四種蛋白水解酶在不同分子篩上的吸附固定

劉 平 邢國文 李宣文 葉蘊華

(北京大學化學與分子工程學院,北京分子科學國家實驗室,教育部生物有機與分子工程重點實驗室,北京 100871)

四種蛋白水解酶在不同分子篩上的吸附固定

劉 平 邢國文a李宣文 葉蘊華*

(北京大學化學與分子工程學院,北京分子科學國家實驗室,教育部生物有機與分子工程重點實驗室,北京 100871)

系統研究了α-胰凝乳蛋白酶、木瓜蛋白酶、枯草桿菌蛋白酶和嗜熱桿菌蛋白酶4種蛋白水解酶在一系列分子篩上的吸附固定.所用分子篩載體包括微孔分子篩:HY、NaY、NH4Y、MCM-22、Hβ沸石,改性Y沸石: HDAY、HNH4DAY以及介孔分子篩MCM-41.結果表明,不僅分子篩的結構與酶的性質對酶的固定化量與固定化酶的活性有重要影響,而且吸附固定化條件如緩沖液的pH值和酶的濃度等對酶的吸附固定化也有顯著影響.在多數情況下,pH值為6時蛋白水解酶在分子篩上的吸附固定化的量較高,隨著pH值進一步升高吸附量降低.探討了蛋白水解酶與不同分子篩之間的相互作用,例如α-胰凝乳蛋白酶在Hβ沸石上吸附固定化量最高,而固定在MCM-22上的α-胰凝乳蛋白酶的活性最高,這顯然與其吸附狀態有關.

吸附;固定化作用;蛋白水解酶;沸石;分子篩;多肽合成

Enzymes are biocatalysts,which exhibit high selectivity and reactivity under mild reaction conditions.However,the low stability of enzymes limits their applications.In order to extend the use of these biocatalysts for practical applications,the technology of enzyme immobilization on suitable supports was developed.Immobilized enzymes are more stable and can be reused more times as compared to free enzymes.Additionally,enzyme immobilization can prevent some detrimental processes,such as enzyme autolysis.Different methods for immobilization of enzymes are critically reviewed[1-2].

Molecular sieves and mesoporous materials with some unique characteristics,such as special pore structure and large surface area,have attracted much attention for enzyme immobilization by adsorption and have found various applications in organic synthesis[3-5].As inorganic support materials,molecular sieves not only offer novel properties including high surface area,hydrophobic or hydrophilic behavior,electrostatic interaction,and resistance to biodegradation,but also can be prepared with cavities ranging from micropore(＜2 nm)to mesopore(2-50 nm)by control the preparation conditions.Macario et al.[6]reported lipase immobilization on zeolitic support and transesterification reaction in a solvent free-system.Carvalho et al.[7]reported the influence of the presence of NaY zeolite on the activity of horseradish peroxidase in the oxidation of phenol.In recent years,mesoporous molecular sieve MCM-41 was widely used as matrix for immobilization of many enzymes such as penicillin acylase[8],cytochrome P-450[9],papain[10],trypsin[11],laccase[12]. Hudson et al.[13]studied the methodology for the immobilization of enzymes onto mesoporous materials.

Proteases constitute one of the most important groups of industrial enzymes,accounting for at least 25%of the total enzyme sale[14].We pay much attention on proteases including αchymotrypsin,papain,subtilisin,and thermoase(or its pure form thermolysin)for peptide synthesis.Various microporous HY, NaY,NH4Y,MCM-22,Hβ zeolites,modified Y zeolites HDAY, HNH4DAY,and mesoporous MCM-41 molecular sieve were selected as matrixes for immobilization of different proteases. With the zeolite immobilized enzymes as catalysts,some oligopeptides were successfully prepared.The immobilization of proteases by adsorption method has been studied and reported by our group previously[15-16].The results showed that immobilized α-chymotrypsin could enhance the reaction rate as well as the yield of Z-Tyr-Gly-Gly-OEt(Z=benzyloxycarbonyl)comparing with free α-chymotrypsin.However,various supports provided different immobilization abilities and activities for immobilized enzymes.The microporous MCM-22 and HY zeolites as supports gave the best yield,which declined to a certain degree after being reused 4-5 times.For the synthesis of sweetener precursor, Z-Asp-Phe-OMe,by using zeolite immobilized thermolysin in tert-amyl alcohol,NH4Y zeolite gave the best yield(64%)and it could be reused 5 times,while HY zeolite was the worst,the yield was less than 10%.A precursor of osteogenic growth peptide fragment(10-14),Z-Tyr-Gly-Phe-Gly-Gly-OEt,was accomplished using immobilized α-chymotrypsin and papain on MCM-22 in cyclohexane.The reaction rate and synthetic yield were enhanced in most cases than free enzyme.These results showed that molecular sieves exhibited different immobilization abilities and activities for immobilized enzymes.In recent years, many research groups focused their attentions on the preparation and application of molecular sieves to immobilize enzymes such aslipase,peroxidase,andpenicillinacylase[6-12],but there are a few reports involved in molecular sieves as matrixes for immobilization of different proteases[10-11]and applications for peptide synthesis[15-16].However,the nature of adsorption interaction between enzyme and molecular sieve and their enzymatic activity have not been demonstrated clearly in the previous study.

In order to elucidate immobilization behaviour of different molecular sieves,the factors influencing immobilization of enzymes,such as the external surface area,the surface properties of supports,enzyme properties,immobilization conditions,and the interaction property between enzyme and matrix,were systematically studied.The activities of some immobilized α-chymotrypsins were determined and compared to each other.

1 Experimental

1.1 Materials

α-Chymotrypsin(EC 3.4.21.1,from bovine pancreas),and subtilisin carlsberg(EC 3.4.21.14,from bacillus licheniformis) were purchased from Sigma Company.Thermoase,i.e.,crude thermolysin(EC 3.4.24.4,from thermoprotedyticus rokko),was obtained from Daiwa Kasei K.K.Company.Papain(EC 3.4.22.2) was provided by Sino-America Biotech.The physical data of the proteases are listed in Table 1[17].Molecular sieve MCM-22, MCM-41 and Hβ zeolites were provided by Research Institute of Petroleum Processing,SINOPEC.Some parameters of molecular sieves are listed in Table 2.

The amino acid residues which were used in this study are of L-configuration.Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPACIUB Biochemical Nomenclature[18].

Electronic spectra were recorded by a Shimadzu UV-265FW UV-Vis spectrophotometer.Lyophilization was performed by a Flexi-DryTMμP(FTS SYSTEMS,INC.USA)drier.

1.2 Preparation of immobilized α-chymotrypsin on MCM-22,MCM-41,Hβ zeolites and determination of enzyme loading

The preparation of immobilized α-chymotrypsin on MCM-22, MCM-41,Hβ zeolites and determination of enzyme loading was carried out according to Ref.[16].3 mg α-chymotrypsin was dissolved in 4 mL PBS(phosphorus buffer solution)[0.05 mol·L-1, pH 6.0(or 4.92,7.5,8.67)](solution A).0.2 mL from solution A was diluted to 3.5 mL by deionized water(solution B).The absorbance of the diluted enzyme solution B at 282 nm was recorded by UV-Vis spectrophotometer.Then 60 mg of MCM-22(or MCM-41,β zeolites)was added to 3.8 mL solution A at 4℃and stirred for 2 h.The mixture was centrifugated to get supernatant(solution C)and 0.2 mL from solution C was diluted to 3.5 mLbydeionizedwater(solutionD).Theabsorbanceofsolution Dwas recorded by UV-Vis at 282 nm as described above.The amount of immobilized enzyme was calculated from the difference between the absorbances of solution B and solution D.The MCM-22(or MCM-41,β zeolites)immobilized α-chymotrypsin waswashedwithPBS,lyophilizedbyFlexi-DryTMμP andstoredat -2℃.Additionally,α-chymotrypsin solutions with other concentrations were also used for preparation of MCM-22 immobilized α-chymotrypsin in order to investigate the dependence of enzyme loading on enzyme concentration.

Table 1 Physical data of different proteases[17]

Table 2 Texture parameters of different molecular sieves

1.3 Preparation of immobilized α-chymotrypsin and thermolysinonHY,NH4Y,NaY,HDAY,HNH4DAY

The preparation procedure was same as our previous work[15].

1.4 Preparation of immobilized papain(or subtilisin, thermoase)on MCM-22 or MCM-41 and determination of enzyme loading

The method was similar to that described in section 1.2.The absorbance of papain,subtilisin,and thermoase solutions were recorded at 278,280,277 nm,respectively(Table 1).

1.5 Determination of the activities of MCM-22,MCM-41andHβ-2zeoliteimmobilizedα-chymotrypsin[16]

Toasolutionof1mL casein(2%,mass fraction)and 2 mL PBS (pH 8.0,0.05 mol·L-1)warmed in a 37℃oil bath for 5 min,6 mg MCM-22(or MCM-41,Hβ-2 zeolite)immobilized α-chymotrypsin was added.The mixture was stirred for 20 min at 37℃and the reaction was stopped by 2 mL 1.2 mol·L-1trichloroacetic acid.Then the reaction solution was centrifugated after precipitation for 10 min and 0.5 mL of the supernatant was diluted to 5 mL by deionized water.The absorbance of the diluted supernatant at 280 nm was recorded by UV-Vis spectrophotometer using the sample without addition of immobilized enzyme as control.

2 Results and discussion

The proteases loading amount on various molecular sieves was investigated.According to the enzyme molecular size and the pore sizes of molecular sieves used(Table 1 and Table 2),the enzymes were located on the external surface of supports instead of the pore channel of molecular sieves.The results are summarized in Table 3.As shown from the results,the amount of adsorbed enzyme was influenced by many factors,such as the structure and property of carriers and enzymes,the pH value of adsorptionbufferetc.Partofresultwasbrieflyreportedin2004[19].

2.1 Influence of surface area of molecular sieves

The molecular sieves have external surface of particles and internal framework surface.However,as we mentioned above, the molecular size of proteases used in this study was larger than the pore size of molecular sieves,therefore the proteases would be adsorbed and located on the external surface rather than in pore channels of these molecular sieves.We used Hβ zeolites with different particle sizes to study the influence of the external surface area on the loading amount of enzyme.The results in Table 2 demonstrated that decreasing the particle size of Hβ from 500 nm to 20-30 nm led to increase of their external surface area and the enzyme loading amount.Moreover,the enzyme loading amount on Hβ was approximately proportional to the external surface area.These results indicated clearly that the surface area is one of the most important factors to determine enzyme loading amount.It is worth to note that Yagiz et al.[20]reported hydrotalcite and 4 different zeolites were used as immobilization materials for lipase,their results showed that the size of zeolite particles did not affect the amount of adsorbed protein.The different results may be caused by using different enzymes.

2.2 Influence of surface characteristics of molecular sieves

In the case of MCM-41,the external surface is larger than those of NH4β and Hβ-1.If enzyme molecules could be adsorbed on the external surface area,the enzyme loading on MCM-41 would be much higher than those on Hβ-1 and NH4β zeolites.However,the amounts of α-chymotrypsin adsorbed onMCM-41 were much lower than that on Hβ-1 and NH4β zeolites.These results indicate that besides the surface area,the surface characteristics are another important factor to determine the enzyme loading amount and immobilized enzyme activity.

Table 3 Effects of pH,supports and enzyme concentration on enzyme loading by adsorption method in 0.5 mol·L-1 phosphorus buffer solution

Fig.1 Schematic structure and composition of NH4β zeolite(a)and Hβ zeolite(b)

Hβ,NH4β are zeolites which are constituted by tetrahedral SiO4and AlO-4with negative charges in their frameworks(Fig.1). Cationic type zeolite has internal electrostatic field between negative charges of zeolite framework and cations.The protease surface has many charged and polarized amino acid residues.

Consequently,in the adsorption process,electrostatic interaction and hydrogen bond formation between enzymes and molecular sieves existed,and the stronger the interaction was,the more enzyme loading by adsorption was,and vice versa.Thus, in the case of α-chymotrypsin adsorption on NH4β zeolite which has internal electrostatic field,the electrostatic interaction occurred and more proteases were adsorbed than that on other molecular sieves.These results are consistent with the suggestion by Takahashi et al.[21]that the ionic surface characteristics of molecular sieves determine their adsorption abilities.

For the Hβ zeolite,the proton has a very strong electron affinity which causes the interaction of proton with negative charged oxygen of zeolite framework to form the acidic OH. These hydroxyl groups would be favorable to form hydrogen bond with protease,the principal drive force of protease adsorption on Hβ and MCM-22 would be interaction of hydrogen bond,even some electrostatic actions exist on the surface.

MCM-41 is composed by neutral SiO4,there are no negative charges and almost no acidic hydroxyl groups in the framework, only the silanols exist on the surface.So there are no electrostatic interaction and strong hydrogen bond between protease and MCM-41.The adsorption of protease on MCM-41 was ascribed by weak hydrogen bond and/or van der Waals interaction,which led to much less proteases adsorption on MCM-41 than that on other zeolites(Table 3).

Surface acidity of molecular sieves also has great influence on enzyme immobilization.Fontes et al.[22]reported that zeolites had dramatic acid-base effect on enzyme activity in low watermedia,which was consistent with our previous work[15].The catalytic effect of HY immobilized thermolysin was the worst of the four kinds of zeolites immobilized thermolysins screened.Thermolysin is a kind of neutral protease which is stable at pH of 6.0-9.0.However,owing to the strong acidity of HY zeolite,the pH value of the solution decreased from 6.98 to 3.22 after the thermolysin buffer solution was stirred for immobilization over HY zeolite in 1 h(Table 4).As a consequence,the activity of HY zeolite immobilized thermolysin became very low and the yield of dipeptide Z-Asp-Phe-OMe was poor.HY zeolite immobilized α-chymotrypsin had a high catalytic activity resulting from the broaderstablepHrange(3-10)of α-chymotrypsincompared with that of thermolysin.In this case,the acidity of HY zeolite had no noticeable influence on the activity of α-chymotrypsin(Table 5). For other zeolites(NaY,NH4Y,HDAY,and HNH4DAY)used, the pH values all approached neutral and were located in the stable pH scope of thermolysin or α-chymotrypsin.Therefore, with them as immobilization matrixes,thermolysin or α-chymotrypsin presented catalytic activity to a certain extent in the reaction(Tables 4,5).

Table 4 pH value changes during thermolysin immobilization on different zeolites

2.3 Influence of buffer pH

When α-chymotrypsin was adsorbed on NH4β,the process was significantly affected by the buffer pH value.The enzyme loading amount dropped down gradually with pH increasing from 4.92 to 8.67(Table 3).This result may be caused by the fact that the number of the positive charges of α-chymotrypsin reduced with the increase of the pH value which was closed to isoelectric point of α-chymotrypsin.Consequently,the electrostatic interaction between α-chymotrypsin and NH4β surface was reduced,and then the α-chymotrypsin adsorbed amount was gradually reduced.

Interestingly,in the case of adsorption of protease on MCM-41,there was almost no pH effect on the adsorption process. This could be explained by weak hydrogen bond and van der Waals interaction between protease and MCM-41 surface.Only the protease polarity and charge changes with pH value did not lead to alter their interaction action with inert surface of MCM-41.

Table 5 pH value changes during α-chymotrypsin immobilization on different zeolites

Fig.2 Effect of concentration(c)of α-chymotrypsin on enzyme loading(Γ)adsorbed on MCM-22

For the adsorption of α-chymotrypsin and papain on Hβ and MCM-22 zeolites,the protease loading was not affected significantly by pH from 4.92 to 7.5 of the buffer,but it dropped significantly from pH 7.5 to 8.67.This may be caused by the changes of ionization state near isoelectric point of protease, when pH increased from pH 7.5 to 8.67,which led to reduce the availability of hydrogen bond formation between protease and molecular sieves.However,in most cases,the loading amount was a little less at pH 4.92 than that at 6.0,because a part of negative charges of molecular sieves were neutralized by H+of buffer to reduce the hydrogen bond and electrostatic interaction between the protease and matrix.

2.4 Influence of protease concentration and adsorption time

The effect of protease concentration and adsorption time on the protease loading amount on matrixes were investigated.Protease loading was enlarged with the increase of enzyme concentration,but the increasing degree dropped when enzyme concentration was beyond 0.75 g·L-1as shown in Fig.2.Fig.3 illustrates that the adsorption process reached equilibrium after molecular sieves were added to enzyme solution and the mixture was stirred for 2 h,since curve B of UV-Vis spectra of α-chymotrypsin solution after adsorption for 2 h was almost overlapped with curve C after adsorption for 24 h.

Fig.3 UV-Vis spectra of α-chymotrypsin solution(pH=6.0, c=1.2 g·L-1)before adsorption(A),after adsorption by MCM-22 for 2 h(B)and 24 h(C)

Table 6 Absorbance(A280nm)of products formed from hydrolysis of casein by immobilized α-chymotrypsin for 20 min at 35℃

2.5 Influence of protease structure

α-Chymotrypsin,papain and subtilisin belong to protease big family,and they hydrolyze peptide bond of protein with different substrates.The loading amount on MCM-22,α-chymotrypsin was similar to papain,but for subtilisin,it less than that of αchymotrypsin and papain at different pH values(Table 3).We suppose that the subtilisin contains less basic amino acid residues than α-chymotrypsin and papain to form less positive charges at pH 6.0,which decreases the electrostatic and hydrogen bond interaction between the protease and supports.The basic amino acid residues of α-chymotrypsin and papain are similar,the difference is only that α-chymotrypsin contains more Lys,while papain contains more Arg to make a little difference of the loading amount on MCM-22.

The activities of α-chymotrypsin immobilized on MCM-22, MCM-41 and Hβ-2 zeolites were determined.The absorbances (A280nm)of products formed from hydrolysis of casein by immobilized α-chymotrypsin for 20 min at 35℃were in the following order:α-CT/MCM-22>α-CT/Hβ-2>α-CT/MCM-41(Table 6).It suggests that the activities of immobilized enzymes were also in the same order:α-CT/MCM-22>α-CT/Hβ-2>α-CT/ MCM-41,because the absorbances(A280nm)of products were proportional to their amount.The activity of MCM-41 immobilized α-chymotrypsin was the lowest mainly because α-chymotrypsin loading adsorbed on MCM-41 was the least.However,the activity of Hβ-2 immobilized α-chymotrypsin was lower than that of MCM-22 immobilized α-chymotrypsin,even enzyme loading on Hβ-2 zeolite was the most among the above three immobilized proteases.This is probably due to different adsorption states of protease on the surface of various molecular sieves. MCM-22 is a microporous zeolite with special structure and external surface[23-24].The surface pockets of MCM-22 would accommodate protease molecules in favorable state where enzyme molecules should have more available active sites to substrates. Our previous study also demonstrated that MCM-22 was a good matrix for immobilization of proteases.A precursor of osteogenic growth peptide fragment OGP(10-14),Z-Tyr-Gly-Phe-Gly-Gly-OEt,was synthesized successfully by immobilized papain and α-chymotrypsin on MCM-22 in cyclohexane.The immobilized protease could be reused for several times.In comparison with free enzyme,most protected fragments and the target protected pentapeptide OGP(10-14)were synthesized by immobilized protease with reaction rates remarkably enhanced, the corresponding product yield was also increased in most cases[16].

3 Conclusions

Immobilization of four different proteases by adsorption method on a series of molecular sieves including microporous HY,NaY,NH4Y,MCM-22,Hβ zeolites,modified Y zeolites HDAY,HNH4DAY and mesoporous MCM-41 molecular sieve was investigated.The results showed that enzyme adsorption and loading on molecular sieves was remarkably affected by many factors.i)The external surface area of molecular sieves is one of the most important factors to determine enzyme loading amount.The adsorbed proteases were located on external surface of supports,and the larger external surface area of molecular sieves was,the more protease loading by adsorption was.As for α-chymotrypsin,enzyme loading amount on nanometer materials Hβ-2 zeolites was the biggest due to its largest area of external surface.ii)Enzyme loading was dependent on the interaction between molecular sieves and proteases,including the hydrogen bond and the electrostatic action among the charge groups on the surface of the proteases and ions from the framework of molecular sieves as well as charged atoms.The stronger the interaction was,the more protease loading by adsorption was.The results herein indicated that the drive forces for enzyme adsorption on NH4β,Hβ and MCM-41 were electrostatic interaction,strong hydrogen bond and weak hydrogen bond(or van der Waals)interaction respectively.iii)Other factors such as pH value of buffer and enzyme concentration etc.also played important role during the adsorption process.In most cases,enzymes adsorbed on molecular sieves were relatively more at pH 6.0,but decreased with further increasing pH value.The amount of thermoase and subtilisin adsorbed on supports was much less as compared to α-chymotrypsin and papain.In addition,enzyme loading increased with increasing enzyme concentration.iv)The activity of immobilized enzyme was relevant to support and the enzyme loading amount.The activity order of α-chymotrypsin immobilized on MCM-22,MCM-41 and Hβ-2 zeolite was:α-CT/MCM-22>α-CT/Hβ-2>α-CT/MCM-41.Our study also indicated that immobilization of different proteases on different molecular sieves could be successfully used for synthesis of peptide in organic solvents.

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Adsorptive Immobilization of Four Proteases on Different Molecular Sieves

LIU Ping XING Guo-WenaLI Xuan-Wen YE Yun-Hua*
(Beijing National Laboratory for Molecular Science,Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Ministry of Education,College of Chemistry and Molecular Engineering,Peking University,Beijing 100871,P.R.China)

The immobilization of four different proteases on a series of molecular sieves,including microporous HY,NaY,NH4Y,MCM-22,Hβ zeolites,modified Y zeolites HDAY,HNH4DAY,and mesoporous MCM-41,by adsorption was systematically investigated.The four proteases were α-chymotrypsin,papain,subtilisin,and thermoase (or its pure form thermolysin).The results showed that the enzyme loading amount and the activity of immobilized enzyme were significantly affected not only by the structures and textures of molecular sieves and the enzyme properties,but also by the adsorptive conditions such as buffer pH values and enzyme concentration.In most cases,the amount of protease loading on molecular sieves was relatively higher at pH 6.0,but declined with further increasing of pH values.The nature of the interaction between protease and molecular sieves is discussed.As for α-chymotrypsin,its loading amount on Hβ zeolites was found to be the highest,whereas the activity of α-chymotrypsin immobilized on MCM-22 was the highest,which is probably due to different adsorption states.

Adsorption; Immobilization;Protease;Zeolite;Molecular sieve; Peptide synthesis

The authors thank Research Institute of Petroleum Processing,SINOPEC for providing β zeolites, MCM-22,and MCM-41.

*Corresponding author.Email:yhye@pku.edu.cn;Fax:+86-10-62751708.

aCurrent address:Department of Chemistry,Beijing Normal University,Beijing 100875,P.R.China.

The project was supported by the National Natural Science Foundation of China(29872002)and the Hong Kong Polytechnic University.

國家自然科學基金(29872002)和香港理工大學資助項目

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