阅读说明：本技术 热稳定性普鲁兰酶复配及其淀粉原料糖化工艺 (Heat-stable pullulanase compound and starch raw material saccharification process thereof ) 是由 聂尧 穆晓清 毕家华 于 2021-06-21 设计创作，主要内容包括：本发明公开了热稳定性普鲁兰酶复配及其淀粉原料糖化工艺,属于酶工程技术领域。本发明提供了一种复合糖化酶制剂,所述制剂含有热稳定性的中性普鲁兰酶和糖化酶。利用该复合糖化酶制剂,本发明进一步提供了一种淀粉料液的糖化方法：质量分数为35％的B淀粉溶液在pH为6.0、温度60℃的糖化条件下,复合酶制剂用量为40U/g的干基B淀粉,普鲁兰酶：糖化酶的复配比例为1：1,糖化时间为120min。DE值提高至91.66％。本发明中公开的复合糖化酶制剂及其淀粉原料糖化工艺将有望克服现有技术存在的工序繁琐、不经济、效率低等问题,促进糖化工艺的发展。(The invention discloses a heat-stable pullulanase compound and a starch raw material saccharification process thereof, belonging to the technical field of enzyme engineering. The invention provides a compound saccharifying enzyme preparation, which contains heat-stable neutral pullulanase and saccharifying enzyme. The invention further provides a saccharification method of starch liquid by using the compound saccharifying enzyme preparation, which comprises the following steps: under the saccharification condition that the pH value of a B starch solution with the mass fraction of 35% is 6.0 and the temperature is 60 ℃, the dosage of a complex enzyme preparation is 40U/g dry-base B starch, pullulanase: the compounding ratio of the saccharifying enzyme is 1:1, the saccharification time is 120 min. The DE value increased to 91.66%. The composite saccharifying enzyme preparation and the starch raw material saccharifying process thereof disclosed by the invention are expected to overcome the problems of complicated working procedures, low economy, low efficiency and the like in the prior art, and promote the development of the saccharifying process.)

1. A compound saccharifying enzyme preparation, characterized in that the preparation contains pullulanase and saccharifying enzyme; the amino acid sequence of the pullulanase in the saccharifying enzyme preparation is shown as SEQ ID NO. 2; the amino acid sequence of the saccharifying enzyme is shown as SEQ ID NO. 4.

2. The complex enzyme preparation as claimed in claim 1, wherein the adding ratio of pullulanase to saccharifying enzyme is (1-8): (1-9).

3. A method of producing starch sugar, the method comprising:

(1) preparing feed liquid by using starch B;

(2) saccharifying the feed solution in step (1) with the complex enzyme preparation described in claim 1 or 2.

4. The method according to claim 3, wherein in the step (1), the feed liquid is obtained by liquefying B starch solution by using alpha-amylase; the mass concentration of the starch solution B is 30-40%.

5. The method according to claim 4, wherein in the step (1), the alpha-amylase is liquefied for 1.5-2.5 h.

6. The method according to claim 3 or 4, wherein in step (1), the B starch is selected from byproducts produced by at least one of corn starch, tapioca starch or wheat starch.

7. The method according to claim 3 or 4, wherein in the step (2), the amount of the complex enzyme preparation is 20-50U/g B starch.

8. The method according to claim 3 or 4, wherein in the step (2), the saccharification is carried out under the reaction conditions of pH 5.5-6.5, temperature 55-65 ℃ and time 90-150 min.

9. Use of the complex enzyme preparation of claim 1 or 2 in starch saccharification.

10. Use of the starch sugar prepared according to any one of claims 3 to 8 in the field of food, pharmaceutical preparation or health care products.

Technical Field

The invention relates to a heat-stable pullulanase compound and a starch raw material saccharification process thereof, belonging to the technical field of enzyme engineering.

Background

The starch sugar is a bulk product produced by taking starch rich in grains, potatoes and the like as raw materials and carrying out a series of reactions such as liquefaction, saccharification and the like under the catalysis of enzyme. During the starch liquefaction and saccharification processes, some by-products are often produced. The B starch is used as a by-product in the production of the vital gluten, has the characteristics of dark color, more impurities, high viscosity, difficult separation and the like due to the fact that the B starch contains pentosan, crude fat and the like, and if the B starch is directly used as a feed, the contained non-starch polysaccharide cannot be decomposed by endogenous digestive enzyme, is not easy to be absorbed by monogastric animals, and can cause environmental pollution after being directly discharged. There have been some reports on the comprehensive utilization of B starch, but no reasonable method has been found so far. Therefore, with the rapid increase of the production of wheat gluten, the development of effective utilization of the leftover B starch slurry becomes an irreparable problem.

The starch sugar production process generally adopts a double-enzyme method, namely, the process comprises enzyme liquefaction and saccharification. The saccharification process is a process of finally producing the glucose syrup by adding saccharifying enzyme, pullulanase and the like after the starch slurry is liquefied into dextrin, controlling the amount and the concentration of the added enzyme and controlling the reaction temperature and time. In the process, the single enzyme preparation is prepared into the complex enzyme preparation, so that the saccharification rate can be obviously accelerated, the generation of saccharification byproducts is reduced, and the yield and the purity of the glucose are greatly improved. At present, the saccharification complex enzyme sold in China is mainly produced by two companies of Novoxin and Jenengke, and the compounded enzyme is acid enzyme. When the compound enzyme preparation is applied to a saccharification process, as the optimum pH value is in a partial acid range of about 4.5-5.5 and the original pH value after liquefaction reaction is in a partial neutral range of 6.0-6.5, the pH value of the liquefied liquid needs to be adjusted to an acid range firstly and then pullulanase is added for saccharification in the transition of liquefaction and saccharification, which inevitably increases the resource consumption and hydrolysis time.

CN202010180787 discloses a pullulanase mutant G692M, which has the advantages of low cost, high efficiency, shortened starch hydrolysis time and the like compared with the acidic pullulanase Prozozyme which is widely applied to industrial production. However, the saccharification effect is generally only 31.67% at the maximum after the saccharifying enzyme is compounded with the saccharifying enzyme at the later stage. Therefore, an economical and efficient saccharification method is needed to provide a reference for comprehensive utilization of B starch.

Disclosure of Invention

[ problem ] to

In the prior art, the saccharification effect of starch is only 31.67% after the pullulanase mutant G692M and saccharifying enzyme are compounded, and a saccharification method for improving the saccharification efficiency of starch B is urgently needed.

[ solution ]

The invention aims to provide a compound saccharifying enzyme preparation and a method for producing starch sugar, thereby overcoming the problems of complicated working procedures, uneconomic performance and the like in the prior art.

In order to achieve the above object, a first object of the present invention is to provide a complex saccharifying enzyme preparation comprising a pullulanase which is a mutant enzyme G692M derived from Bacillus thermovoran US105 and a saccharifying enzyme which is a thermophilic neutral saccharifying enzyme derived from Aspergillus flavus HBF 34.

In one embodiment, the pullulanase in the saccharifying enzyme preparation has a nucleotide sequence of a gene shown as SEQ ID NO. 1 and an amino acid sequence of a gene shown as SEQ ID NO. 2; the nucleotide sequence of the saccharifying enzyme is shown as a gene shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4.

In one embodiment, the pullulanase and the saccharifying enzyme are added in a ratio of (1-8): (1-9).

In one embodiment, pullulanase and saccharifying enzyme are added in a ratio of 1: 1.

a second object of the present invention is to provide a method for producing starch sugar, comprising:

(1) preparing feed liquid by using starch B;

(2) and saccharifying the feed liquid by using the composite saccharifying enzyme preparation.

The compound saccharifying enzyme preparation can improve the DE value of a product in the saccharifying process, greatly shorten the saccharifying period and improve the efficiency.

In one embodiment, in the step (1), the liquefaction time of the alpha-amylase is 1.5-2.5 h.

In one embodiment, in step (1), the B starch is selected from, but not limited to, byproducts produced by at least one of corn starch, tapioca starch, and wheat starch.

In one embodiment, in the step (1), the feed liquid is prepared by liquefying a starch solution B with a mass concentration of 20-50% by using alpha-amylase. More preferably, the feed liquid is obtained by liquefying B starch solution with the mass concentration of 30-40% by using alpha-amylase. Further preferably, the feed liquid is obtained by liquefying starch solution with mass concentration of 35% by using alpha-amylase.

In one embodiment, in the step (2), the dosage of the compound saccharifying enzyme preparation is 20-50U/g B starch; preferably 35-45U/g B starch; more preferably 40U/g B starch.

In one embodiment, in step (2), the saccharification is carried out at a pH of 5.5-6.5; preferably at a pH of 6.0.

In one embodiment, in step (2), preferably, the saccharification conditions comprise: a temperature of 50-70 ℃, preferably about 55-65, more preferably 60 ℃; the saccharification time is 90-150min, preferably about 120 min.

The invention also provides application of the complex enzyme preparation in starch saccharification.

The invention also provides application of the starch sugar prepared by the starch saccharification method in the fields of food, medicine preparation and health care products.

The invention has the beneficial effects that:

(1) the composite saccharifying enzyme preparation can improve saccharifying efficiency. For example, the DE value of 35% B starch solution is increased to 91.66% after the composite saccharifying enzyme preparation provided by the invention acts for 120min under the saccharifying condition that the pH is 6.0 and the temperature is 60 ℃.

(2) The composite saccharifying enzyme preparation provided by the invention can be used for saccharifying without adjusting the pH of the feed liquid to an acidic range, so that the saccharifying period can be greatly shortened, and the consumption of resources can be reduced.

Drawings

FIG. 1 influence of the amount of saccharifying enzyme added on the saccharification DE value;

FIG. 2 Effect of pullulanase addition on glycation DE values;

FIG. 3 effect of saccharification temperature on saccharification DE value;

FIG. 4 Effect of saccharification pH on saccharification DE value;

FIG. 5 Effect of saccharification time on saccharification DE value;

FIG. 6B Effect of starch concentration on glycation DE value.

Detailed Description

Enzymes referred to in the examples below

Alpha-amylase: is purchased fromAvailable high temperature resistant alpha-amylase Supra.

Saccharifying enzyme: thermophilic neutral saccharifying enzyme derived from Aspergillus flavus HBF 34.

Pullulanase: mutant enzyme G692M from Bacillus thermoleovoran US 105.

Method for determining DE: the DE value of the saccharification is determined by the 3, 5-dinitrosalicylic acid (DNS) method (Jianhua Bi, Shuhui Chen, Xianghan Zhao, Nie Yao, Yan xu. computation-aided engineering of stage-branching and purification throughput from Bacillus thermophilus and Biotechnology,2020,104, 7551-7562). In the experimental group, 200. mu.L of 2% (w/v) pullulan solution and 200. mu.L of enzyme solution were mixed, reacted at 70 ℃ for 20min, and immediately left in an ice water bath for 5min to terminate the reaction. The control group was incubated at 70 deg.C for 20min and then supplemented with 200. mu.L of 2% (w/v) pullulan solution. After cooling, 600. mu.L of DNS reagent was added to both the experimental and control groups and boiled in a boiling water bath for 5 min. And (5) measuring a corresponding light absorption value at 540nm after cooling in an ice water bath, and calculating the concentration of the reducing sugar by using a standard curve.

DE(％)＝(C×V)/1000w×100％

In the formula: c is the reducing sugar concentration (mg/mL) determined by using a standard curve; v is the volume after dilution, mL; w is the B starch content, g.

Example 1: effect of saccharifying enzyme addition amount on saccharified DE value

(1) Preparing feed liquid: firstly, a certain amount of starch B is dried in an oven at 40 ℃ for 30min and then dried at 105 ℃ to constant weight. Preparing a 30% B starch reaction system by using 0.2mol/L, pH 6.0.0 sodium carbonate-sodium bicarbonate buffer solution, uniformly stirring, and liquefying for 2 hours by using alpha-amylase.

(2) Saccharifying feed liquid: and (2) cooling the liquid material liquefied in the step (1) to 65 ℃, dividing the liquid material into 7 groups, adding 30U/g of pullulanase of dry base B starch into each group, and adding 20, 25, 30, 35, 40, 45 and 50U/g of saccharifying enzyme of dry base B starch into each group. And (5) accurately timing the reaction for 60min, sampling, inactivating enzyme, diluting by a certain multiple, and determining the DE value by using a DNS method.

The results are shown in FIG. 1: when the addition amount of the saccharifying enzyme is 20-40U/g B starch (calculated on a dry basis), the degradation rate of the substrate is accelerated along with the increase of the enzyme amount of the saccharifying enzyme, and the DE value is gradually increased; when the amount of the saccharifying enzyme added reached 40U/g, the DE stabilized at about 55% because the substrate was saturated with the saccharifying enzyme added. Thereafter, even if the amount of the saccharifying enzyme is further increased, the reaction rate is not increased and the DE value is not increased.

Example 2: effect of pullulanase addition on glycation DE value

(1) Preparing feed liquid: the same as in example 1.

(2) Saccharifying feed liquid: and (2) cooling the feed liquid liquefied in the step (1) to 65 ℃, dividing the feed liquid into 7 groups, and adding 40U/g dry base B starch saccharifying enzymes into each group, wherein the adding amount of the pullulanase is 20, 25, 30, 35, 40, 45 and 50U/g dry base B starch, namely the adding ratio of the saccharifying enzymes to the pullulanase in the system is 2:1, 8:5, 4:3, 8:7, 1:1, 8:9 and 4:5 respectively. And (5) accurately timing the reaction for 60min, sampling, inactivating enzyme, diluting by a certain multiple, and determining the DE value by using a DNS method.

The results are shown in FIG. 2: when the addition amount of the pullulanase is 20-40U/g B starch (calculated on a dry basis), with the increase of the pullulanase enzyme amount, the degradation rate of a substrate is accelerated, and the DE value gradually increases to 76%; when the amount of pullulanase added reached 40U/g, the DE value remained at a stable level because the substrate was saturated with pullulanase added. Thereafter, even if the amount of the enzyme added is further increased, the reaction rate is not increased and the DE value is not increased.

Example 3: effect of saccharification temperature on saccharification DE value

(1) Preparing feed liquid: the same as in example 1.

(2) Saccharifying feed liquid: respectively cooling the liquid material liquefied in the step (1) to 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃, respectively adding 40U/g dry-base B starch complex enzyme, wherein the ratio of saccharifying enzyme to pullulanase in the complex enzyme is 1: 1. and (5) accurately timing the reaction for 60min, sampling, inactivating enzyme, diluting by a certain multiple, and determining the DE value by using a DNS method.

As shown in FIG. 3, when the saccharification temperature was 50 ℃ to 60 ℃, the DE value gradually increased with the increase in the saccharification temperature; when the saccharification temperature reaches 60 ℃, DE reaches a maximum of 76.24%; then, as the temperature is increased, the DE value decreases faster. The reason why the temperature rise promotes the DE value of the saccharification liquid to rise is probably that the effective collision frequency of enzyme molecules on a substrate in unit time is increased along with the continuous rise of the saccharification temperature, and the enzyme activity is also continuously increased; when the saccharification temperature exceeds 60 ℃, an excessively high temperature may change the active center of the enzyme as saccharification proceeds, thereby decreasing the enzyme activity and sharply decreasing the DE value. Thus, 60 ℃ is the optimum saccharification temperature.

Example 4: effect of saccharification pH on saccharification DE value

(1) Preparing feed liquid: the same as in example 1.

(2) Saccharifying feed liquid: reducing the temperature of the feed liquid liquefied in the step (1) to 60 ℃, respectively adjusting the pH to 5.0, 5.5, 6.0, 6.5 and 7.0, adding 40U/g dry base B starch compound enzyme, wherein the ratio of saccharifying enzyme to pullulanase in the compound enzyme is 1: 1. and (5) accurately timing the reaction for 60min, sampling, inactivating enzyme, diluting by a certain multiple, and determining the DE value by using a DNS method.

As shown in FIG. 4, the DE value of saccharification was at a higher level at a pH in the off-neutral range of 5.5 to 6.5 and reached a maximum of 76.89% at a pH of 6.0 under otherwise identical saccharification conditions. The activity of the enzyme is closely related to the pH, and the glycated DE drops dramatically below or above the optimum pH, probably because the pH changes the spatial conformation of the enzyme molecule. The enzyme protein can exert the activity to the maximum extent only under proper pH value. Therefore, the pH value of 6.0 is selected as the proper pH value for hydrolyzing the starch B by the complex enzyme preparation. The pH of the liquefaction reaction is between 6.0 and 6.5, so the compound enzyme preparation can exert good saccharification effect under the condition of not adjusting the pH of the liquefaction reaction liquid.

Example 5: effect of saccharification time on Complex enzyme saccharification

(1) Preparing feed liquid: the same as in example 1.

(2) Saccharifying feed liquid: and (2) reducing the temperature of the liquefied material liquid in the step (1) to 60 ℃, adding a complex enzyme of 40U/g dry base B starch, wherein the ratio of saccharifying enzyme to pullulanase in the complex enzyme is 1: 1. and (3) accurately timing the reactions for 30, 60, 90, 120, 150 and 180min respectively, sampling, inactivating the enzymes, diluting by a certain multiple, and determining the DE value by using a DNS method.

As shown in FIG. 5, the DE value gradually increased with the increase of the reaction time under a certain condition when the saccharification time was 30-120 min, and the DE value of the saccharified liquid reached 85.89% at 120 min; thereafter, the rate of decomposition of the feed liquid was reduced, and the DE value was stabilized at a saccharification level of 85%.

Example 6: effect of starch concentration on glycation DE value

(1) Preparing feed liquid: respectively preparing B starch reaction systems with different mass fractions (20%, 25%, 30%, 35%, 40%, 45% and 50%) by using 0.2mol/L, pH 6.0.0 sodium carbonate-sodium bicarbonate buffer solution, uniformly stirring, and liquefying to a certain DE value by using alpha-amylase.

(2) Saccharifying feed liquid: and (2) cooling the liquid material liquefied in the step (1) to 60 ℃, adding a composite enzyme of 30U/g dry base B starch, wherein the ratio of saccharifying enzyme to pullulanase in the composite enzyme is 1: 1. and (5) accurately timing the reaction for 60min, sampling, inactivating enzyme, diluting by a certain multiple, and determining the DE value by using a DNS method.

The results are shown in FIG. 6: when the mass fraction of the starch B is 20-35%, with the increase of the content of the starch B, the degradation of a substrate is accelerated, and the DE value is increased gradually; after 35%, the DE value remained at a steady level, about 91%, because the enzyme was saturated with the substrate, and even if the amount of the substrate was increased, the reaction rate did not increase and the DE value did not increase.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Industrial and technical research institute of south Jiangnan university in Suqian City

<120> heat stability pullulanase compound and starch raw material saccharification process thereof

<130> BAA210778A

<160> 4

<170> PatentIn version 3.3

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Val Glu Leu Lys Gln Ala Ile His Thr Leu His Glu Asn Gly Leu Arg

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ccgggcgtgg tgattgcgtc cccgtccaaa tccgatccgg attattttta tacctggacc 240

cgtgattccg gcctggtgat gaaaaccctg gtggatctgt ttcgtggcgg cgatgcggat 300

ctgctgccga ttattgaaga atttatttcc tcccaggcgc gtattcaggg catttccaat 360

ccgtccggcg cgctgtcctc cggcggcctg ggcgaaccga aatttaatgt ggatgaaacc 420

gcgtttaccg gcgcgtgggg ccgtccgcag cgtgatggcc cggcgctgcg tgcgaccgcg 480

atgatttcct ttggcgaatg gctggtggaa aatggccata cctccattgt gaccgatctg 540

gtgtggccgg tggtgcgtaa tgatctgtcc tatgtggcgc agtattggtc ccagtccggc 600

tttgatctgt gggaagaagt gcagggcacc tcctttttta ccgtggcggt gtcccatcgt 660

gcgctggtgg aaggctcctc ctttgcgaaa accgtgggct cctcctgccc gtattgcgat 720

tcccaggcgc cgcaggtgcg ttgctatctg cagtcctttt ggaccggctc ctatattcag 780

gcgaattttg gcggcggccg ttccggcaaa gatattaata ccgtgctggg ctccattcat 840

acctttgatc cgcaggcgac ctgcgatgat gcgacctttc agccgtgctc cgcgcgtgcg 900

ctggcgaatc ataaagtggt gaccgattcc tttcgttcca tttatgcgat taattccggc 960

cgtgcggaaa atcaggcggt ggcggtgggc cgttatccgg aagattccta ttataatggc 1020

aatccgtggt ttctgaccac cctggcggcg gcggaacagc tgtatgatgc gctgtatcag 1080

tgggataaaa ttggctccct ggcgattacc gatgtgtccc tgccgttttt taaagcgctg 1140

tattcctccg cggcgaccgg cacctatgcg tcctccacca ccgtgtataa agatattgtg 1200

tccgcggtga aagcgtatgc ggatggctat gtgcagattg tgcagaccta tgcggcgtcc 1260

accggctcca tggcggaaca gtataccaaa accgatggct cccagacctc cgcgcgtgat 1320

ctgacctggt cctatgcggc gctgctgacc gcgaataatc gtcgtgatgc ggtggtgccg 1380

gcgccgtggg gcgaaaccgc ggcgacctcc attccgtccg cgtgctccac cacctccgcg 1440

tccggcacct attcctccgt ggtgattacc tcctggccga ccatttccgg ctatccgggc 1500

gcgccggatt ccccgtgcca ggtgccgacc accgtgtccg tgacctttgc ggtgaaagcg 1560

accaccgtgt atggcgaatc cattaaaatt gtgggctccg tgtcccagct gggctcctgg 1620

aatccgtcct ccgcgaccgc gctgaatgcg gattcctata ccaccgataa tccgctgtgg 1680

accggcacca ttaatctgcc ggcgggccag tcctttgaat ataaatttat tcgtgtgcag 1740

aatggcgcgg tgacctggga atccgatccg aatcgtaaat ataccgtgcc gtccacctgc 1800

ggcgtgaaat ccgcggtgca gtccgatgtg tggcgttaa 1839

<210> 4

<211> 612

<212> PRT

<213> Artificial sequence

<400> 4

Met Val Ser Phe Ser Ser Cys Leu Arg Ala Leu Ala Leu Gly Ser Ser

1 5 10 15

Val Leu Ala Val Gln Pro Val Leu Arg Gln Ala Thr Gly Leu Asp Thr

20 25 30

Trp Leu Ser Thr Glu Ala Asn Phe Ser Arg Gln Ala Ile Leu Asn Asn

35 40 45

Ile Gly Ala Asp Gly Gln Ser Ala Gln Gly Ala Ser Pro Gly Val Val

50 55 60

Ile Ala Ser Pro Ser Lys Ser Asp Pro Asp Tyr Phe Tyr Thr Trp Thr

65 70 75 80

Arg Asp Ser Gly Leu Val Met Lys Thr Leu Val Asp Leu Phe Arg Gly

85 90 95

Gly Asp Ala Asp Leu Leu Pro Ile Ile Glu Glu Phe Ile Ser Ser Gln

100 105 110

Ala Arg Ile Gln Gly Ile Ser Asn Pro Ser Gly Ala Leu Ser Ser Gly

115 120 125

Gly Leu Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Phe Thr Gly

130 135 140

Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala

145 150 155 160

Met Ile Ser Phe Gly Glu Trp Leu Val Glu Asn Gly His Thr Ser Ile

165 170 175

Val Thr Asp Leu Val Trp Pro Val Val Arg Asn Asp Leu Ser Tyr Val

180 185 190

Ala Gln Tyr Trp Ser Gln Ser Gly Phe Asp Leu Trp Glu Glu Val Gln

195 200 205

Gly Thr Ser Phe Phe Thr Val Ala Val Ser His Arg Ala Leu Val Glu

210 215 220

Gly Ser Ser Phe Ala Lys Thr Val Gly Ser Ser Cys Pro Tyr Cys Asp

225 230 235 240

Ser Gln Ala Pro Gln Val Arg Cys Tyr Leu Gln Ser Phe Trp Thr Gly

245 250 255

Ser Tyr Ile Gln Ala Asn Phe Gly Gly Gly Arg Ser Gly Lys Asp Ile

260 265 270

Asn Thr Val Leu Gly Ser Ile His Thr Phe Asp Pro Gln Ala Thr Cys

275 280 285

Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu Ala Asn His

290 295 300

Lys Val Val Thr Asp Ser Phe Arg Ser Ile Tyr Ala Ile Asn Ser Gly

305 310 315 320

Arg Ala Glu Asn Gln Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Ser

325 330 335

Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Thr Thr Leu Ala Ala Ala Glu

340 345 350

Gln Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Ile Gly Ser Leu Ala

355 360 365

Ile Thr Asp Val Ser Leu Pro Phe Phe Lys Ala Leu Tyr Ser Ser Ala

370 375 380

Ala Thr Gly Thr Tyr Ala Ser Ser Thr Thr Val Tyr Lys Asp Ile Val

385 390 395 400

Ser Ala Val Lys Ala Tyr Ala Asp Gly Tyr Val Gln Ile Val Gln Thr

405 410 415

Tyr Ala Ala Ser Thr Gly Ser Met Ala Glu Gln Tyr Thr Lys Thr Asp

420 425 430

Gly Ser Gln Thr Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu

435 440 445

Leu Thr Ala Asn Asn Arg Arg Asp Ala Val Val Pro Ala Pro Trp Gly

450 455 460

Glu Thr Ala Ala Thr Ser Ile Pro Ser Ala Cys Ser Thr Thr Ser Ala

465 470 475 480

Ser Gly Thr Tyr Ser Ser Val Val Ile Thr Ser Trp Pro Thr Ile Ser

485 490 495

Gly Tyr Pro Gly Ala Pro Asp Ser Pro Cys Gln Val Pro Thr Thr Val

500 505 510

Ser Val Thr Phe Ala Val Lys Ala Thr Thr Val Tyr Gly Glu Ser Ile

515 520 525

Lys Ile Val Gly Ser Val Ser Gln Leu Gly Ser Trp Asn Pro Ser Ser

530 535 540

Ala Thr Ala Leu Asn Ala Asp Ser Tyr Thr Thr Asp Asn Pro Leu Trp

545 550 555 560

Thr Gly Thr Ile Asn Leu Pro Ala Gly Gln Ser Phe Glu Tyr Lys Phe

565 570 575

Ile Arg Val Gln Asn Gly Ala Val Thr Trp Glu Ser Asp Pro Asn Arg

580 585 590

Lys Tyr Thr Val Pro Ser Thr Cys Gly Val Lys Ser Ala Val Gln Ser

595 600 605

Asp Val Trp Arg

610