Microb Ee.ol (1986) 12:121-127 MICROBIAL ECOLOGY �9 1986 Springer-Verlag

Screening for a "New" Enzyme in Nature: Haloperoxidase Production by Death Valley Dematiaceous Hyphomycetes

Jennie C. Hunter-Cevera and Lynn Sotos

Cetus Corporation, 1400 Fifty-Third Street, EmeryviUe, CA 94608, USA

�9 Abstract. Haloperoxidases are enzymes that have the ability to halogenate a broad range of substrates [10]. To find a biologically produced halope- roxidase that could function at a pH greater than 3.0 and at a temperature greater than 19~ dematiaceous hyphomycetes were isolated from the Death Valley desert and screened for their ability to produce such an enzyme. A qualitative assay using bromophenol red was employed in situ over a 12-day fermentation period. Several dematiaceous hyphomycetes, such as Dreschlera haloides and Ulocladium chartarum, produced halo- peroxidases that were active in broth culture at 19, 25, and 34~ at pH 7.0 and 8.0.

Introduction

There has been considerable interest in recent years in enzymatic halogenation [8, 9]. Haloperoxidase enzymes which have been used in halogenation reaction Studies include chloroperoxidase derived from the fungus Caldariomyces fu- mago, bromoperoxidase from algae, lactoperoxidase from milk, thyroid pcr- Oxidase from thyroid, myeloperoxidase from leukocytes, and horseradish per- Oxidase from horseradish [7]. Chloroperoxidase is the haloperoxidase of choice, since many industrial applications of halogenation involve chloride ions. The Conversion of propylene to propylene oxide via chlorohydrin is one example [10]:

Alkene + X- + H + + H202 Haloperoxidase

X = CI,Br,I Alkene halohydrin + H20

Alkene halohydrin + OH-

Halohydrin epoxidase

Alkene epoxide + H20 + X-

Of the several known haloperoxidases that are mentioned above, only mye- loperoxidase derived from leukocytes and chloroperoxidase from C. fumago are able to utilize chloride ions. A limitation of the C. fumago chloroperoxidase, in industrial applications, is that the enzyme has a pH optimum of around 3.0 and has little activity and stability above pH 7.0. Thus, the enzyme preparation

122 J.C. Hunter-Cevera and L. Sotos

Table 1, Death Valley sample description, pH, and temperature

Tem- pera-

Sample description pH ture *F

1. Dried orange peel with salt crust, Keanan Mine 5.5 102 2. Desert flower roots and soil, Keanan Mine 8.8 89 3. Rock shale with small yellow flower, Keanan Mine 8.3 93 4. Desert flower on trail, Keanan Mine 6.5 87 5. Dried plant material under rock, Keanan Mine 4.6 61 6. Red soil and roots of desert, Keanan Mine 7.5 83 7. Crusty soil, Salt Creek 8.0 90 8. Porous rock with salt crust, Salt Creek 8.3 87 9. Dune sand, Salt Creek 9.5 85

10. Sandy soil under pickleweed, Salt Creek . 8.7 77 l 1. Carbonated mud under shaded bank, Salt Creek 8.6 74 12. Soil under desert holly, Furnace Creek 8.7 89 13. Sandy soil under pickleweed, Furnace Creek 10.0 94 14. Soil, Travertine Hot Springs 8.8 105 15. Soil under bush, Travertine Hot Springs 9.9 99 16. Dried picldeweed, Badwater 8.5 96

o b t a i n e d by f e r m e n t a t i o n m u s t first be ac id i f ied and, a f te r p r o d u c t f o r m a t i o n , n e u t r a l i z e d wi th base for p r o d u c t r e c o v e r y o r fu r the r c o n v e r s i o n . T h e two p H a d j u s t m e n t s can s igni f icant ly inc rease the cos t o f the h a l o g e n a t i o n reac t ion . In a d d i t i o n , the e n z y m e is n o t s table at h igh t e m p e r a t u r e s . M y e l o p e r o x i d a s e op- e ra tes in the des i r ed neu t ra l p H range, bu t the e n z y m e is n o t read i ly o b t a i n a b l e in q u a n t i t i e s for indus t r i a l use.

In ou r search to f ind h a l o p e r o x i d a s e s tha t (a) u t i l ize c h l o r i d e or b r o m i d e ions ac t i ve ly a t an o p t i m u m p H a b o v e 3.0, (b) are read i ly o b t a i n e d v i a f e r m e n t a t i o n , a n d (c) a re s table at h ighe r t e m p e r a t u r e s , we s a m p l e d e c o s y s t e m s w h e r e b i o - phys ica l p a r a m e t e r s such as p H , t e m p e r a t u r e , a n d sa l in i ty were fair ly high. O n e such e c o s y s t e m in Ca l i f o rn i a is the D e a t h Val ley deser t whe re these b io - phys ica l p a r a m e t e r s occu r in the ex t r eme .

M a t e r i a l s

Sampling

A variety of samples were collected from several sites within Death Valley (Table 1). Soils were collected with sterile metal spatulas. Plant parts were cut aseptically with sterile scapels. Rocks and miscellaneous materials were handled with sterile gloves. All samples were placed in Nasco Whirlpak collection bags. Samples were stored overnight at 5"C.

Recording of Biophysical Parameters. Soil or air temperatures were recorded with a thermometer at each sampling site. The pH of soils was determined by mixing a one-to-one w/v ratio of soil and distilled water, permitting this mixture to stand for 30 min, and recording the pH with a glass electrode after stirring the soil water suspension with a wood stick. Salinity was not recorded, since the sites we sampled were well documented [4].

Screening for a "New" Enzyme in Nature 123

Table 2. Fungal isolation agars

1. Modified rose bengal [5]

Peptone i 0.0 g MgSO4.7 K20 0.5 g K2HPO4 1.0 g Agar 20.0 g Distilled water to 1.0 L Dextrose 5.0 g Streptomycin 30.0 mg 1% Rose Bengal 3.3 mi pH 7.3

2. Modified rose bengal plus 6.0% NaC1, pH 8.0

3. Soil infusion [6]

A. Supersoil 400.0 g Distilled water 900.0 ml

Mix well and let heavy soil grains settle Decant or filter supernatant Autoclave 30 min

B. Make up an equal volume to part A Dextrose 1% K~HPO4 0.3 M Distilled water agar 3%

Mix A and B Adjust pH with KOH to alkaline pH Autoclave 20 min

Sterile addition: Penicillin G 0.05 g/liter Streptomycin 0.025 g/liter

4. Soil infusion plus 6.0% sea salts, (Instant Ocean Aquarium Systems, Eastlake, Ohio), pH 8.2

5. Alkaline water agar

Agar 20.0 g K2HPO4 26.0 g Tap water to 1.0 liter Sterile addition: Penicillin G 0.5 g/liter Streptomycin 0.025 g/liter Adjust pH to alkaline pH with NaOH

6. Alkaline water agar plus 6% NaCl

Isolation and Identification of Dematiaceous Hyphomycetes. Dematiaceous hyphomycetes were isolated from Death Valley samples by aseptically implanting bits of soil, plant debris, and plant parts into a variety of fungai isolation agars (Table 2). Agar pH was adjusted to pH values that roughly corresponded with sample pH. The difference in salinity (0 vs 6%) represents two of the three salinity zones present in Death Valley, i.e., xerophyte and phreatophyte [4]. Penicillin G (50 rag/liter) and streptomycin (130 mg/liter) were added to the isolation agars to retard bacterial COntaminants. Plates were incubated right side up at 25 and 35"C and examined every 48 hours for colony development. Isolates were transferred several times to assure pure colony development and then identified according to Barnett and Hunter [1] and Ellis [2, 3].

124 J .C . Hunter-Cevera and L. Sotos

Table 3. Germination and fermentation broths

1. FA-I

Glucose 15.0 g Yeast extract 3.0 g Microelement solution* 1.0 ml Distilled water 1.0 liter Filter sterilize and add: Thiamine 100 t~g Biotin 5.0 ~g

*per liter distilled water KH2PO 4 0.g g CUSO4' 5H20 0.64 g FeSO4' 7H20 0.11 g MnC12.4H20 0.8 g ZnSO4-7H20 0.15 g

2. FP [i l]

Tryptone 5.0 g Male extract 3.0 g Glucose 1.0 g Yeast extract 3.0 g Distilled water i .0 liter

3. 1=-2

KI"I 2 P O 4 2.0 g MgSO4"7H20 2.0 g CaSO4.2H20 0.25 g Yeast extract 1.0 g Brown sugar 5.0 g Distilled water 1.0 liter

4. CFM

Potato-dextrose broth 24.0 g Yeast extract 3.0 g Microelement solution* 1.0 ml Distilled water 1.0 liter

*Same as in FA-I

5. 305

Staley's F200 soy flour 30.0 g Glucose 30.0 g CaCO3 7.0 g Antifoam 60 0.5 g Distilled water 1.0 liter

Final concentration

Fermentation. Following the growth of the selected fungus on a seed plate, a uniform segment of mycelia and spores was aseptically removed and transferred to a 250 ml flask containing 50 ml of a germination broth (Table 3), which often included a small amount ofagar (0.2 g/liter) to prevent mycelial clumping and pellet formation. The inoculated germination broths were shaken for 3-5 days at 200 rpm on a rotary shaker at 25*(2. If a gelatinous pellet formed during incubation, the material was aseptically harvested and ground to a mushy or applesauce consistency in a sterile Waring Blendor for 5-30 see in short 5 sec bursts.

Each fungal isolate was inoculated into fermentation broths as a 5% inoculum. A total of 48 flasks per isolate were inoculated. Fermentation broths (Table 3) were dispensed as 15 ml per 125 ml flask and buffered at pH's 7.0 and 8.0. Incubation temperatures employed were 19, 25, and

Screening for a "New" Enzyme in Nature 125

34~ with humidity set at 70%. Rotary shaker speed was 200 rpm. The total incubation period was 12 days. Individual flasks were harvested on days 5, 7, 9, and 12 for assaying ofhaloperoxidase activity.

Qualitative Assay for Haloperoxidase

Fifteen milliliters ofa substrate reagent (40 ml of 0.2% phenol red in 95% ethanol, 0.3 M potassium Phosphate buffer pH 7.0, and 0.05 M potassium bromide per liter distilled water) and one drop of freshly prepared 0.3% hydrogen peroxide were added directly to the fermentation medium. I~romination of phenol red is evidenced by a color change from red-orange to blue-violet.

4

Br Br o~. . , j~o l - i + 4Br-+ 2H202 haloperoxidase e ~

~SO)H ~SOI H

The extent of color change was noted 1 hour after the addition of reagent. Depending on the level of hydrogen peroxide present in the fermentation broth, fresh hydrogen peroxide was added at 1, 3, 5, and 8 hours after the initiation of the reaction, and the extent of color change was monitored at hours 2, 4, 6, 9, and 24. Intensity of the reaction was visually estimated as +, + +, and + + +. Haloperoxidase activity present in the growth medium was also estimated semi-quantitatively by following the reaction spectrophometricaily with a Perkin Elmer Lambda 3 spectrophometer set at 595 nm. For the spectrophotometric determination, the fermentation broth was centrifuged to remove fungal cells, and the optical density of the supernatants was measured with respect to diluted fermentation broth supernatant containing hydrogen peroxide but no phenol red.

Results

Biophysical Parameters

The p H a n d t e m p e r a t u r e s o f the s a m p l e s e x a m i n e d a re l i s t ed in T a b l e 1. S a m p l e pI-I v a r i e d f r o m 4 . 6 - 1 0 . 0 . T e m p e r a t u r e was d e p e n d e n t u p o n the t i m e o f d a y the s a m p l e was co l l ec ted , a n d i f t he s a m p l e was in d i r ec t sun l igh t o r shade . The t e m p e r a t u r e s v a r i e d f r o m 61-1050F .

Isolation of Dematiaceous Hyphomycetes

R e p r e s e n t a t i v e m e m b e r s o f the d e m a t i a c e o u s h y p h o m y c e t e s were i s o l a t e d f r o m all 16 s a m p l e s e x a m i n e d . Speci f ic g e n e r a i s o l a t e d in o r d e r o f d e c r e a s i n g n u m b e r s Were: 20 Ulocladium, 17 Alternaria, 12 Embellisia, 9 Cladosporium, 4 Stach- Ybotrys, 2 Helminthosporium, 2 Dreschlera, a n d 2 Botrytis. In a d d i t i o n to the a b o v e gene ra i so l a t ed , 15 d a r k p i g m e n t e d p y c n i d i a l f o r m s were a lso i so l a t ed .

Soil i n fus ion a g a r p l u s 6% sea sa l t s p r o v e d to be t he b e s t aga r for i s o l a t i o n o f D e a t h Va l l ey d e m a t i a c e o u s h y p h o m y c e t e s . T h e rose benga l aga r s were n o t su i tab le for the i s o l a t i o n o f these fungi , s ince t he fungal c o l o n i e s o v e r g r e w t o o r ap id ly to cu l t u r e in p u r e f o r m . T h e w a t e r aga r s we re h igh ly se lec t ive , e spec i a l l y With the a d d e d sea sal ts .

126 J.C. Hunter-Cevera and L. Sotos

Table 4. Haloperoxidase-producing dematiaceous hyphomyeetes and pycnidial forms isolated from Death Valley samples

Final Sam- HP ~ broth Temp pie Fungus description Germ Ferm activity pH *C Day

1. Cladosporium sp. CFM CFM + + ~8.0 25 7 Ulocladium chanarum FA-1 FA-I ++ -~8.0 34 7

2. Embellisia alli FA-1 CFM + ~8.0 25 9 Ulocladium chartarum FP FA-I + + + ~8.0 25 7 Ulocladium chartarum CFM FP + + = 8.0 19 9

3. Embellisia alli 305 CFM + + ~ 7.0 34 7 Ulocladium chartarum 305 CFM + =7.0 25 7 Fungus with dark 305 CFM + + + -~7.0 25 7

setose pycnidia

4. Ulocladium chartarum 305 CFM + + + =7.0 34 9 Fungus with dark 305 CFM + + ~ 7.0 25 9

ostiolate pycnidia and beige ooze

6. Ulocladium sp. CFM CFM + + =7.0 25 7 7. Ulocladium sp. CF FP ++ =8.0 25 9 9. Embellisiasp. FA-I FA-1 ++ =8.0 25 7

fungus with dark 305 CFM + =8.0 25 9 pycnidia

15. Dreschlerahalodes FA-I FA-1 + + + =7.0 25 7 16, Fungus with dark 305 CFM + + =7.0 25 9

setose pycnidia and globose spores

Sample--see Table 1; Germ--germination broth (see Table 3); Ferm--fermen- ration broth (see Table 3); HP activity--+ light violet/<l speetrophotometer reading 595 nm; HP activity-- + + blue violet/> 1 spectrophotometer reading 595 nm; HP activity-- + + + dark blue violet/>2 spectrophotometer reading 595 nm; Final broth pH--assayed at this pH; Temp *C--incubation temperature for best HP production; Day--day which best HP production was observed a HP = Haloperoxidase

Product ion o f Haloperox idase by Death Valley Demat iaceous H y p h o m y c e t e s

O u t o f the total 83 d e m a t i a c e o u s h y p h o m y c e t e s a n d da rk pycn id ia l fo rms isolated, 44 were e x a m i n e d for the p r o d u c t i o n of b r o m o p e r o x i d a s e a t p H s 7.0 a n d 8.0, a n d at t e m p e r a t u r e s o f 19, 25, a n d 34~ Ta b l e 4 lists the 16 isolates tha t p r o v e d pos i t ive for b r o m o p e r o x i d a s e a n d the c o n d i t i o n s u n d e r wh ich " be s t v i sua l ac t i v i t y " was observed .

All o f the " p o s i t i v e s " were capab le o f p r o d u c i n g ha loperox idase ac t ive at 25 a n d 34"C, a n d at pHs 7,0 a n d 8.0. P r o d u c t i o n o f h a l o p e r o x i d a s e was obse rved in several o f the f e r m e n t a t i o n m e d i a tested.

F o u r of the seven genera i so la ted were capab le o f p r o d u c i n g ha lope rox idase wi th ac t iv i ty u n d e r the c o n d i t i o n s tested. It appears tha t Ulocladium char tarum is one o f the m o r e u b i q u i t o u s h a l o p e r o x i d a s e - p r o d u c i n g d e m a t i a c e o u s h y p h o - mycetes . It is o f in te res t to no t e tha t several o f the da rk pycn id ia l fo rms were

Screening fnr a "New" Enzyme in Nature 127

capable of producing haloperoxidase. At this time, we have not identified these Pycnidial forms as to their exact taxonomic position.

Nine of the 16 samples examined yielded fungi that were capable of producing haloperoxidase. Samples associated with live or decaying plant material con- tained a greater number of haloperoxidase-producing dematiaceous hypho- mycetes than did soil samples containing no visible plant material. Only one sample (#5) containing plant material did not yield any isolates positive for haloperoxidase. However, the pH of this sample was 4.6. Since we did not isolate or test fungi under low pH conditions, it is not known what "positives" existed in low pH samples. Nor do we know if the alkaline-tolerant fungi isolated are capable of producing haloperoxidase at acidic pHs of 3-5.

In conclusion, the Death Valley desert proved to be an ecosystem well suited for isolating alkaline-tolerant dematiaceous hyphomycetes capable of producing haloperoxidases active above pH 3.0 and at 19~ We are currently purifying several o f these haloperoxidases and the results will be reported elsewhere.

Acknowledgments. The authors wish to thank Michele Fonda for her assistance in the collection of Samples, Angela Belt for her technical assistance in identification of the fungi, and Saul Neidleman and John Geigert for support of this ecological search for alkaline-tolerant dematiaceous hypho- rnYcetes.

References

1. Barnett HL, Hunter BB (I 972) Illustrated genera of imperfect fungi, 3rd ed. Burgess Publishing Co, Minneapolis, Minnesota, p 241

2. Ellis MB (1971) Dematiaceous hyphomycetes. Commonwealth Mycological Institute, KEW, Surrey, England, p 608

3. Ellis MB (1976) More dematiaceous hyphomycetes. Commonwealth Mycological Institute, KEW, Surrey, England, p 507

4. Hunt CB (1966) Plant ecology of Death Valley, California. United Slates Government Printing Office, Washington, DC p 68

5. Hunt CB and Durrell LW (1966) Distribution of fungi and algae. In: Hunt CB (ed) Plant ecology of Death Valley, California. United States Government Printing Office, Washington, DC p 68

6. Miller VV (1945) Studies on the Fusarium wilt of muskmelon. I. Pathogenic and cultural studies with particular reference to the cause and nature of variation in the causal organism. Can J Research C 23:16--43

7. Morrison M, Schonbaum GR (1976) Peroxidase-catalyzed halogenation. Ann Rev Biochem 45:861-888

8. Neidleman SL, Amon WF, Geigeft J (1981) Method for producing epoxides and glycols from alkenes. US Patent 4,247,641

9. Neidleman SL, Amon WF, Geigert J (1981) Preparation ofep0xides and glycols from gaseous alkenes. US Patent 4,284,723

lO. Neidleman SL, Geigert J (1981) Biological halogenation and epoxidation. Biochem Soc Symp 48:39-52

11. ]?ansey F, Jambor WP, Gadebuseh HH, Donovick R (1966) Hamycin: in vitro and in vivo studies. Antimicrob Agents Chemother (1961-1970) 399-404