(25) Food Preservation Science VOL. 29 NO. 1 2003 〔Article〕 25

Partial amino acid sequence of I produced by sp. 11- 4 isolated from Vietnamese fish sauces

NISHIYAMA Yoshitaka", MAEDA Haruko*2, NAGASHIMA Toshio WATANABE Toshihiro*3 and MURA Kiyoshi*1

* 1 Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture 1 -1 - 1, Sakuragaoka, Setagaya-ku, Tokyo 156 - 8502 * 2 Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture 1 -1 - 1, Sakuragaoka, Setagaya-ku, Tokyo 156 - 8502 * 3 Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture 196, Yasaka, Abashiri-sh, Hokkaido 099 - 2493

Bacillus sp. 11- 4 metalloprotease (protease I) , which shows -like specificity, was purified from the culture supernatant of the strain. The N-terminal amino acid sequence of the protease I was determined up to 43 residues from its N-terminus. Further, five derivative peptides were obtained by V 8 protease digestion and their amino acid sequences were analyzed. Of the fragments, three derivatives showed identical N-terminal sequence with the parent protease I. On the other hand, the remaining two derivatives showed sequences, which seemed to match the internal region of parent protease I. Adapting the partial amino acid sequence information to a BLAST database search, the protease I showed significant similarity with the neutral metalloproteases, which possess the zinc- binding motif HEXXH, produced by some clostridia, bacilli, streptococci and staphylococci. The listed metalloproteases commonly possessed the hidden Markov model domain for family protease. The functional features of the protease I determined previously, such as molecular masses, isoelectric points and pH optimums, showed agreement with those of thermolysin family . Consequently the partial amino acid sequence analysis suggests that the protease I is defined as a thermolysin-like metalloprotease. (Received Jul. 25, 2002 ; Accepted Oct. 4 , 2002)

Fish sauce is a popular seasoning in Southeast other hand, some protease-productive bacteria are Asia, typified by Nuoc mam in Vietnam, Nam pla in known to exist in fish sauce. CHAIYANAN et al .2) Thailand and Patis in the Philippines. In Japan, fish isolated a novel halobacillus, which grew in a sauces are also used in some provinces, such as medium containing 10-20% salt, from a fish sauce Shottsuru in Akita, Ishiru in Ishikawa and Ikanago production line. The strain produced three types of in Kagawa prefecture. Because fish sauce has a proteases with molecular masses of 100, 42 and 17 distinctive flavor, it is rarely used in unprocessed kDa . Of the proteases , the 100 - and 17 - kDa form. But fish sauce came into widespread usage as proteases were defined as serine protease, whereas a hidden flavor in snack foods, basting for beef broil the 42–kDa protease was identified as and fish sausages, for their idiosyncratic flavor. The metalloprotease . The halobacillus has been flavors of fish sauces might be enhanced due to successfully employed to improve fermentation in amino acids and peptides derived from fish industrial production of fish sauce in Thailand. proteins1). In general, fish sauce is produced from Previously we isolated some protease-productive raw fish by adding 15-20% (w/w) concentration of bacilli from fish sauces collected in factories and salts and then matured for a half to one year. In markets in Vietnam3). The strains grew in medium fish sauce production, it was thought that the containing a high concentration of NaC1 ranging degradation of proteins was due to fish-produced from 0 to 2. 5 M, indicating that the bacilli was protease (s) in muscles or digestive tracts. On the able to grow in the Vietnamese fish sauce. The

* 1 E-mail:NISHIYAMA Yoshitaka;[email protected], MURA Kiyoshi;[email protected] * 3 E-mail:NAGASHIMA Toshio;[email protected], WATANABE Toshihiro;[email protected] 26 Food Preservation Science VOL. 29 NO. 1 2003 (26)

major extracellular neutral protease (protease I) was as the previous report4), the fractions containing

purified from one of the bacilli, Bacillus sp. 11- 4 protease I were collected and concentrated into a isolated from Vietnamese fish sauce4). The molecular minimum volume for the next step. mass of the protease I was determined as 33. 5 kDa 3. N-terminal and internal amino acid sequence

by sodium dodecyl sulfate-polyacrylamide gel analysis

electrophoresis (SDS-PAGE) . Because the protease I The N-terminal amino acid sequences of the required calcium ions and was inhibited by chelator protease I and derivative peptides from the protein as EDTA and 1 , 10-phenantrolin, the protease I were determined with the direct protein sequencing

was defined as metalloprotease . Further , the method described by HIRANOand WATANABE5).The

protease I hydrolyzed native collagen, and specific protease I and derivative peptides were separated substrates for collagenase such as FALGPA and z- by SDS-PAGE using the method described by

Gly-Pro Leu-Pro-H2O •`. AcOEt, consequently the LAEMMLI6). The peptides on the gels were

protease I seemed to be collagenase-like protease. electroblotted onto polyvinylidine difluoride (PVDF)

However, some properties of the protease I differed membrane using semidry blotting apparatus (Nippon

from those of general . In this study, Eido, Tokyo, Japan) . The derivative peptides of the the purified protease I was applied to partial amino protease I were prepared by proteolytic digestion

acid sequence analysis. The sequences exhibited with Staphylococcusaureus V 8 protease (Wako Pure

significant similarity with the amino acid sequences Chemical, Osaka, Japan) , during electrophoresis

of some metalloproteases belonging to the according to the methods described by CLEVELANDet

thermolysin family , but not with those of al .7) After the membrane was stained, the visible

collagenases. To identify the protease purified from bands were cut out. The N-terminal amino acid

Bacillus sp. 11- 4 , the properties of protease I were sequences of the protease I and derivative peptides

compared with those of the metalloprotease obtained were determined using a pulsed liquid phase protein

by a database homology search. sequencer (Model 492, Applied Biosystems, Foster City, USA). Materials and Methods 4 . Database homology search

1 . Strain The N-terminal and internal amino acid sequences

Bacillus sp. 11- 4 was used. The strain was found were applied to a database homology search

isolated from commercial fish sauces obtained from using BLAST (hap : //www.ncbi.nlm.nih.gov/BLAST the market at Fue City in Vietnam as previously /) . The Clustal W alignments' of the amino acid

reported3).

2. Purification of protease I

The protease I was purified as previously

reported4). Bacillus sp. 11- 4 was incubated in Luria-

Bertani (LB) medium at 37•Ž for 12 hrs with shaking.

Four ml of the culture of the strain was inoculated

into 400ml of LB medium, and then the medium

was incubated at 37•Ž for 18 hrs with shaking. The

culture supernatant was collected by centrifugation

(9, 000 •~ g , 10min, 4•Ž ) was concentrated by

ultrafiltration using hollow fiber (with a molecular

size of 10,000 Amicons) into a twentieth. The

concentrate was fractionated by ammonium sulfate

saturation at 90%. The precipitate was dissolved in Fig. 1 SDS - PAGE banding profiles of purified Bacillus

20 mM Tris-HC1 (pH 7. 2) containing 1 mM CaC12, sp. 11- 4 protease I and the V 8 protease-derivative fragments dialyzed against the same buffer, and then applied The proteolytic digestion of the protease I was performed in the to a CM-Sepharose CL- 6 B column equilibrated with stacking gels as described in Materials and Methods. Lane 1 , the same buffer. The absorbed protease I was molecular weight standard proteins ; 2 , parent protease I ; 3 , eluted by a linear gradient of NaCl ranging from 0 V 8 —protease digested fragments of the protease I. The fragment to 250 mM. Based on the protease assay described names are indicated in right hand of the gel. (27) 〔Article〕 Amino acid sequence of Bacillus protease 27 sequences were created with MacVector nucleotide applied to N-terminal amino acid sequence analysis. and protein analysis software. The hidden Markov As shown in Table 1 , the N-terminal amino acid model database search was sequence of parent protease I was determined as A -A-T-T-G-S-G-Y-G-V-L-D-D-Y-K- performed using Pfam9) (http : //www. sanger. ac. uk/Software/Pfam/). T-L-N-T-Y-S-S-N-G-T-Y-Y-L-Y-D -V-T-K-P-M-N-G-V-I-E-T-F-T . The Results and Discussion database search for the determined sequence with The purified protease I demonstrated a peptide the BLAST program enumerated Clostridium band with a molecular mass of approximately 33. 5 perfringens lambda toxin and some metalloproteases kDa on SDS-PAGE as shown in Fig. 1. The peptide such as elastase, bacillolysin and aureolysin (Table was electroblotted onto PVDF membrane and then 2 ) . According to the sequence of listed

Table 1 N-terminal amino acid sequences of purified protease I and derived fragments obtained by V 8 protease digestion developed by SDS-PAGE

The band names A through E are corresponded to Fig. 1.

Table 2 List of the proteins obtained by BLAST search using determined partial amino acid sequences 28 Food Preservation Science VOL. 29 NO. 1 2003 (28)

metalloproteases, the molecular masses of the domain, and for the C-terminal third part as nascent translational products were estimated as 56 thermolysin metallopeptidase catalytic domain. More ～ 62 kDa. The N-terminal 25-30 residues of these than half of the metalloproteases possessed the proteases exhibited typical signal-peptide sequence. HEXXH motif, and the triad (two histidine and one The products could primarily convert into pro- glutamic acid residues) could be acting as catalytic protease by eliminating the signal peptide, and residues. The motif HEXXH sequences were also finally mature into active metalloenzyme by observed in the C-terminal third part domain of additional excision of the pro-regions10),11). Consequently, listed proteins as shown in Fig. 2. This observation the molecular masses of the mature-form proteases could indicate that the protease I also possessed a were approximately 33-35 kDa, and the values zinc- in the molecule. Furthermore, the coincided with the molecular mass of Bacillus sp. 11 thermolysin family proteases contain four calcium- -4 protease I. As shown in Fig. 2, the N-terminal binding sites, which provide thermal stability for the amino acid sequence of Bacillus sp. 11- 4 protease I enzyme12),13).Previously MAEDA et al .4) reported that showed a good match with those of matured protease I was inactivated with zinc-specific chelator analogues. From these data, the protease I might be 1 , 10—phenantorolin, further that active protease I primary synthesized as a precursor having the required calcium ions. These observations support enhanced peptide at their N-terminus. the prediction that the protease I would belong to To obtain more primary structure information, the the thermolysin family. The bacillolysin is known as protease I separated by SDS—PAGE was digested the thermolysin family protease produced by bacilli. with V 8 protease and then the derivative peptides The primary structure information of the bacillolysin were sequenced. As shown in Fig. 1 , five is available in six strains of bacilli. Although the derivatives were obtained. Of the five derivatives, determined partial sequences of the protease I the amino acid sequences of the fragment A, B and showed some similarity with these sequences (data C were exactly identical to the N-terminal amino not shown) , the identical sequence was not obtained acid sequence of the parent protease I (Table 1) , from these data. Thus the protease I may be indicating that the fragments obtained were lacking defined as a relative to bacillolysin, but could not be their C-terminal regions. On the other hand, the identified as bacillolysin. According to the previous sequences of fragment D and E were different from report4), the protease I hydrolyzed collagen and a that of the N-terminus of the parent peptide. The specific substrate to collagenase. Evidence for sequences were significantly similar with those of collagen digestion was also found in some some metalloproteases, likewise the N-terminal thermolysin family proteases. A member of the sequence of the parent peptide, based on the results family, pseudolysin, exhibits broader specificity, of the BLAST searches (Table 2 ) . Figure 2 shows acting on large molecules such as elastin, laminin, multiple alignments of the amino acid sequences of proteoglycan , immunoglobrin A and G , and Clostridium perfringens lambda toxin, Staphylococcus collagen14). The broad specificity of the pseudolysin epidermidis elastase, Bacillus subtilis bacillolysin and is considered to be attributable to a wider active aureolysin, with determined site cleft. Furthermore, the Clostridium perfringens partial amino acid sequence of Bacillus sp. 11- 4 lambda toxin, which had been found to be a protease I. The amino acid sequences of the member of the thermolysin family proteases, also homologous metalloproteases with the protease I degrades some large molecules such as fibronectin, were applied to the hidden Markov model protein fibrinogen, immunoglobrin A and collagen, but not family searches using Pfam9). As a result, the N- elastin. terminal third part of the proteases matched with Recently, supplements of protease-containing visceral15) the consensus domain sequences for thermolysin or proteasesl16),17) to accelerate the fermentation metallopeptidase propeptide regions. The prediction process have been attempted for mass-production of agreed with the previous reports about signal-and fish sauce. LOPETCHRATand PARK.described that the pro-peptides in N-terminus of each protease10),11). effective in fermentation of fish sauce were Further, the domain searches lined up the heat stable and salt tolerant, based on the data candidates for the centermost third part of the about the characterization of the fermentation stage proteases as thermolysin metallopeptidase a—helical of fish sauce production using Pacific Whiting. Thus (29) 〔Article〕 Amino acid sequence of Bacillus protease 29

Fig. 2 Multiple alignment of various thermolysin family metalloproteases and determined partial amino acid sequences of the Bacillus sp.11- 4 protease I

The white letters indicate the identical or near relative residues. The shaded residues in the partial sequence of the protease I indicate the residues exhibiting the match with at least one of the other proteases. Fragments D and E correspond to the names defined in Fig. 1. Fragments A, B and C are omitted to describe, because they shows identical sequences with the N-terminus of parent protease I. The positions, which were predicted as cleavage sites with signal peptidases, are indicated with arrowheads. The N-termini of the mature proteins are indicated by the arrow. lambda, Clostridium perfringens lambda toxin ; elast, Staphylococcus epidermidis elastase ; bacillo, Bacillus subtilis bacillolysin ; aureo, Staphylococcus aureus aureolysin. 30 Food Preservation Science VOL. 29 NO.1 2003 (30)

the heat-stable thermolysin family proteases could 10) SABAT, A., KOSOWSKA, K., POULSEN, K., KASPROWICZ, play a beneficial role in the industrial production of A., SEKOWSKA, A., VAN DEN BURG, B., TRAVIS, J. and fish sauce. To determine their structural and POTEMPA, J. : Two allelic forms of the aureolysin functional features, the precise primary structure for gene (aur) within Staphylococcus aureus , Infect. the Bacillus sp. 11- 4 protease I should be clarified. Immun., 68, 973•`976 (2000)

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プチ ド断片 を調製 し,そ のア ミノ酸配列 を分析 した。得 ベ トナ ム産 魚 醤 よ り分 離 され たBaoillus sp.11-4が られた断片の うち3つ は プロテ アーゼ1のN末 端 ア ミノ 産 生 す るプ ロテ ア-ゼ1の 部 分 ア ミノ酸 配列 酸配列 と一致 したが,残 る2つ の断片 は内部領域 と考 え 西 山 由 隆*1・ 前 田 治 子*2・ 永 島 俊 夫*3 られ る配 列 を示 した。得 られ た 部分 ア ミノ酸 配 列 を 渡 部 俊 弘*3・ 村 清 司*1 BLASTデ ー タベ ース検索 した結果,ク ロス トリジウ ム

* 1 東京農業大学応用生物科学部栄養科学科 属,バ チルス属,ス トレプ トコッカス属 お よびス タフィ

(〒156-8502東 京 都 世 田 谷 区 桜 丘1-1-1) ロコ ッカス属が 産生す る中性金 属 プロテアーゼ と相 同性 * 2 東京農業大学応用生物科学部生物応用化学科 を示 した。 これ らの プ ロテ アーゼ は亜 鉛 結 合 モ チ ー フ

(〒156-8502東 京 都 世 田 谷 区 桜 丘1-1-1) HEXXE配 列 を持 ち,サ ーモ リシ ンフ ァミリープ ロテ ア * 3 東京農 業大学生物 産業学部食 品科学科 ーゼ に共通の 隠れマル コフモデル ドメイ ンを有 していた。 (〒099-2493北 海道網走市八 坂196) また,さ きに報告 された プロテアーゼ1の 特性 はサーモ リシ ンファ ミリープ ロテ アーゼのそ れ らと類似 していた。 Bacillus sp.11-4培 養 液 中 か ら コ ラ ゲ ナ ー ゼ 様 の 特 異 以上 の結 果か ら,プ ロテ アーゼ1は 部 分 ア ミノ酸 配列 の 性 を 示 す 金 属 プ ロ テ ア ー ゼ(プ ロ テ ア ー ゼ1)を 精 製 し 結果 を基 にサ ーモ リシン様 プロテ アーゼであ る と推 定 さ た 。 精 製 プ ロ テ ア ー ゼ1は,N末 端 ア ミ ノ 酸 配 列 分 析 を れ た。

行 い,そ のN末 端 か ら43残 基 ま で の ア ミ ノ 酸 配 列 を 決 定 (平 成14年5月25日 受 付,平 成14年10月4日 受 理) し た 。 さ ら に,V8プ ロ テ ア ー ゼ 処 理 に よ っ て5つ の ベ