieve high yield of HP by using both homologous and heterologous recombinant approaches. An overview of recombinant production protocols followed in common host organisms is explained in following sections with reference to figure 1. 3.2.1 HP production by Recombinant Fungi: Fungal overproduction of HPs by overexpression of multiple gene copies by fungi has been described under this subtitle. Schuurs et al. tried to develop over expression in S.commune, by introducing an additional gene copy with the help of modified pUC 20 plasmid. Instead of increasing production levels of Sc3 HP, a contrary effect of silencing of both the genes was observed i.e. both endogenous and introduced one [97]. The result was production of a wettable phenotype with the absence of expressed HPs on conidial surfaces. In a detailed study on gene silencing with the help of nuclear run on transcription assay and study of DNA methylation, it was concluded that silencing occurred at transcription levels by methylation of rDNA as well as methylation of genomic DNA. This method of developing over expression in fungi, first received success for Trichoderma reesei, where HFB I class II HP was produced using this method. With a few exceptions, production of HFB I using this method proved to be the best attempt made so far, for an industrially scalable production of HPs. Using this method 600 mg/L of HFB I was obtained. Three gene copies of hfb1 were introduced into T.reesei. VTT-D-74075 and VTT-D-98692 were the two over-expressing strains grown on glucose for production of HFB I. Most of the protein was cell wall bound so it was isolated by breaking the cell membrane followed by purification using hydrophobic interaction chromatography [98]. Unlike the parent strain low concentration of HFB I was found in culture broth due to over expression and saturation of HFB I in cell walls of T. reesei.

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a) HP production by recombinant Aspergillus niger: Aspergillus niger is a good candidate for recombinant production of biomolecules because of its competent secretary systems. Expression of HPs in A.niger was initially examined for ABH1 from Agaricus bisporus [99]. In initial experiments production levels of ABH1 were investigated. Production levels were found to be low. To check the problems at levels of protein production (due to low expression at RNA level or protein level, problems in secretary machinery or volatility of sandwiched protein), different methods of cultivation were followed [99]. Solidified minimal medium was sandwiched in between porous polycarbonate membrane and inoculated with recombinant A.niger spores. Colonies grown for 3 days were shifted on to polyvinylidene-difluoride (PVDF) membrane, which immobilized proteins secreted from colonies of microorganisms. Immuno-detection studies indicated that ABH1 secretion was more in comparison with shaken liquid culture. Use of PVDF was the key factor of this process as without application of grown colonies on PVDF, presence of ABH1 remained undetected. Overall yield of ABH1 was not mentioned in this work. This process development was effective in checking secretion of recombinant protein by any fungus and also for examination of any effect of proteases on the yield of the recombinant protein secretion [100]. b) HP production by recombinant Trichoderma reesei: T.reesei proved to be an effective host for heterologous production of HPs. Recombinant production of class I HP DEWA (detergent wettable A) from Aspergillus nidulans in T.reesei was successfully achieved. A T.reesei transformant DEWaHY68 was developed producing high amounts of DEWA. Submerged fermentation was performed in 2 liter fermenter, and secreted HP was separated using methanol/chloroform precipitation followed by treatment with trifluroacetic acid. Cell wall bound DEW A was removed by treating mycelial cells with hot SDS. Total protein concentration of the batch was 220mg/L of which HP accounted for 15% w/w. Overall yield of HP was 33mg/L. This level of DEWA concentration in T.reesei was certainly high compared to Sc3 production using a high producing strain

14 T.reesei RUT C30 [101]. This process of HP production was found to be promoter dependent, cel7A and hfb2 were the two promoters used with lactose as a carbon source. Production of DEWA was observed under hfb2 promoter and failed in the presence of cel7A promoter. The conclusion of this study was that impairment of DEWA synthesis in the presence of cel7A was either during translation or in the secretary pathway of DEWA [101]. Presence of HP producing fungi in malt is responsible for gushing in beer. To check whether purified HPs (from gushing inducing fungi) can induce the phenomenon in beer and carbonated mineral water different fungi were screened using a bioinformatics approach for presence of putative HP genes. Fusarium species was mainly found to contain HP genes and were found responsible for causing gushing in beers and carbonated beverages. HPs from Fusarium graminearum and Fusarium poae have been produced in T.reesei. T.reesei VTT D-99676 which is devoid of HFB II genes was used for experiment. Putative HP genes from both Fusarium species were isolated and introduced with the help of expression vector pMS186. Positive transformants were grown on Trichoderma minimal medium provided with lactose as carbon source in shake flask under submerged condition for 4-7 days. HPs were purified from the culture filtrate using a surfactant (Berol 532) in a combined recovery process of aqueous two phase extraction and foam flotation. Presence of HPs was analysed by SDS-PAGE and RP-HPLC. These two HPs were characterized as GZHYD5 (Fusarium graminearum teleomorph Gibberella zeae hydrophobin 5) and FPHYD5 (Fusarium poae hydrophobin 5) using MALDI-TOF Mass Spectrometry. Despite of the same recombinant process using same T.reesei strain (VTT D-99676) yield of FpHYD5 was more than GzHYD5. Production concentration of FpHYD5 was 18 mg/L of culture broth and Yield of GzHYD5 was 1 mg/L of culture broth. 1.5 mg of FpHYD5 was separated from 20 grams of mycelium produced in one liter of culture broth. Isolated HPs were further tested for their beer and carbonated water gushing properties. Minimum concentration of 1 µg in 0.33 liter of Beer was responsible for beer gushing. Purified HPs also induced gushing in carbonated water with a minimum concentration of 1 µg [102].

15 c) HP production by recombinant Saccharomyces cerevisiae: Saccharomyces cerevisiae is a yeast strain which does not produce and secrete HPs. Hydrophobicity of cell surface of yeast cells is limited. To improve attachment of yeast cells to surfaces and efficient production of ethanol, beer etc. greater hydrophobicity of yeast cells is desirable. To improve the hydrophobicity of cell surfaces, HP HFB I was expressed in to S.cerevisiae. This attempt was successful which made the cell surface hydrophobic and it was examined by studying surface activity of yeast cells, aqueous two phase separation of yeast cells in apolar phase and adsorption profile of yeast cells on different hydrophobic surfaces [103]. This property of S.cerevisiae to express HPs was further explored for cloning and expression of two HPs from Fusarium culmorum along with wheat lipid transfer protein (LTP1500) [104]. Production of HPs using S.cerevisiae was not the focus of the reported study. This study was aimed at understanding qualitative effects of HPs on beer gushing. Class I HP FcHyd3p and class II HP FcHyd5p (Fusarium culmorum hydrophobin 5 protein) from F.culmorum were cloned into S.cerevisiae DSM 3820 strain. Gushing properties of successfully cloned yeast strains with HPs and LTP from wheat were evaluated by producing beers using these strains. LTP expressing strains did not show gushing and have been proved to be stabilizers of beer foam. In comparison with class I HP FcHYD3p, class II HP FcHYD5p had shown prominent gushing in beer. These findings specifically indicated the different role of class I and class II HPs with respect to foam stabilization or destabilization in beers [104]. A fed batch process was developed for recombinant production of HFB II in Saccharomyces cerevisiae, CBS 128322. The process was performed with two scales 1.67 liter and 10 liter with and without antifoam. Production levels were increased in presence of antifoam. At 1.67L maximum yield of HFB II was 260mg/L and maximum yield at 10 L was 307.6 mg/L. When antifoam was not added, foam formed during fermentation was removed by foam fractionation methods. Enrichment and recovery of the HPs on small scale was good. Percentage recovery of HFB II at 1.67 L was 98.1 % with enrichment of 54.6 and at 10 L was 49.5% with enrichment of 2.4. It indicated that there was scope for large scale production of HFB II by controlled foam fractionation. Apart from foam fractionation, use of antifoam has given promising results [105].

16 d) HP production by recombinant Pichia pastoris: Pichia pastoris has been explored in recent years for production of HPs [106]. In comparison with S.cerivisiae, cellular systems of P.pastoris are efficient for all posttranslational modifications of eukaryotic proteins and secrete correctly folded proteins into the culture medium [106]. The first attempt was made to express class II HP FcHyD5p from F.culmorum in P.pastoris under alcohol oxidase 1 (AOX1) promoter. Two recombinant strains were developed one with histidine tag and another without tag. These transformed strains were grown on BMMY medium in shake flask culture. FcHyD5p was purified using preparative isoelectric focusing system and analysed by SDS-PAGE. HP was analysed by amino acid sequence analysis method and its surface activity was tested by checking foam stability. Wild type P.pastoris X33 strain produced foam which remained stable for only two hours, whereas foam produced by both transformed strains was stable for 72 hours. Gushing properties of recombinant FcHyD5p isolated from culture broth was examined with beer and carbonated water. 2 milligram freeze dried FcHyD5p was added in 500 mL of beer and 330 mL of carbonated water. Around 50% loss in gushing was experienced by addition of 2 mg FcHyD5 in beer and carbonated water. FcHyD5p tagged with histidine appeared to cause less gushing with lesser volume loss as compared to transformed FcHyD5p without histidine tag. This process was successful in expression, isolation, and identification of class II HP (FcHyD5p) but the yield was not evaluated [107]. Expression of FcHyD5p was repeated in P.pastoris to check gushing in beers with addition of hop oil component [108]. Addition of hop oil content was responsible in reduction of gushing volume in beers as well as carbonated water. To widen the scope of the study mentioned above, different surface active molecules along with HPs were expressed in P.pastoris and their effect on gushing in beers was examined [109]. This study included class I HP FcHyd3p from F.culmorum, class II HP HFB II from T.reesei, non-specific lipid transfer protein (nsLTP1) from barley and alkaline foam protein (AfpA) from F.graminearum. In addition to all these molecules FcHyd5p was also used to treat beer. Comparison of two class II HPs HFB II and FcHyd5p showed that effect of hop oils was negligible in case of HFBII on beer volume loss due to gushing. Foam stability study of all expressed proteins indicated that FcHyd5p and HFBII were the most

17 foam stabilizing proteins in comparison with class I FcHyd3p, nsLTP1, and AfpA . Orientation of the work for recombinant production of HPs and study of effect of these proteins on beer gushing was neither focused on production yield of the process nor on the optimization of the process. These results helped to understand feasibility of successful heterologous production of different HPs in P.pastoris. Extension of application of P.pastoris as a host for heterologous production of class I HP HGFI from Grifola frondosa underlines the need of expression attempts made for different HPs in Pichia [110]. rHGFI was expressed in P.pastoris using vector pPIC9 with an alcohol oxidase 1 (AOX 1) promoter. Product yield was 86 mg/L of fermented culture medium with two step purification process. First step was ultrafiltration using 4K pore size hollow fiber membrane with minimum pressure of 0.06 MPa to avoid aggregation of rHGFI at the membrane. Second step was use of RP-HPLC (Vydac C4 reversedphase column) for purification of rHGFI. Maximum secretion levels of rHGFI were 120 mg/L of culture medium. This study emphasizes the fact that P.pastoris is an efficient host for large scale production of HPs from different sources. The same recombinant production strategy was applied for production of rHFB I from T.reesei in P.pastoris [101]. rHFB I was purified by a two-step process of ultrafiltration and RP-HPLC. Product yield was 120 mg/L (92 % recovery),which was higher than that obtained for rHGFI from G.frondosa. Results of contact angle measurements were similar to those of wild type HFBI protein. Emulsification capacities of rHFB I were examined for stabilizing oil droplets in water and these capacities found to be more than rHGFI of G.frondosa. Comparative gushing potential of rHGFI and rHFB I was tested and it was found that rHFBI is the better gushing inducer than rHGFI. The process developed for rHFB I gave higher yield of HP in comparison with rHGF I for the suitable application in food products using P.pastoris as a host system [111]. rHFBI production in P.pastoris has been attempted with a different promoter than AOX1 to avoid risk of methanol handling. Promoter glyceraldehyde-3-phosphate (GAP) has been employed in the process of production of HFBI from T.reesei in P.pastoris SMD1168H [102]. Constitutive production of rHFBI was achieved by supplying glucose as a carbon source. Two forms of rHFB I were produced in this work. One form was produced using vector pGAPZalphaB without putative signal sequence of HFB I while the

18 second was produced using vector pGAPZB with wild signal sequence of HFBI present in T.reesei. Purification strategy was based on foaming capacities of secreted HPs. Foam catcher was attached with bioreactor to collect foam produced in fermentative broth due to use of stirrer. Surface pressure was measured for both the HPs. It was increased from 0.07mN/m to 34mN/m for the amount of 100µg rHFB I and when the amount of protein injected was doubled to 200µg, measured surface pressure was 60mN/m [112]. Foam separation as a method of purification of rHFBI from T.reesei has been recently tried by production in P.pastoris using AOX1 promoter [103]. Development of a recombinant production strain was the same as that mentioned previously by Niu B. et al.[101]. Maximum yield of the process was 300 mg/L and attained by direct loading of culture broth on to His-tag affinity chromatography column. Yield of the foam separation method using CO2 (110mg/L) and foaming with air (360mg/L) was comparable with that previously reported method by Niu B. et al [101]for foaming with air i.e. of 70% purity [113]. Rod A and Rod B HPs from Aspergillus fumigates have been expressed in P.pastoris successfully with the production yield of 200-300 mg/L [114]. Two vectors have been constructed including pPICZαA and pPICZB and transformed into P.pastoris X33 strain. AOX1 promoter was used as mentioned earlier. Fermentation was repeated at 50 ml scale and 500 ml scale in a fed batch process. Process was run initially on glycerol in batch mode till the 250g/L of wet biomass concentration achieved and then process was turned on to methanol in fed-batch mode. Rod A and Rod B HPs have been produced with the concentration of 329 and 262 mg/L respectively. 3.2.2.2 HP production by recombinant Bacteria (Escherichia coli): Escherichia coli have been successfully used for expression of HPs. It is the simplest and easy to transform model system for recombinant production of HPs. Despite its ease of gene transfer method; this system is not effective at the production levels of HPs which are 10 to 100 times lower [115] [116][117][118] in comparison with wild type and other recombinant production processes using P.pastoris. The first reported expression of HP in E.coli was of cerato-ulmin (CU) from fungal pathogen

19 Ophiostoma ulmi. Plasmid vector pUC 19 was modified to pCU4 by insertion of CU gene through expression vector pGEX-2T. CU has been produced as a translational fusion protein with glutathione Stransferase (GST) and hence Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used as an inducer for CU production. Presence of CU was detected using western blotting and the resulting concentration of CU in culture medium was 80 ug/L. This production yield was too low for considering large scale production of Cerato-Ulmin. The production of CU in E.coli is important in view of development of a two-step purification procedure of affinity chromatography followed by RP-HPLC. [118] The yield, however, was negligible in comparison with that from wild mould (140 mg/L) [118]. HYDPt-1 (Hydrophobin of Pisolithus tinctorius-1) HP from ectomycorrhizal basidiomycete Pisolithus tinctorius have been produced in E.coli as histidine tagged protein [119]. This study was aimed at producing recombinant HYDPt-1 HP to develop antibody for immuno-localization of this HP during ectomycorrhizal symbiosis with Eucalyptus globulus. E.coli strain M15 was transformed with vector pREP4 containing HYDPt-1 gene and grown on Luria-Bertani medium. IPTG was used as an inducer for expression of HP. Purification was followed on to Ni-NTA sepharose column followed by RP-HPLC. Product concentration of the process was low up to 1 mg/L of bacterial culture medium. The main reason for reduced production is toxicity of HYDPt-1 for E.coli. Antibody production using recombinant HYDPt-1 was successful. It helped to study relationship of expression of this HP and symbiotic association of P. tinctorius with E.globulus [119]. Recombinant DGH1 (Dictyonema glabratum hydrophobin-1); class I HP of Dictyonema glabratum has been expressed in E.coli [52]. Concentration of DGH1 has not been mentioned in this study. It might be less than 1 mg/L as reported for HYDPt-1 [119].

The first reported fungal allergen HCh-1 of

Cladosporium herbarum has also been produced in E.coli with high level expression vector pQE30. Production of HCh-1 (Hydrophobin of Cladosporium herbarum-1) was induced by IPTG and purified with Ni-NTA column chromatography as performed for other HPs produced in E.coli [13][52]. Product recovery was 9mg/L of HCh-1and 2 mg/L HYP 1(Hydrophobin -1). However this product showed only a

20 28% similarity to the amino acid sequence of HCh-1, and failed to interact with IgE. This confirmed that HCh-1 is a rare allergen produced by fungi [13]. A kilogram scale industrial production of HP has been reported using E.coli as a recombinant host [120]. Produced HP was a fusion protein with combination of Dew A from Aspergillus nidulans, yaaD and truncated form of yaaD from Bacillus subtilis. Two fusion proteins have been produced namely H*Protein A which contained yaaD and DewA; the other was H*Protein B contained truncated yaaD and Dew A. Vector pQE60 was used for both transformations and expressions of proteins in E.coli. Fermentation was carried out by growing E.coli cells on LB medium in 20-l fermenter. Production of recombinant fusion HPs was induced by addition of IPTG. Purification of proteins was carried out using Nickel Sepharose column chromatography. To check physiochemical property of fusion HPs like selfaggregation; analytical ultracentrifugation was followed [121][122]. Interfacial tension and emulsification capacity of fusion HPs was also checked. H*Protein A and H*Protein B have been proved to be fusion HPs, mimicking all properties of naturally occurring HPs. They have formed dimmers at room temperature with acidic pH, while they have formed monomers at 5°C with alkaline pH [123]. Application of both fusion proteins in laundry industry was tested by studying emulsification capacities with different oils and findings suggested that minute amount of fusion HPs was found to be sufficient to boost emulsifying power in comparison with 300ppm of an commercial surfactant. Reported study was the most efficient industrially scaled up process of engineered HP retaining all natural surfactant activities [120]. Recombinant HGFI was also produced in E.coli strain BL21 with transforming vector pET-28a [115]. The recombinant process was similar to that mentioned earlier for HYDPt-1 and DGH1 [119] [52]. Yield of the process was not mentioned. Heterologous production of class I HP Hyd2 of entamopathogenic fungus Beauveria bassiana was also attempted in an E.coli host system using expression vector pTW1N1. It showed minimum production of HP .The total protein yield was 7-10mg/L in which actual recombinant mHyd2 concentration was of 200-260µg/ml [116].

21 In summary P.pastoris was found to be the most efficient host for production of hydrophobins with a good yield due to presence of desired foldases/chaperons for proper folding of hydrophobins [106]. E.coli was proved to be a poor hydrophobin producer except for production of fusion hydrophobin. Despite successful production of HFBII from T.reesei in S.cerevisiae; production of HP is not completely established in this host system. Recombinant production of HPs in different host organisms has been summarized in table 4 with yields. 4. Future Perspectives: HPs have been isolated from more than a thousand ascomycete and basidiomycete fungi. Identification of HP in different species of fungi and their isolation, characterization will be an enduring process. It will open avenues for developing processes with increasing yield; as process development for HPs is species specific. Study of production attempts made in wild strains indicated that yield did not exceed a few milligrams per liter. A Detailed understanding of the molecular mechanism of hydrophobin expression, transportation to the cell surface, role in sporulation, and mechanism of 3D rodlet structure formation etc. would be of great help in designing of efficient production methods [70][124][126]. For example, understanding of role of HP like protein in expression of enzyme chitinases in species L.lecanii, helped to design media with chitin for efficient expression of HP like protein (HfbL) [93]. This media designing resulted in 14 % more production of HfbL [93]. Exploring different fungi from natural hydrophobic environment for production of HPs would also be an effective strategy to improve production yields [74][75]. Production of HPs using recombinant methods are more effective in comparison to wild type methods for both the classes of HPs. Currently large scale production of HPs is limited by poor yields. Use of recombinant methods for their bulk production would be desirable. Current methods of production seem to be adequate for small scale applications like medical devices, biosensors, protecting nanoparticles, and drug formulations [70] [71]. Study of chaperons and foldases that are involved in folding of HPs in wild type fungi would help to attain improved yield during scale up using recombinant hosts [125].

22 Exceptionally BASF succeeded in bulk production of HPs H*Protein A and H*Protein B in kilogram scale [120]. This promising example augurs well for development of genetically engineered strains for increasing production yields of HPs.

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33

RECOMBINANT PRODUCTION OF HYDROPHOBINS

E.coli

Design of vector with GST

P.pastoris

Design of vector with AOX1 promoter

S. cerevisiae

Process is not well developed

Induction of HP production by IPTG Transformation in X33 P.pastoris strain

Purification by Ni2+ Affinity

Batch/Fed Batch production induced by methanol

Chromatography

Analysis by SDS-PAGE

Yield estimation and Surface active study

Analysis by SDS-PAGE/Western Blot/ MS

Purification by RP-HPLC/Ultrafiltration /Affinity Chromatography

Yield determination and Surface activity study

Figure 1: Overview of Recombinant production processes in common Host organisms

34 Table 1. Major studied Hydrophobins from Ascomycetes

Species

A. fumigates

A.nidulans

A.oryzae Cladosporium fulvum

Cladosporium herbarum Fusarium Verticillioids

Fusarium graminearum Fusarium culmorum Grifola frondosa Magnaporthe grisea

Metarhizium anisopliae Neurospora crassa Paracoccidioides Brasiliensis Xanthoria ectaneoides Xanthoria parietina Beauveria bassiana Trichoderma atroviridae

Claviceps fusiformis Claviceps purpurea Cryphonectria parasitica Ophiostoma Ulmi Ophiostoma quercus Trichoderma harzianum Species

Trichoderma reseesi

Hydrophobin

Rod A Rod B Rod C Rod D Rod E Rod A Dew A Dew B Dew C Dew D Dew E Rol A HYPB HCf 1 HCf 2 HCf 3 HCf 4 HCf 5 HCf 6 HCh 1 Hyd 1 Hyd 2 Hyd 3 Hyd 4 Hyd 5 FgHyd5p FcHyd5p FcHyd3p HGF I MPG1 MPH1 MHP1 HYD1 HYD2 EAS NC2 PbHYD1 PbHYD2 XEH1 XPH1 Hyd2 HFB22b HFB1A HFB1a CFTH1 CPPH1 Cryparin Cerato-ulmin Cerato-ulmin QID3 SRH I Hydrophobin

HFB I HFB II HFB III

Class

Protein Isolated

Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class II Class II Class I Class I Class I Class I Class II Class II Class II Class II Class I Class I Class I Class II Class II Class I Class II Class I Class II Class I Class I

+ + + + (R) + + (R) +(R) +(R) +(R) + + -

Class I Class I Class I ClassI* ClassI* ClassI* Class II Class II Class II Class II Class II Class II

+ +

Class

Class II Class II Class II

+ +(R) -

+ + + + Protein Isolated + + +

Mol. Wt. (kDa) 16 14

References

35

[5],[6]

[7],[8]

[8] 10

[9],[10],[11],[12]

[13] [14]

12

[15] [16], [17], [18], [19]

8 15

[77] [20]

[21] 8.2

[22],[23],[24],[25] [26]

10 10 13.8

[27]

36.5 85 18.6 7.6

[75] [30] [31],[32] [33] [34] [35], [69]

Mol. Wt. (kDa) 7.5 7.2 7.5

[70] [28]

References

[36], [37], [38], [39], [40], [41], [42], [43],[44]

36 HFB IV HFB VII

Class II Class II

-

+ : Protein isolated from native wild fungi -

: Proteins studied using genomics or bioinformatics tools and not isolated from native fungi

R : Protein isolated by recombinant process *Indicates Separate Clade of Class I Hydrophobins.

37

Table 2. Major studied Hydrophobins from Basidiomycetes

Species

Hydrophobin

Protein Isolated

Molecular Weight (kDa)

Agaricus Bisporus

ABH1/HYPA ABH2/HYPC ABH3 HYPB CoH1 CoH2 DGH 2 DGH 3 DGH 1 FVH 1 FV-HYD 1 Le.hyd 1 HYDPt-1 HYDPt-2 HYDPt-3 Fbh-1 POH 1 POH 2 POH 3 Vmh 1 Vmh 3 Vmh 2 PN1 SC3

+ + + + + + + + + + + + + + +

8-9 9.1 19

[46],[47],[48],[49]

10

[50]

14

[52]

SC 1 SC 4 SC 6 Hyd 1

+ +

14.4 15

+

23

Coprinus cinereus Dictyonema glabratum

Flammulina Velutipes Pisolithus tinctorius

Pleurotus ostreatus

Pleurotus nebrodensis Schizophyllum commune

Tircholoma terreum

References

[53],[54] [55], [56], [109]

12 9 20 10 9 17 13.5 13.6

[51],[57],[58]

[59] [45], [46] [60],[61],[62],[63],[6 4],[65],[66],

[67]

+ - Protein isolated from native wild fungi -

: Proteins studied using genomics or bioinformatics tools and not isolated from native fungi

38 Table 3.Native Production Processes

Sr.No. 1 2 3 4 5 6 7 8 9 10 11 12 13

Hydrophobin Cerato-Ulmin SC3 ABH3 CFTH1 HFBII HGFI PLHYD PLHYD PfaH1 PfaH2 HfbL HfbL HFBII

Fungi Source Ceratocystsis ulmi Schizophyllum Commune Agaricus bisporus claviceps fusiformis Trichoderma reesei Grifola frondosa Paecilomyces lilacinus Paecilomyces lilacinus Paecilomyces farinosus Paecilomyces farinosus Lecanicillium leccani Lecanicillium leccani Trichoderma reesei

(-) Indicates Information is not available

Class II I I II II I I I II II

Production Process SmF SmF SmF SmF SmF SmF SmF SSF SmF SmF SSF SSF Fungal Biofilm Bioreactor

Yield 140 mg/L 60mg/L 2 mg/L 3.5mg/L 71 mg/L 300 mg/L 1.1 mg/L 1.3 mg/L Negligible Negligible 48.6 mg/L

39 Table4. Recombinant Production Processes

Sr. Hydrophobin No. 1 ABH1

Class Fungi Source

Fungi Host System

Yield

Purification Methods Sandwich Culture Method

I

A. bisporus

Aspergillus niger

I

A. nidulans

Trichoderma reesei

Not Mentioned 33 mg/L

2

Dew A

3

GzHYD5

F.graminearum

Trichoderma reesei

1 mg/L

4

FcHYD5

F. poae

Trichoderma reesei

18 mg/L

5

HFB I

II

T. reesei

Yeast S. cerevisiae

6

FcHyd3p

I

F. culmorum

S. cerevisiae

7

FcHyd5p

II

F.culmorum

S.cerevisiae

8

HFBII

II

T.reesei

S.cerevisiae

9

FcHyd5

II

F. culmorum

Pichia pastoris

10

HGFI

I

G.frondosa

Pichia pastoris

86 mg/L

11

HFBI

II

T. reesei

Pichia pastoris

12

HFBI

II

T.reesei

Pichia pastoris

Not determined 300 mg/L

Not Determined Not Determined Not Determined 1.67L:260 mg/L 10L: 307.6mg/L Not determined

110 mg/L

Promoter dependent process (cel7A and hfb2 ) Aqueous Two Phase extraction Aqueous Two Phase extraction -----------Foam Fractionation

AOX1 promoter and Histag recombinant hydrophobin production AOX1 promoter Purification by Ultrafiltration GAP promoter AOX1 promoter with Histag Affinity Chromatography Foaming with CO2

13 14

Rod A Rod B

I I

A.fumigatus A.fumigatus

Pichia pastoris Pichia pastoris

360mg/L 329 mg/L 262 mg/L

Foaming with Air AOX1 promoter with Histag Affinity Chromatography

15

Cerato-ulmin

II

O.ulmi

Bacteria E.coli

80ug/L

16

HYDPt-1

I

P.tinctorius

E.coli

1 mg/L

17

DGH1

I

D.glabratum

E.coli

1 mg/L

Glutathione-agarose affinity chromatography Ni-NTA sepharose chromatography Ni-NTA sepharose chromatography

40

Sr. Hydrophobin No. 18 H*Protein A

Class Fungi Source

Host System

A.nidulans B. subtilis

E.coli

19

H*Protein B

A. nidulans B. subtilis

E.coli

20

HGFI

G.frondosa

E.coli

21

Hyd2

B.bassiana

E.coli

(----) Indicates information is not available

Yield

Production/Purification Methods (Kilogram Ni-Sepharose Scale) Not chromatography mentioned (Kilogram Ni-Sepharose Scale) Not chromatography mentioned Not Electroelution from PAGE Mentioned 260 ug/ml Chitin bead column chromatography