Purification and characterization of restriction endonuclease BcoI from a soil isolate of Bacillus coagulans

FEMS Ml~oblology Letters 66 (1990) 153-156 Pubhshed by Elsevier

153

FEMSLE 03786

Purification and characterization of restriction endonuclease BcoI from a soil isolate of Bacillus coagulans S.M. L e u n g l, K . M . K a m 3 K.Y. C h a n 2 a n d P.C. Shaw l Department of I Bz~chenustryand 2 Biology. The Chinese Umversl(y of HongKong, and 3 Institute of Patholo~. Sm FrogPunJ#ckey Club Chnw.HongKong

Recmved10 April 1989 Resasmnreceivedand accepted 7 July 1989 Key words: Type II restriction endonuclease; Aval; Isoschizomer

1. S U M M A R Y A soil isolate identified as Bacdlus coagulans is found to produce Bcol, an isoschizomer of Aval and with the same cleavage site This thermal stable enzyme, BcoI, ts produced at high level and can be Isolated by passing the crude bactenai lysate through a DEAE-celhilose column.

2. I N T R O D U C T I O N Restriction endonucleases have become indispensable tools in the cloning and mapping of genes. Their production cost ts governed by the ease of strain cultivation and enzyme isolation. Therefore, many resmction enzyme coding genes have been cloned into Eschertehla colt to express them m large quantity and to avoid contannnatmg enzymes of the source strains [1]. In this communication, we report the isolation of a strmn of Bacdlus coagulans which produces an isoscluzomer of AvaL A one-step chromatoCorrespondence to PC Shaw, Department of Biochermstry. The Clunese Umverstty of Hong Kong, Shaen, N T, Hong Kong

graphic procedure was used to purify the enzyme. Properties of this enzyme are reported. 3. MATERIALS A N D M E T H O D S 3 1 Strata :solatwn and charactertzatJon Soil samples were obtained from the campus of the Chinese Umverstty and incubated at 6 0 ° C for 18 h. Soil (1 g) was then inoculated into 50 ml Luna-Bertam (LB) medium containing (%, w / v ) 1~ bactenai tryptone (DlfCO); 0.5% bacterial yeast extract (Difco); 1% NaC1 and shaken overnight at 55°C. The culture was then spread on LB agar plates and incubated overnight at 55 ° C. Upon restreaking for purity, several soil isolates were screened for the sources of restriction enzymes based on [2]. One of them was found to cut lambda D N A to discrete restriction fragments. The strlun was characterized according to [3-5]. The soil Isolate was identified to be Bacillus coagulans according to the criteria shown in Table 1 and was designated strain SM 1. 3.2. Purification of restriction enzyme Bacterial cells of B coagulans SM 1 were grown overmght at 5 5 " C in 500 ml LB medmm and harvested by eentnfugation at 5000 rpm (Beckman JA 14 rotor), 4 ° C for 20 min.

0378-1097/89/$03 50 © 1989 Federauon of European MicrobiologicalSocieties

The cell pellet (5 8) was suspended in 15 ml extract buffer (EB), 10 mM Tris-HCl, pH 7 . 5 : 5 mM /]-mercaptoethanol; 1 m M EDTA; 5% glycerol; 1 m M phenylmethylsulfonyl-fluonde, and disrupted by son|cation (160-170 watts output) tn an ice-water bath for periods of 20 s allowing 2-3 rain between bursts. The cell debris was removed by centnfugatmn at 34000 rpm (Beckman 42 1 rotor) for I h at 4 ° C. The supernatant (12 ml) was collected and loaded directly onto a DEAE-cellulose column (1.8 × 8.0 cm, Sigma, medium mesh). The restriction enzyme was duted wflh a 0-0.2 M hnear gradient of NaCI in 200 ml EB at a flow rate of 15 m l / h (2 ml/fractmn) Fractions contammg restnctmn enzyme aeUwty were pooled and dmlyzed against EB and stored at 4 ° C

3 3 Enzyme assay Enzyme assays were performed as previously descnbed [6,7]. Definmon of enzyme activity was at 3 7 ° C and according to [6,7]. 3.4. Determination of recognmon and cleavage sites The recogmtton sequence and cleavage site for Bcol was deternuned by a method modified from Brown and Snuth [11] 2.4/~g single-strained M I 3 m p l 8 D N A and 5.0 ng M13 sequencing pnmer (17 mer) at the - 4 0 regmn were nuxed in a buffer contammg 0.1 M Tns-HCI (pH 8.0) and 0.1 M MgCI2 and annealed at 55 ° C for 20 min and then divided rote five samples. Four samples of 2.5/d each were used for convenuonal dtdeoxy-sequencTable l ] d c n t t f l e a l t o n o f t h e r e s l r l c l t o n ¢ n z y a l c p r o d u c i n g soil i s o l a t e +' growth or wllh reaction. -. no growth or without reaction

3 5. Heat mactmauon To deterrmne heat hablhty, 9/~1 of medmm salt buffer containing 2 U of BcoI was heated at 6 5 ° C for 20 nun and cooled on ice before 250 ng (1 ,al) lambda D N A was added and the tube was incubated at 3 7 ° C for 1 h. 3 6. Assay for optmlal temperature Six eppandor[ tubes each with 10/~1 of medmm salt buffer, 250 ng lambda D N A and 0.2 U punfred enzyme were incubated for 30 min at 45, 55, 65, 75 and 8 5 ° C respectwely. The extent of D N A dtgesUon was compared by agarose gel elcctrophotcsls.

4. RESULTS

Growlh at 37 a C aerobic .+ ann©tobit -

Koser cllrat¢ G|uco,s¢

430C aerobic + anaerobic • 56 ° C aerobic ' + 65° C aerobic 7~[ NaC| -Catalase + Oradase +

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m g reacUons using [a-3sS]dATP~s as labelled nudeotide. The remaining 5.0 pl sample was incubated at 3 0 ° C containing dCTP, dTTP, d G T P (0.125 mM) together with 5 /LCI [a-35S]dATPaS (7.7 pmol, Amersham, code SJ.304) and 1.0 unit Klenow enzyme. After 5 rain, unlabelled dATP (1 #l, 0.5 raM) was added and the incubation continued for 10 rain. The reaction was terminated by heating at 6 5 ° C for 10 rain. The volume of the mixture was made up to 10 td by adding 1 td 10 × medium salt buffer and 0.6 #l distilled water. The mixture was dwlded into two samples" The first sample (I) was incubated with Bcol (approximately 0.15 unit) at 3 7 ° C for 30 rain. The second sample (II) was treated m the same way as sample I except after 15 nun incubatmn, klanow enzyme (1.0 unit) was added and incubatmn contmued for 15 rain. Both samples were analyzed alongside with the dldeoxynucleot~de sequencing ladders.

+

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with spore

4.1. Purtfzcatton of B¢ol A one step punhcaUon protocol was dcvdoped for the resmctton enzyme. Fl a. 1 shows the ¢lutlon profile of the crude Bcol lysate. Starting with 5 g of wet ceils, 6,53 × 104 U of punhed enzyme was recovered. This enzyme was found to produce the same restriction pattern on lambda D N A as AvaL To test for the source of contarmnatlng activity, 1/~g of lambda D N A was digested overnight wRh

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Fraction no. Fig 1 DEAE-ccllulosc¢lulion profile o[ crude B ~aguians lysate and enzymatic ac~:Palyon lambda DNA ;~1 fractions wllh reslnctlon enzymeactroty Insert 0 5 U of enzyme was mcubat¢dwith250 ng non-methylatedlambda DNA at g7°C for 30 nan, 1 punfied Bcol 2 Aval (New Eng Btolabs)3 LambdaDNA control 100 U of enzyme at 370C. Upon gel clectrophorcsis, no alternations in the charactensttc banding pattern was observed. Lambda DNA fragments produced by overmght digestion were purified by phenol/chloroform extracUon and preclpnatcd by cthanol and subjected to figation according to [6]. DNA fragmanrs were shown to be rchgatablc. 4.2. Properties of Bcol Bcol (0.5 umt) was used to digest 250 ng DNA from methylated lambda, non-methylated lambda. adenuvlrus 2, SV-40, pBR322, T 7 and oX174 at 37°C for 1 h. The dlgesuon patterns obtained were tdenucal to that of Aval. The optimal reaction occurred at lugh salt buffer (100 mM NaCI, 50 mM Tns-HCI, pH 7.5, 10 mM MgCI 2, 1 mM diduothreltol). The cleavage site of Bcol was identical to AvaI winch cleaved between the first C and the second pynmidine of the pahndromic sequence 5"CPyCGPuG-3' (Fig. 2). This enzyme was not inactivated by treating at 65°C for 20 nun. Enzyme assays at different

temperatures showed that it was functional at 75 ° C although the optimal reaction temperature was at 65°C Judging from the amount of lambda fragments created, acUvlty of Bcol at 7 5 ° C was

I

C

A

T

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[I

Fig 2 Recognmonand cleavagessaes of Bco] The recoojntwn and cleavage stes were deterauned as descnbed m MATERIALSAND M~THODSUSlllgMI3 rapt8 as thc substrdte DNA

156 about 50% compared to its optimal activity at 6 5 ° C . T h e D N A denatured at 8 5 ° C and no discrete b a n d was observed on the gel.

5. D I S C U S S I O N Saree the discovery of the type II restriction endonuclease, AvaI, from Anabaena vartabths by M u r r a y el al. [8], a n u m b e r of isosehizomers were tsolated from other sources [9,10]. T h e i r p u n f l c a tton usually revolve multtple chromatographic steps. These laborious protocols tnevitably c o m promise the final yield and mcrease the productton cost. O n the other hand, BcoI from B. coagulans S M 1 can be isolated tn reasonable yield by a simple one-step c h r o m a t o ~ a p h t c procedure Similar to Aval [7 l, Bcol ts not reactivated by treattng 1t at 6 5 ° C for 20 n'nn. O n the other hand, the buffer system for BcoI is high salt w h d e that for AvaI is recommended to be m e d m m salt [7]. O u r bacterial strata grows at 56 ° C and appears to produce little u n w a n t e d enzymes. Actually, we find that crude cell lysate is already able to dtgest l a m b d a D N A w i t h high specificity. Since BcoI is thermal stable, the enzymattc reaction can be performed at high temperature and reduces the task due to other c o n t a m i n a t i n g enzymes. As a result, our B coagulans strain is a good candidate for the productton of restrtctton enzyme with the same specifictty as AvaI and its isoschizomers.

ACKNOWLEDGEMENT T h i s w o r k is partially supported by a Project Research G r a n t from the Institute of Science and Technology of the Chinese Untversity of H o n g Kong.

REFERENCES [1] Geoffrey. G W (1988) Trends Gen¢t. 4 (! 1). 314-318 [2] Schtldkraul. I (1984) m Genetic En~neerm 8 Pnnclples and Methods (Setlow. J K and Hollaender, A, cds ) vol 6, pp 117-140, Plenum Press, New York [3] Buchanan. R E and Gibbons. N E (Co-eds) (1974) Ikrgey's Manual of Determmauve Bactenology. 8th ed, Williams and Wllkn~s. Balnmore [4] Cowan, S T and Steel, K J (1974) Manual for the Idcnhficatlon of Medical Bactcrta. 2nd ed Cambridge UmveTsay Press [5] Lennetle. E H. Balows. A, Hausler. W J and Shadomy. H J (1985) Manual of Chmcal M.crobtology. 4th ed ASM, Washington DC [6] Mamaus, T , Fntsch. E F and Sambrook, J (1982) Molecular Cloning. A Laboratory M~mual. Cold Spnng Harbor Laboratory [71 New England Btolabs Catalog (88-89) [8] Murray. K. Hughes. S G . Brown. J S and Bruce. S A (1976) Btochem J 159, 317-322 [9] [,an. R H and Doohnle. W F (1980) FEBS Leu 121 (2). 200-202 [I0] Reaston.J, Duyvcsteyn,M G C and Waard, de A {1982) Oene 20. 103-n0 Ill] Brown, N L and Smith. M (1980) m Methods m Enzymolog~y (Grossman, L and Moldave, K, eds ), vol 65, pp ~.91-404. Academic Press, New York