CN112266907B

CN112266907B - Sclerotium rolfsii endogenous sclerotium rolfsii hydrolase and application thereof

Info

Publication number: CN112266907B
Application number: CN202011172161.0A
Authority: CN
Inventors: 曾伟主; 周景文; 谭润卿; 陈坚; 堵国成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2022-03-15
Anticipated expiration: 2040-10-28
Also published as: CN112266907A

Abstract

The invention discloses an endogenous sclerotium rolfsii sclerotium gum hydrolase of sclerotium rolfsii and application, belonging to the technical field of enzyme engineering. The invention excavates 14 potential beta-glucanase genes from sclerotium rolfsii, performs heterologous expression on potential hydrolase genes in pichia pastoris, performs functional verification, and screens to obtain sclerotium rolfsii hydrolase with positive hydrolysis effect on sclerotium rolfsii and a coding gene thereof. The sclerotium rolfsii glue hydrolase can hydrolyze sclerotium rolfsii glue under the condition of pH 6.0, so that the number average molecular weight is 2.748 multiplied by 10⁷Da is reduced to 1.923 multiplied by 10⁷Da, overcoming the restriction of the three-dimensional structure of the sclerotium rolfsii glue and directly cutting glycosidic bonds.

Description

Sclerotium rolfsii endogenous sclerotium rolfsii hydrolase and application thereof

Technical Field

The invention relates to an endogenous sclerotium rolfsii glue hydrolase of sclerotium rolfsii and application thereof, belonging to the technical field of enzyme engineering.

Background

Sclerotium rolfsii gum (also known as Scleroglucan, Scleroglucan) is a non-ionic water-soluble microbial exopolysaccharide formed by alternately connecting beta-D-1, 3 glucopyranose and beta-D-1, 6 glucopyranose residues, and a main chain unit connected with 3 beta-1, 3 glycosidic bonds is connected with a side chain of 1 beta-1, 6 glycosidic bond on average. The repeated unit structure makes the branching degree of sclerotium rolfsii gum reach about 0.33. In addition to having the bioactivity characteristics common to most beta-1, 3 glucans, the high branching frequency makes it highly water soluble. Due to the unique chemical structure and high molecular weight characteristic, the sclerotium rolfsii gum has good water solubility, pseudoplasticity, moisture retention, salt resistance and viscosity stability, and is widely applied to the industries of food, biomedicine, petrochemical industry, cosmetics and the like.

Currently, sclerotium rolfsii gum is mainly produced in a microbial fermentation mode. Sclerotinia sclerotiorum rolfsii is the most important production strain, and can be produced by fermentation by using glucose, sucrose, fructose, xylose, molasses and the like. Research aiming at the production of sclerotium rolfsii gum mainly focuses on the aspects of improving the yield and the productivity of sclerotium rolfsii gum and the like. Sclerotium rolfsii gum produced by the sclerotium fungi fermentation method has higher molecular weight, so that the solubility, the fluidity and the dispersibility of the sclerotium rolfsii gum are poorer, and the difficulty is brought to the industrial application of the sclerotium rolfsii gum. The activity of sclerotium rolfsii gum is closely related to viscosity and molecular weight, when the molecular weight is too large, the water solubility is poor, and when the molecular weight is too low, the physiological activity of sclerotium rolfsii gum can be lost. How to select a proper degradation method to modify sclerotium rolfsii glue to obtain an ideal molecular weight and improve the water solubility of the sclerotium rolfsii glue on the basis of keeping the activity, so that the industrial application of the sclerotium rolfsii glue is expanded.

It has been shown that the beta-1, 3 glucanase genes BGLM, PGLA, EXG1 and BGLF from Bacillus circulans, Paenibacillus, Saccharomyces cerevisiae and Alkaliphilic nocardiopsis are expressed in colibacillus and Pichia pastoris through recombination, and there is almost no hydrolysis to sclerotium rolfsii after treatment with 4 enzyme crude enzyme solutions. In addition, the sclerotium rolfsii gum can form a rigid triple helix structure after being dissolved in water, glucose groups on side chains are uniformly distributed on the outer side of the helix structure, and helix aggregation is prevented through the action of electrostatic force, so that the polysaccharide structure can be stabilized, and the gelling capacity of the sclerotium rolfsii gum can be reduced. The triple helix structure is very stable to heat and the sclerotium rolfsii gum can be dissolved to form a random coil single-chain structure only when dispersed in dimethyl sulfoxide (DMSO) or at a pH value of more than 12.5. The three-dimensional structure of the sclerotium rolfsii gum enables a glucoside connecting bond to be wrapped, the combination and hydrolysis efficiency of glucan hydrolase is greatly influenced, and early researches find that the conventional beta-glucanase can not play a role in hydrolyzing the sclerotium rolfsii gum so as to improve the molecular weight of the sclerotium rolfsii gum. Therefore, the screening of the hydrolase with the sclerotium rolfsii gum degradation capability has important significance for preparing the sclerotium rolfsii gum with low molecular weight.

Disclosure of Invention

The preservation number is CCTCC NO: the sclerotium rolfsii WSH-G01 of M2017646 is screened to obtain sclerotium rolfsii hydrolase with sclerotium rolfsii hydrolysis capability and a gene for coding the hydrolase, and a research basis is provided for large-scale production of small molecular weight sclerotium rolfsii.

The first purpose of the invention is to provide a sclerotium rolfsii hydrolase, which contains an amino acid sequence shown in SEQ ID NO. 1.

It is a second object of the present invention to provide a gene encoding the sclerotium rolfsii hydrolase.

In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The third purpose of the invention is to provide an expression vector carrying the gene.

In one embodiment, the expression vector is pPIC 9K.

It is a fourth object of the present invention to provide a recombinant microbial cell expressing said sclerotium rolfsase.

In one embodiment, the microorganism is pichia pastoris.

In one embodiment, the pichia is pichia GS 115.

The invention also claims the application of the sclerotium rolfsii hydrolase in the aspect of preparing the low molecular weight sclerotium rolfsii.

The invention also provides a method for hydrolyzing sclerotium rolfsii gum, which is to add the sclerotium rolfsii gum hydrolase into a system containing sclerotium rolfsii gum and react for at least 2 hours at 28-30 ℃.

The invention also provides an enzyme preparation containing the sclerotium rolfsii hydrolase.

The invention also provides application of the sclerotium rolfsii hydrolase in the fields of food, biomedicine, petrochemical industry and cosmetics.

In one embodiment, the use includes, but is not limited to, the preparation of polysaccharides, pharmaceuticals, or daily chemical products.

Has the advantages that: the invention excavates 14 potential beta-glucanase genes from sclerotium rolfsii, performs heterologous expression on 10 potential hydrolase genes which are successfully amplified in pichia pastoris, performs functional verification, and screens to obtain the sclerotium rolfsii hydrolase which has a positive hydrolysis effect on sclerotium rolfsii and a coding gene thereof. The beta-1, 3-glucanase expressed by the sclerotium rolfse in pichia pastoris has the enzyme activity of 8.64U/mL, and can carry out forward hydrolysis on sclerotium rolfse to ensure that the number average molecular weight is 2.748 multiplied by 10⁷Da is reduced to 1.923 multiplied by 10⁷Da shows that the enzyme can overcome the restriction of the three-dimensional structure of sclerotium rolfsii gum and directly cut glycosidic bonds under an acidic condition (about pH 6).

Drawings

FIG. 1 phylogenetic tree analysis of Sclerotinia sclerotiorum WSH-G01;

FIG. 2 extracellular enzyme activity in two enzyme-producing medium systems;

FIG. 3 differential gene GO enrichment and KEGG database enrichment;

FIG. 4 is a SDS-PAGE gel of the target polysaccharide hydrolase; 1, pPIC 9K; 2, GME9629_ g; 3, GME9263_ g; 4, GME2879_ g; 5, GME10818_ g; 6, GME8597_ g; 7, GME7035_ g; 8, GME9860_ g; 9, GME3016_ g; 10, GME9076_ g; 11, GME2686_ g;

FIG. 5 Effect of endogenous beta-1, 3-glucanase on Sclerotinia sclerotiorum molecular weight; 1, GME 7035; 2, GME 2686; 3, GME 9076; 4, GME 9263; 5, GME 9860; 6,2g/L sclerotium rolfsii gum.

Detailed Description

(1) Bacterial strains and plasmids

The sclerotium rolfsii WSH-G01 is obtained by screening a subject group to which the inventor belongs, is currently preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university with the preservation number of CCTCC NO: m2017646 and disclosed in patent publication No. CN 108441429B.

Escherichia coli JM109 is a commercial strain used for replication of plasmids for expression of recombinant enzymes.

Pichia pastoris GS115 is a commercial strain, is used for expression and fermentation of recombinase,

plasmid pPIC9K is a commercial plasmid used for secretory expression of proteins in pichia pastoris.

(2) Culture medium

Sclerotium rolfsii seed culture medium (g/L): glucose 30.0, KH₂PO₄ 1.0，NaNO₃3.0, yeast powder 1.0, KCl 0.5, MgSO₄·7H₂O 0.5，pH 4.0。

Sclerotium rolfsii fermentation medium (g/L): glucose 55.0, KH₂PO₄ 1.0，NaNO₃3.0, yeast powder 0.5, KCl 0.5, MgSO₄·7H₂O0.5, citric acid 1.5, pH 4.0.

Pichia pastoris MD medium (1L): 20g of agar, 100mL of 10 XYNB solution (final YNB solution concentration of 13.4g/L), 100mL of 10 Xglucose solution (final glucose concentration of 20g/L), and 2mL of 500 Xbiotin (final biotin concentration of 4X 10)^-4g/L)。

Pichia pastoris induced expression BMGY medium (1L): yeast extract 10g, peptone 20g, K₂HPO₄ 3g，KH₂PO₄11.8g, 100mL of 10 XYNB solution (final concentration of YNB solution 13.4g/L), and 1mL of 500 XHioin (final concentration of biotin 4X 10)^-4g/L), 10mL of glycerol.

Pichia pastoris induced expression BMMY medium (1L): yeast extract 10g, peptone 20g, K₂HPO₄ 3g，KH₂PO₄11.8g, 100mL of 100 XYNB solution (final concentration of YNB solution 13.4g/L), and 1mL of 500 XBiotin (final concentration of biotin 4X 10)^-4g/L), 5mL of methanol.

(3) Primary reagent

The column type RNA rapid extraction kit, the column type plasmid small quantity extraction kit, the PCR product recovery kit, the T4 DNA ligase, all restriction enzymes, kanamycin antibiotics, ampicillin antibiotics, G418 sulfate and the like are purchased from the company of Biotechnology engineering (Shanghai). Other chemical reagents were purchased from Shanghai national drug group.

(4) Detection method

Detecting the enzyme activity of the crude enzyme liquid of the beta-glucanase: measuring a certain amount of fermentation liquor, centrifuging at 4 ℃ at 4000 r/min for 10min, and collecting supernatant, namely crude enzyme liquid. 1mL of reaction solution containing 500. mu.L of crude enzyme solution, 500. mu.L of 0.5% laminarin and 20mmol of HAc-NaAc buffer solution (pH 6.0), water-bathing at 30 ℃ for 30min, adding 1mL of DNS solution to terminate the reaction, heating at 100 ℃ for 5min, cooling to room temperature, collecting supernatant, and measuring OD₅₄₀The enzyme activity was calculated using the inactivated enzyme as a control. 1 enzyme activity unit is defined as the amount of enzyme required to release 1.0. mu. mol reducing sugar (based on glucose) per minute under the reaction conditions.

The dry weight of the sclerotium rolfsii and the extraction and yield of crude polysaccharide are determined in Liu L, Zang YJ, Xu CZ, et al.A modified CTAB method for isolating genomic DNA from funcus with uncovered polysaccharose, Chin Biotechnol,2014,34(5): 75-79.

Detection of recombinant protein SDS-PAGE: centrifuging 1mL recombinant protein fermentation broth at 12000 r/min for 5min, removing supernatant, adding appropriate amount of distilled water to make the bacterial liquid OD₆₀₀About 5.0, carrying out ultrasonic disruption, mixing 30 mu L of protein disruption solution with 10 mu L of loading buffer solution, and heating at 99 ℃ for 15 min. 10% Bis-Tris Protein Gels was electrophoresed in MES buffer at 120V.

Example 1 analysis of polysaccharide degrading enzyme Gene in Sclerotinia sclerotiorum WSH-G01

1. Sclerotium rolfsii culture

Picking single sclerotium of sclerotium rolfsii WSH-G01, inoculating on PDA plate, standing at 30 deg.C for 4 days until rich hypha grows out. Scraping 1cm²Inoculating the hypha into a 250mL shake flask filled with 50mL sclerotinia sclerotiorum seed culture medium, and culturing for 72h under the conditions of 28 ℃ and 220r/min to obtain a first-stage seed solution. Transferring the primary seed solution with the inoculation amount of 5% into a 500mL shake flask filled with 100mL seed culture medium, and culturing at 30 ℃ and 220r/min for 72h to obtain a secondary seed solution. Inoculating the secondary seed liquid into a fermentation medium in an inoculation amount of 5% for fermentation.

2. Extraction of sclerotium rolfsii genome

Inoculating the strain to potato glucose agar (PDA) culture medium, activating for 4d, scraping fresh hyphae, transferring to PDA culture medium covered with glass paper, and scraping fresh hyphae on the glass paper for DNA extraction after 5 d. Since sclerotium rolfsii produces a large amount of extracellular polysaccharide and DNA extraction is difficult, the experiment refers to a Cetyltrimethylammonium bromide (CTAB) method improved by Liuli and the like for DNA extraction.

3. Sclerotium rolfsii gene analysis

Selecting the concentration range obtained by extraction in the step 2 to be between 50 and 150 mu g/mu L, and OD₂₆₀/OD₂₈₀DNA samples ranging between 1.8-2.0 were subjected to genome sequencing. The PacBio platform is used for whole genome sequencing, genome splicing is carried out by combining the sequencing result of the Illumina platform, and the sclerotinia rolfsii WSH-G01 genome with the size of 38Mb is obtained, wherein the genome comprises 116 Contigs and 42 Scaffolds, and the GC content is 46.51%. Based on the obtained whole genome sequence, the near-source relationship between the sclerotinia rolfsii WSH-G01 and other typical white rot fungi is analyzed according to NCBI retrieval and data processing (figure 1).

Comparing with typical white rot fungus with polysaccharide degradation activity, it is found that the sclerotium rolfsii WSH-G01 is close to Schizophyllum commune and Coprinopsis cinerea okayama relatives, and the exopolysaccharide produced by Schizophyllum commune is highly similar to sclerotium rolfsii gum in structure. After functional annotation of the genes associated with the glucan degrading enzyme of strain WSH-G01, it was found that 200 genes associated with the glucan degrading enzyme were possessed, including 68 genes of the Carbohydrate-binding-domain family, 14 genes of Carbohydrate esterase (Carbohydrate esterase), 82 genes of Glycoside hydrolase (Glycoside hydrolase), 30 genes of Glycoside transferase (Glycoside transferase), and 6 genes of Glycoside lyase (Polysaccharide lyase) (table 1). The result shows that the sclerotinia rolfsii WSH-G01 has the degradation potential of sclerotium rolfsii glue.

TABLE 1 sclerotium rolfsii WSH-G01 polysaccharide degrading enzyme gene distribution

Example 2 enzyme Activity analysis of Sclerotinia sclerotiorum WSH-G01 under different enzyme production systems

Two different culture systems of sclerotium rolfsii shown in Table 2 were designed, and the seed solution of sclerotium rolfsii WSH-G01 cultured according to step 1 of example 1 was inoculated with 5% of inoculum size into a high-yield enzyme system and a low-yield enzyme system, respectively, and cultured under the same environment (30 ℃).

TABLE 2 cultivation systems for different enzymes

The activity of the beta-glucanase in the fermentation process is detected, on the premise of ensuring that the biomass in different culture systems is the same, when the culture is carried out for 3 days, the enzyme activity difference of extracellular beta-glucan hydrolase is obvious, the enzyme activity is hardly detected in a low-yield enzyme culture system, and the enzyme activity of the beta-glucanase in a high-yield culture system reaches 8.62U/mL (figure 2). The method shows that in a high-yield enzyme culture system, the thalli is easy to induce and express polysaccharide hydrolase when polysaccharide is used as a sole carbon source so as to obtain enough energy substances. The design of two culture systems with obviously different enzyme production activities provides a theoretical basis for the excavation of the sclerotinia sclerotiorum glue hydrolase gene.

Example 3 transcriptome sequencing and analysis of Sclerotinia sclerotiorum WSH-G01 in two enzyme production systems

High enzyme activity (HE) and low enzyme activity (LE) samples with obvious enzyme production difference when cultured for 3d in the embodiment 2 are selected, total RNA is extracted respectively, and an original cDNA library is constructed.

Extracting RNA of sclerotium rolfsii: the extraction is carried out according to the extraction method of the column type RNA extraction kit of the biological engineering (Shanghai) corporation.

Construction of a sclerotium rolfsii cDNA library: the reverse transcription operation was performed by using a high-quality sclerotium rolfsii WSH-G01 RNA as a template and referring to the method of a reverse transcription kit of TaKaRa Co.

Sequencing and optimizing the data, and performing sequence analysis by taking the obtained genome as a reference, wherein the alignment rate of the HE sample and the LE sample respectively reaches 69.28% and 70.95%. Finally, 2584 genes (P) with significant differences are screened from 9086 genes by comparing the gene expression level differences of HE and LE samples<0.01&|log₂FC | ≧ 1), wherein 1054 of the expression genes are significantly up-regulated and 1530 of the expression genes are significantly down-regulated.

Functional annotation is carried out on the obtained 2584 significant difference genes by GO enrichment, and 32 GO enriched entries are obtained. As can be seen from fig. 3A, the genes with the most significant enrichment difference have molecular activity functions, and the two types of genes with the highest enrichment ratio have molecular catalytic functions and molecular binding functions. The glucan hydrolase generally has the catalytic activity of glycosidic bond hydrolysis and the substrate binding activity simultaneously in structure, and the significant differential enrichment of the two genes indicates that the sclerotium rolfse genes with differential expression are in samples of two different culture systems. Analysis using KEGG revealed that the differential genes were enriched to 130 Pathway of KEGG, with 21 being significantly enriched, with the most significantly enriched metabolic Pathway being the carbohydrate metabolic Pathway (fig. 3B). The change of the carbon source metabolic pathway of the sclerotinia rolfsii WSH-G01 in different culture systems is probably caused by the fact that hyphae in an HE sample produce more sclerotium rolfsase.

TABLE 3 endogenous Sclerotinia sclerotiorum glue hydrolase genes predicted in Sclerotinia sclerotiorum WSH-G01

Note: FPKM, Fragments Per Kilobase Per Million.

Based on the sequencing result of WSH-G01 transcriptome of sclerotium rolfsii, gene annotation of genome databases and transcriptome difference analysis under two enzyme-producing conditions are compared, and 14 suspected sclerotium rolfsii heteroside hydrolase genes including 1 exoglucanase (Exo-glucanase) gene and 13 endoglucanase (Endo-glucanase) genes are excavated from 2584 genes with significant difference obtained by analyzing in transcriptome data (Table 3).

Example 4 recombinant expression of Sclerotinia sclerotiorum hydrolase in Pichia pastoris

Extracting total RNA of strains in a high-yield enzyme culture system, and constructing a cDNA library by using reverse transcriptase. According to the CDS sequences of 14 hydrolases obtained through prediction, primers are designed to amplify gene fragments of 14 hydrolases, and 10 gene fragments (shown as SQE ID No. 2-11 respectively) are successfully amplified. The 10 obtained fragment DNAs are homologously recombined to a pichia pastoris expression vector pPIC9K, and the pichia pastoris GS115 is transformed by an electrical transformation method after the recombination vector is linearized.

Transformation of pichia pastoris and transformant screening: the pichia pastoris expression vector pPIC9K containing the target polysaccharide hydrolase gene is linearized, transformed into pichia pastoris GS115 competent cells in an electrotransformation mode, screened on an MD plate, and cultured for 3d at 30 ℃. Selecting YPD plate with well-grown single colony up to 2mg/mL G418, screening for His resistant to high concentration G418⁺Transformants, further Mut⁺After type selection, positive transformants were verified by PCR.

The positive transformants were selected and inoculated into a tube containing 2mL YPD medium, cultured at 30 ℃ and 220r/min for 24 hours, transferred at 2% inoculum size to a 250mL shake flask containing 50mL BMGY medium, and cultured at 30 ℃ and 220r/min to OD₆₀₀Centrifuging to collect thallus, washing with normal saline twice, inoculating into 250mL shake flask containing 50mL BMMY culture medium, culturing at 30 deg.C and 220r/min, adding methanol to the culture medium every 24 hr to final concentration of 0.5%, sampling, centrifugingRespectively collecting supernatant and thallus, analyzing the expression amount of target protein and optimal protein harvesting time, and detecting and identifying the expression of recombinant protein by SDS-PAGE and activity experiment.

As a result, as shown in FIG. 4, a total of 10 recombinant strains having an expression ability were obtained. All the extracellular expression proteins were subjected to the measurement of the hydrolase activity using laminarin as a substrate, and it was found that 5 of 10 soluble expressed recombinant enzymes had the laminarin hydrolase activity and were each a β -1, 3-glucanase, and the amino acid sequences were obtained by translation from the gene sequences thereof, respectively, as shown in table 4.

TABLE 4 potential endogenous Sclerotinia sclerotiorum hydrolase Activity on laminarin hydrolysis

N.a means no enzyme was detected.

Example 5 hydrolysis of Sclerotinia sclerotiorum Gum by endogenous hydrolase

The 5 endogenous glucanases with laminarin hydrolytic activity prepared in example 4 were used to hydrolyze sclerotium rolfsii, the specific steps were: 1mL of the reaction solution contains 500. mu.L of crude enzyme solution and 500. mu.L of sclerotium rolfsii gum, the initial pH of the reaction system is 6, the reaction system is inactivated in a water bath at 30 ℃ for 6h and in a boiling water bath at 100 ℃ for 5min, and the molecular weight of the polysaccharide is determined by using high performance liquid chromatography (HPGPC) with the inactivated enzyme as a control. Detecting by using Shodex SB-806M HQ gel chromatographic column under the following detection conditions: differential detector, mobile phase 0.1mol/L NaNO₃The flow rate of the solution is 0.6mL/min, the column temperature is 40 ℃, and the sample injection amount is 50 mu L.

The results are shown in FIG. 5. After the sclerotium rolfsii gum is hydrolyzed by hydrolase GME9860, the average molecular weight of the sclerotium rolfsii gum is reduced to a certain extent, the time of peak emergence is delayed from 10.382min to 11.036min, and the number average molecular weight is 2.748 × 10⁷Da is reduced to 1.923 multiplied by 10⁷Da, and the other 4 pairs of hydrolases (shown in SEQ ID NO. 12-15) are smallThe sclerotium gum has no degradation effect. In addition, the dispersity of sclerotium rolfsii gum after enzyme hydrolysis of GME9860 shown in SEQ ID No.1 is higher than that before treatment, which shows that degradation of high molecular weight polysaccharide and generation of part of low molecular weight polysaccharide occur in an enzyme reaction system, so that the molecular weight distribution of polysaccharide becomes wider.

Example 6 preparation of enzyme preparation by endogenous hydrolase of sclerotium rolfsii

Collecting the sclerotium rolfsii hydrolase GME9860 expressed in the example 4, concentrating, mixing the concentrated enzyme solution with auxiliary materials and/or protective agents, and then carrying out spray drying to obtain the solid enzyme preparation of the sclerotium rolfsii hydrolase.

The auxiliary materials and/or the protective agents are as follows: one or more of maltodextrin, water-soluble starch, sucrose, trehalose or sorbitol, and the addition amount of the concentrated enzyme solution is 100-300 g/L.

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> university of south of the Yangtze river

<120> sclerotium rolfsii endogenous sclerotium rolfsii hydrolase and application

<160> 15

<170> PatentIn version 3.3

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<213> Sclerotium rolfsii WSH-G01

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

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Ser Pro Gly Ala Ser Gly Pro Val Arg Ser Cys Asn Ala Asn Asn Gly

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Pro Ala Ala Ser Gly Ala Gln Asn Ala Cys Phe Ala Ser Gly Gly Asn

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Leu Ala Phe Ser Cys Ser Asp Tyr Gln Pro Ile Ile Ile Ser Asn Thr

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Leu Ser Tyr Gly Phe Ala Gly His Gly Asp Thr Ala Thr Asp Ser Cys

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Ser Met Ile Val Gln Ala Ile Asn Ala Gly Gly Ile Thr Ala Thr Asp

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Phe Asp Ile Tyr Thr Pro Gly Gly Gly Val Gly Asp Tyr Asn Ala Cys

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Thr Ala Gln Tyr Gly Ala Pro Ser Gln Gly Trp Gly Ala Gln Tyr Gly

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Gly Val Ser Ser Asp Ser Gln Cys Asp Glu Leu Pro Ser Val Leu Gln

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Ile Ser Gly Cys Tyr Pro Ala Ser Ile

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ggtggctgca cgagctcgac gcaatccatc gtgctcgacg gcaactggcg ctggacacac 180

gaaaccgacg gttatactaa ctgctacacc ggcaatgcat gggacaccag catttgcacg 240

agccccactg tctgcgctga ggattgtgct ctcgacggtg ccgcctatga gagcacctat 300

ggtatcacta cgagcggcga tagcttgaag ctcgacttcg tcactggaag caacgttggc 360

tctcgtgttt acctcatgaa cgaggccaat acagaatatc aaatgttcaa gctccttaac 420

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gctcaccctt gcactgtcac tgagcagact gagtgcgaca gctatgtctc cggcgagtgc 780

ggagctctca gcggaagcgc ccgttactcc ggttactgtg acaaggacgg ttgcgattac 840

aacacctggc gattgggcaa tgagaccttc ttcggtcctg gtcttactgt tgacactaac 900

agcgttgtga ctgttgtcac ccagttcatc actgatgacg gtacatcctc tggcacgctc 960

tctgagatcc gccgcatcta cgtccaggac ggcaaggtga tccagaacgc aaacactaac 1020

cagcctaaca ttcccaccac caactctatc tctacggact tctgcacggc tgagaagacc 1080

gagttcggcg acaccaacta cttccagacc gttggcggtc tccctcaatt gggcaaggct 1140

ttgggcgatg gtgttgtcct cgttatgtct atctgggatg atcatcaggc cgatatgctc 1200

tggctcgatt ctcttgaccc ctctactggc agtgcctcta cccctggtgt ctctcgtggc 1260

ccttgctcga ccacctctgg cgtccccgct accgtcgaag gcaactcgcc caacgattac 1320

gttgtcttct ccaacatccg ctggggtgac ctcgactcca cctactcttc tggatcttct 1380

ggttccactg gctcctccag tacctctgtc gccgccacct ctacggcttc caagaccgcc 1440

acctcgacta gcgccgccgc gacatccacg tctacctcta cggctgtcgc tgagtacggc 1500

cagtgcggtg gagagaactg gaccggctcc acgacctgcg ccagtggtct tacctgcgtg 1560

gaggtgaacg cctactactc ccaatgccag tcagtgtag 1599

<210> 4

<211> 1584

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 4

atgtcttctg taccagaacc cgagatcaag gataaacttc cttctgattt tatctggggc 60

tttgccactg cctcattcca aattgaaggg tctaccgaca gagatggtag agggccctcc 120

atctgggacg aattttcgcg gactcccggg aagattcaag atgggcgtaa tggagatgta 180

gctactgata gttataaccg ttacaaggag gacatccagc tactcaagga ttatggcgtt 240

aagtcttatc gtttctccat ctcgtggtca cgtatcatcc cgcttggtgg ccgcaatgat 300

cctatcaacg aagctggaat caagttctat tccgacttga ttgatggtct gctcgaagcc 360

gaaatcattc catttgtcac tctttatcac tgggaccttc cccaaggatt gcatgataga 420

tatggtggat ggcttaacaa agacgaaatt gcagcagatt atgagaatta cgctcgcgtc 480

tgcttcaaag ccttcggcga ccgcgtaaaa tactggctca ccatgaatga gccatggtgc 540

atttcaattc ttggctatgg ccgtggcgtc tttgctcccg gacggtcgtc tgaccgcgag 600

cgctctgccg aaggtaacag cagcacagag ccctggatcg tcggccacag cgtgcttctt 660

gcccatgcac gagcggtcgc tgcctatagg aaggacttca aacccacaca gaagggtgtc 720

attggtatca cgcttaacgg agatatggcc ttgccttgga atgatgagca aaagaacatc 780

gatgctgctc agcatgctct cgattacgca atcggatggt ttgctgaccc catttatctg 840

ggccattacc ccgagtatat gcgctcaatt ctcggtgatc gcctaccaga atttactccc 900

gaagaatggg ccgttgtcaa gggctcgagt gacttctatg ggatgaacac gtatacaaca 960

aatctctgca aggccggtgg cgatgacgag ttccagggct ttgtcgaata caccttcact 1020

cgtcccgatg gcactcagct tggcacacaa gcgcactgcg cctggttgca ggactatgcc 1080

cccggattca ggatgctctt gaattacctt tggaagaaat ataaacaccc catctacgtc 1140

accgaaaatg gattcgccgt ccgcaacgag gatgccatgc ctcgcgatca ggctattcat 1200

gacgccgacc gagtggccta cttcaagggt acgaccaagg ccctctggga ggctgtgaaa 1260

ttggatggcg tcgaagtcaa ggcatatttc ccgtggagct tcttggacaa tttcgagtgg 1320

gctgatggct acgttactcg cttcggcgta acatatgtag actatgaaac ccaggagcgc 1380

ttcccgaagg acagtggcaa gttcttggta gactggcaca aggcttacgt tgccaaagag 1440

tcagaggcgt catcatcatc atcatcgaag acctcggcaa aaccggcggg ggccaagaca 1500

gcaaacaagc gcaacagcat cccctttacc actcctcttg ccccgctgtg gaaaaagctt 1560

ttctcgttga aggccaaggc gtga 1584

<210> 5

<211> 1926

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 5

atggtctctc tgcttttcac gtatgcggct cttgccagca cttggctttt tggatctgtt 60

ctcggagcga cgaccgtaac tatcagttcg agtgcgagtc attcaattcc tacgacgtta 120

tgggggcaga tgtttgagag tggcgacgga gggctttatg gcgagctcct acagaacaga 180

gctttccagc aggtgactcc gggcagcggc gcagcgctga acgcctggtc tgcggtcaac 240

agctccagca tctctgtggt gtcagatgct gtttctagtg ccctaccaaa cgcgcttcaa 300

gtctccgtgc ctagcggtca cactggttat tccgggtttg ccaacagcgg ctactggggt 360

atcaaggtca attcagcctg gacatacacc gcctctttct actaccgctt cccgtcatac 420

tcaacctggt ccggatccgc cgtggttgct ctgacaggca gtagcggcac cgtctacggc 480

tcggccgtgg tgaacctgca aggcagccag acctcatgga aacaggtcac ggtcaccttc 540

acaccccaca cgtcggcgag ctcaacaagc aactcattca cggtctcact cctcaatgcg 600

ggtggccaga cggttcactt tgctatgttt tctttgttcc caccgaccta caacaatagg 660

gcaaatggaa tgcgaatcga tatcgcagag acgttggcgg ctgctaagcc gactttcttc 720

aggttcccag gtggaaacaa cttgggccag actcctgcga cgaggtggca gtggaatgca 780

acggtcggcc cccttgttaa ccgtcctggt cgtgccggtg attggggtta tgttaacaca 840

gacggacttg gactccttga atacttgtac ttcatcgagg atgcgggcat ggaacccatc 900

atggcagttt attcgggcta ttcactagga gggaccagca tcgcccagaa tgatctcggc 960

ccatacgttc aacaagccat tgatcaaatc aatttcgtca ttggagaccc cagtcaaagc 1020

tctgctgccg cattaagagc ttctcttgga cgttctgaac cattcagctt aacatatgtg 1080

gagattggca atgaagattt tgccgaccag tcaacctatg tttataggtg gcaaacaatt 1140

gtcgatgcat tatctagcga gttcccaaat ctcaaattta ttgctacctc ctactcgacg 1200

ggacctaccc ttacgcccaa acccctgcag tgggataacc atgtgtatca atcacctaca 1260

tggtttgctc agaatgtttt cctgtatgac agctatgcga gaaacggcgt tacatatttc 1320

caaggagaat atgcagccat gcaagtcgcc tctaccaata cagctaacgt ctggggttcc 1380

actggtcgcc tttcttatcc cactattcag agcagtgttt ccgaggccgc ttatatgact 1440

ggtctcgaga gaaactctga catagtgttc gcggcttctt atgcgccttt gctcatgaac 1500

accaactatc agtcctggac acccaacatg gtcaatttcg atgctggcaa cgtctacccg 1560

tcgacatctt actacgttca acaactcttc ggagaatatc gaggagacgt tttccttccc 1620

agcactctcc catcttcttc aggaacatta ttctggagcg tcgtgaggaa ccagtcttcc 1680

aaccaggtct acattaagat tgccaacgtc ggatcctcca ccagcacctt gaactttgtt 1740

ctgccatact cgactgtttc ttctactggc acggctgtcg tcctaacagg gtctgagacc 1800

gcctcgaaca cgcccagcaa cccgaatgca gctgttccga gcacctcgac gatctccacc 1860

gggaagtctt tcagctacaa cagtcccgcg tggtctttca gcgtattggt tgtgaccgca 1920

tactag 1926

<210> 6

<211> 1653

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 6

atgcctcgaa ctagtttcat attactgtgg gcatctatcc taggtttgtc tgggcaaggt 60

ttggggtgga ccggaggaac ccgtctggtt ctccagcccg gtgcaacatc cacaccggtt 120

cagcttcgct cagatttcct atcttttagc atagaacccg cctactgggt cgaatttttt 180

gggacggcca gcgagccaaa ttcattcagc ttgagactgc ttgagtacat tgctaatcgc 240

actgggaccc cacctatcat ccggccagga ggaatcacga tggactccat gatttttgag 300

tcaaatggaa cagatcctac tagagtcagc tcaccgaatg gcggtgttta tgaaactatc 360

tacggtccag cctggttcaa atcgtttgat aatttcccaa atggcactcg ctttgttggt 420

actcttaatt tcgggaacaa ctcgcttgag attgcaaaca acgaagcact ggcttgggct 480

cagtatgtcg gttctgagaa gatttttgtt tttgagttgg gaaacgagcc tacaaattat 540

gcacgatgga cacaagacag tcctcttggc accttggaat acgtggacga gtggacaaac 600

tggaccaaag ccatcgacaa caatttgcct gcaggacaag ttccagcgga accccgctgg 660

tggggttcta gcgcaacaac tgataccggc tcagggacaa cagatgtcct tcctgcggcc 720

atcatacctc tgggaattga caatagcagc aacatcctgg aatattctat tcacagttat 780

gcatggtcca catgtgatcc cactaggaat gcgcttgcga ctatcccaaa cctgctaaat 840

catagcagta ttgttgcata tgcgaacacg ttcatgagac cgagcgagac ggtggcactg 900

gcgtcaggat ctggcatctg tatcggcgag ttcaatagtg tcagctgcag cggcaagccc 960

aacgtcacag acactttcac ccaagcactt tggactgtag acacttcttt taactatgcc 1020

gccatcaacg cttcacatgt ctttctccat caaggggcaa ctttagtctt ccaatctggc 1080

agccagagca ataccgcagg ggcagacggc actcctggct ggagcgcata tgatttatgg 1140

taccctgtca atagtactct tcgcggacct caacgggtca atccgggcta cgtttctcag 1200

ctactaatgg ccgaagccat cggtgatagt ggcaatagcc atttggctac cctggctact 1260

cctcaaggga tctctaacga ttcattttca gcgtacgcca tatatgacaa ttctgccacc 1320

tctaattcaa cggatcccgc tcgtctggtt cttttgaata tgtctccata tctacttaat 1380

gtgacctcac ccgcaccgcg tcaatatgtg acgatcgata tatcagcttt tgctggtgca 1440

catggcgcaa cactcaagag gatgacagct cctagtgccg atgaattgaa ttctacctta 1500

gttacgtggg caggacagag ctggggaact ggggaacctc aaggtgatgt acatgtggag 1560

gatgttcatg gaggtcaagt cgtcattcaa caatcggagg ccgtgcttgt tttcctctct 1620

cgtgaccctt cagaaaaata tcgtcaatat tag 1653

<210> 7

<211> 2280

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 7

atggggcttg ggtcgtcctg ctcggcggaa gtaaactctg gcactgctgc tgccagtgac 60

cccttctggt tgcagaatat taaacaccag ggaacgtctg cattcaactc caacccctcc 120

tcttataccg tcttcagaaa cgtcaaggac tatggtgcca agggtgatgg tgtaaccgac 180

gatacagctg cgatcaactc tgccatctct tcaggcagcc gctgcggtgg cggaacatgc 240

ggttcttcta ccgtgacccc tgctgttgtc tttttcccgc aaggcaccta cgtcgtctca 300

tcaccaatca ttgcttatta ctttactcaa ctcattggtg acgcaagaca tcctcccacc 360

atcaaagctt cttccagctt ctctggtatt gctgtcatag atgctgatcc atacatggcc 420

ggtggtgcca attggtacac aaatcaaaac aatttcttcc gttctgtgcg caatttcgtc 480

atcgatctta ccgccgtgcc tgcctcggcc tctgccactg ggttacattg gcaggtttcg 540

caggcgacct cgttaatcaa catcgtcgtc aacatgtcca ctgcatcagg aaacaaccac 600

caaggcatct tcatggaaaa cggaagcggt ggatacatgg gcgacatcgt ctttaatggt 660

ggcaaatacg gtgtttggct tggcaatcag caattcactg tccgcaacat caccgtcaac 720

aacgcagcca cagctatctt tgctgactgg aattggggct ggactttcca gggcgttacc 780

attagcaact gtcaagtcgg attcgatata ttgactggcg ggactaccca ggccaatcaa 840

ggtgtcggag cggaggccat catcgacgct gtcgttacaa acacccctat ctttgttcgc 900

agctcgaaag cgtccagcgg atcagtaaca gggtctctcg taatcaacaa tgcgaagctg 960

aacaacgttc ccaccgcagt tggcgttgtc ggcggtgctg tcgttctctc tggtactact 1020

ggaacaacga cgatctcctc ctggggccag ggcaacatct acaagggcac atctacctct 1080

ggaacgttca ctcaaggctc gattgctgct cctacaaaag ccagctccct cctagacagt 1140

gcgggacgca tcgtgcagaa gacccaccct cagtactctg actgggcggt atcacaattc 1200

gtgagcgtca agagcaacgg tgccaagggt gacggatcca cagatgacac cgcggctttg 1260

caagccatct tcaaccagta tgccggatgc aagatcatct tcttcgatgc cggaacttac 1320

attgtcactt caacactcaa aatcccggct ggcgtccgaa tcgttggaga ggcctggtcc 1380

gttatcgccg gcaaaggctc cgctttccag aatatcaaca acccgactcc agttgtgcaa 1440

attggtacct ccggctctac cggtatcgtc gagatcacgg acatcatctt caaaaccatc 1500

ggctacgcgc ccggagctat cattgttgaa tggaacgtac acgaaccatc tggccaacag 1560

gccggtgcag gtacatggga tacccacatc atcctcggtg gcactgctgg tacccagctt 1620

cagacatcac aatgcccgtc cggttcccag aataccggaa cctgtaccac ggcctatctc 1680

ggtctacatc ttaccactag ctcatctgcg tacctcgagg gcatgtgggt ctggctcgcc 1740

gaccatgatc tcgacagcag tgggcaaaca cagctgacga tattcagtgg ccgtggtatt 1800

ctctctgagt ctcaaggacc agtctggctc gttggaacag cgtcggaaca ccacgtcctc 1860

taccagtaca acctcgttaa tgcaaagaac cactacatgg gtctcatcca gactgagacg 1920

ccgtactatc agccatcacc cgccccgccc gctcccttct ccatcaacag cgcgtaccat 1980

gacccgtcat tcgctagcga caccccagca gcctggggac taaacgtaca gtcatcgaca 2040

gacattattg tcttcggggc tggccactac tcgttcttcc aaaactacgg acaaaactgc 2100

ctcaccacct tcaattgcca aaaccagatc gtcaatgtcg attcggcatc ttcgatctgg 2160

atctactccc tcgcgaccgt cgccactact aagcaggtca gcatgagcgg gacgggtatc 2220

attccggaga gcgccaacct taacggcttc caagaaactg taacattctg ggagccttga 2280

<210> 8

<211> 1077

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 8

atgaaatcac tcatctatac tgcattatca tcgttgcttg ggctgtcata cgcctaccct 60

gcgccttcaa acgctgagtc attgggagtg tctacaaaca taagcctaga cgccggctcg 120

ttcttcccac ctcccggggc accatgcttc cctgctcttg gatttcagac gccttctaat 180

gtccccagcc acctgaacgg atggtggtgt gatcccgtca ccgagcatgc tttcctgggc 240

tttagctatg aagttactgc ttgtcaagat atatcaaccc tcaaaaagga atttgcagat 300

attcgttatc atttcaacag tcgatacatt cgtctgtacg gtgtctgtga taatgatggc 360

ttctacgatg atattgttga agctgcttgg gaaaataact tgggcgttca tgctcttgtc 420

tggtttggat ttgatggcac tgatcaatgg atcggacgcc gcgataccct atttgctacc 480

ctacactcga atcccaaggc taaatttgtc acccgtgttg tccaattcgg gtctgagccc 540

ctctttgatg acgtgctacc acaccagcaa cttgcagagc aagtgatact ggcaaaacag 600

aatctgtcgt ccttgcacat caacgtcact gtcagcgaac ttgcttatgg ataccaggag 660

cgcggcggcg ccgaggatgt cttgtccgcc atcgatagca ttaacgcgca catgttgccc 720

ttctttgcgc agaatgcttc tactgccttc aattcgtggc cgagcgttct tgacgacatg 780

gactggtttg tcaacaatgg tcaaggcaag aagatctact tcgacgagaa cggctggccg 840

tctgttacct cacctagcgt acaaaataac tcgatatatg ctgttgctga tgttcagcag 900

gaacaggact acttcctcct cctcgagtca aaatgcgaat acctccggga taacgcaccc 960

gttggcggcg tcggctggtt cgctcacatc tacagcgaca acatggagcc tggttatggc 1020

atctacgata cgtcgggcca gatgaaattc ccattcaacc caaagcctca ctgctag 1077

<210> 9

<211> 750

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 9

atgagctgcg acgactgcta ttcaggtgcc cgtcacgagg gccacttcga ggtaatcgga 60

ggtgtaaact gttatgttgc cactcccgaa aaggagtatg acgaaagcgc tgttataatc 120

ttcctgtccg atgtctttgg tgttcagttc attaacaatc agctgcttat atccgacttc 180

gcaaagaacg ggtttaagac agtggcgccg gacatgtttg atggggaccc tgtaccagca 240

atcgtagtag aaggcaacct aagcacacca caaaactggg acagagccac ctggactgct 300

aagcatggtt tcgaagtcac taggccgctt gttgacaagg ttattgatga cctcaaatcg 360

aaaggcataa ctagatttgc cgccaacggc tactgctacg gcgctagaat tggtttcgat 420

ctcgcttttg agaacattgt caaagccgta gttgtatcac atccctcacg cctaactgtt 480

cccgacgatt ttgagaaata caaatccacg tcgaatgcac ctctgctgat caactcttgc 540

accatagacc agcaatttga tcatgacgcc cagaagggag cagacgccat tatgggtgcg 600

ggccaattcg aacctggata tctgagagaa tatttcgatg gatgcagaca cggcttcgct 660

gtgagagctg acttgaataa ccctcaggct gtggctggaa aagagggcgc attcaaagca 720

acagtccaat tcttgcaaaa acacctatga 750

<210> 10

<211> 762

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 10

atgaagctca ccacttccca tgtccttgct ctcctggctg cggccacggg cgcgtcagct 60

cgtcagatta cagtgtacaa tggctgcccg ttcactatct ggcccgctat gttcactggt 120

gtcctttcaa cggcgccctc ttacactacc ggttgggagg ccgcggccta cacccatgtc 180

acctttgatg tcccagacaa ctggactgca ggtcgcatct ggggtcgtcg tgactgtgac 240

ttcagctcag tccaaggccc aacttcctgc cttgatggag gttgcaatgg cggtctcgtc 300

tgcgatcttt ccacgggcac gggtgttccc cccgcgaccc tcgccgagtt cactctctcg 360

acgtccatcg acaactatga tgtctctctt gtcgacggtt acaatttgcc catgagaatc 420

gacaacacca acggctgcgg tgtagcttct tgccccgttg atctcggccc cgactgtccc 480

gctgctctcc agggtcctta cgactctact ggttttcctg ttggctgcaa gagtgcttgc 540

gatgctaacc tcagcggaga tccctccaac tcccctaact gctgctcggg tcaatatgac 600

acgcctgcga catgccccag ctccggcgtg gcatactact cctacttcaa ggacaactgc 660

ccagactcgt acgcatacgc ttacgatgag tcgagcggca ccgctctctg gacctgcacg 720

ggcctgccca actacaccgt caccttctgc cctgccagtt ag 762

<210> 11

<211> 810

<212> DNA

<213> Sclerotium rolfsii WSH-G01

<400> 11

atgttgttca agtcgctctt cgctgttgtt tctctttctg tatcagctgt ctccgctggc 60

aaacgtggtc ttgcatggcc atggtacaac agtcctttgg accctggtgt cttcaacaac 120

ggagacggtg aagtggtagc catctacgat tgggagacct atgctcctcc ctctaccaat 180

ggcaatggtg gactgggctt catcggcacg cagcgttgca tggattgctc ctctagccca 240

atcgatgagc ttcagtcccg gtggaaagca cagggatggg caaccgtctt tagcttgaac 300

gagcccgacc agaacggcat ctctgccagc gaggcagcct cttggtacat cgaatacatc 360

aacccgttgg atatcaagaa ggccctccct gctatcagct ccagcacatc atcgggtcaa 420

ggcctttcct ggctagcaga aatggtctcc gcttgcgctg gagcctgtta tgccgattat 480

atcaatcttc actggtatgg taactccttc tcagagttcg agagctatgt caccgaggcg 540

cacaaccaat tccctcaata caccgtcgtc gtctcagagt ttgctcttca gaacccctct 600

ggtggtgcaa ctgcccagta cgacttcttc caacaagcct ttgcttggct tgatgatcaa 660

gaatgggtga cgctgtactt ccccttcgtg gcaacgtcgc cttcgctcat gcaggccaac 720

gatgcctcga gttactccta tgttggcacg ggctcctgct tgttcaccaa cgcaggtcaa 780

ccttcgagtg tgggtgacct tatgtactaa 810

<210> 12

<211> 527

<212> PRT

<213> Sclerotium rolfsii WSH-G01

<400> 12

Met Ser Ser Val Pro Glu Pro Glu Ile Lys Asp Lys Leu Pro Ser Asp

1 5 10 15

Phe Ile Trp Gly Phe Ala Thr Ala Ser Phe Gln Ile Glu Gly Ser Thr

20 25 30

Asp Arg Asp Gly Arg Gly Pro Ser Ile Trp Asp Glu Phe Ser Arg Thr

35 40 45

Pro Gly Lys Ile Gln Asp Gly Arg Asn Gly Asp Val Ala Thr Asp Ser

50 55 60

Tyr Asn Arg Tyr Lys Glu Asp Ile Gln Leu Leu Lys Asp Tyr Gly Val

65 70 75 80

Lys Ser Tyr Arg Phe Ser Ile Ser Trp Ser Arg Ile Ile Pro Leu Gly

85 90 95

Gly Arg Asn Asp Pro Ile Asn Glu Ala Gly Ile Lys Phe Tyr Ser Asp

100 105 110

Leu Ile Asp Gly Leu Leu Glu Ala Glu Ile Ile Pro Phe Val Thr Leu

115 120 125

Tyr His Trp Asp Leu Pro Gln Gly Leu His Asp Arg Tyr Gly Gly Trp

130 135 140

Leu Asn Lys Asp Glu Ile Ala Ala Asp Tyr Glu Asn Tyr Ala Arg Val

145 150 155 160

Cys Phe Lys Ala Phe Gly Asp Arg Val Lys Tyr Trp Leu Thr Met Asn

165 170 175

Glu Pro Trp Cys Ile Ser Ile Leu Gly Tyr Gly Arg Gly Val Phe Ala

180 185 190

Pro Gly Arg Ser Ser Asp Arg Glu Arg Ser Ala Glu Gly Asn Ser Ser

195 200 205

Thr Glu Pro Trp Ile Val Gly His Ser Val Leu Leu Ala His Ala Arg

210 215 220

Ala Val Ala Ala Tyr Arg Lys Asp Phe Lys Pro Thr Gln Lys Gly Val

225 230 235 240

Ile Gly Ile Thr Leu Asn Gly Asp Met Ala Leu Pro Trp Asn Asp Glu

245 250 255

Gln Lys Asn Ile Asp Ala Ala Gln His Ala Leu Asp Tyr Ala Ile Gly

260 265 270

Trp Phe Ala Asp Pro Ile Tyr Leu Gly His Tyr Pro Glu Tyr Met Arg

275 280 285

Ser Ile Leu Gly Asp Arg Leu Pro Glu Phe Thr Pro Glu Glu Trp Ala

290 295 300

Val Val Lys Gly Ser Ser Asp Phe Tyr Gly Met Asn Thr Tyr Thr Thr

305 310 315 320

Asn Leu Cys Lys Ala Gly Gly Asp Asp Glu Phe Gln Gly Phe Val Glu

325 330 335

Tyr Thr Phe Thr Arg Pro Asp Gly Thr Gln Leu Gly Thr Gln Ala His

340 345 350

Cys Ala Trp Leu Gln Asp Tyr Ala Pro Gly Phe Arg Met Leu Leu Asn

355 360 365

Tyr Leu Trp Lys Lys Tyr Lys His Pro Ile Tyr Val Thr Glu Asn Gly

370 375 380

Phe Ala Val Arg Asn Glu Asp Ala Met Pro Arg Asp Gln Ala Ile His

385 390 395 400

Asp Ala Asp Arg Val Ala Tyr Phe Lys Gly Thr Thr Lys Ala Leu Trp

405 410 415

Glu Ala Val Lys Leu Asp Gly Val Glu Val Lys Ala Tyr Phe Pro Trp

420 425 430

Ser Phe Leu Asp Asn Phe Glu Trp Ala Asp Gly Tyr Val Thr Arg Phe

435 440 445

Gly Val Thr Tyr Val Asp Tyr Glu Thr Gln Glu Arg Phe Pro Lys Asp

450 455 460

Ser Gly Lys Phe Leu Val Asp Trp His Lys Ala Tyr Val Ala Lys Glu

465 470 475 480

Ser Glu Ala Ser Ser Ser Ser Ser Ser Lys Thr Ser Ala Lys Pro Ala

485 490 495

Gly Ala Lys Thr Ala Asn Lys Arg Asn Ser Ile Pro Phe Thr Thr Pro

500 505 510

Leu Ala Pro Leu Trp Lys Lys Leu Phe Ser Leu Lys Ala Lys Ala

515 520 525

<210> 13

<211> 358

<212> PRT

<213> Sclerotium rolfsii WSH-G01

<400> 13

Met Lys Ser Leu Ile Tyr Thr Ala Leu Ser Ser Leu Leu Gly Leu Ser

1 5 10 15

Tyr Ala Tyr Pro Ala Pro Ser Asn Ala Glu Ser Leu Gly Val Ser Thr

20 25 30

Asn Ile Ser Leu Asp Ala Gly Ser Phe Phe Pro Pro Pro Gly Ala Pro

35 40 45

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

50 55 60

Leu Asn Gly Trp Trp Cys Asp Pro Val Thr Glu His Ala Phe Leu Gly

65 70 75 80

Phe Ser Tyr Glu Val Thr Ala Cys Gln Asp Ile Ser Thr Leu Lys Lys

85 90 95

Glu Phe Ala Asp Ile Arg Tyr His Phe Asn Ser Arg Tyr Ile Arg Leu

100 105 110

Tyr Gly Val Cys Asp Asn Asp Gly Phe Tyr Asp Asp Ile Val Glu Ala

115 120 125

Ala Trp Glu Asn Asn Leu Gly Val His Ala Leu Val Trp Phe Gly Phe

130 135 140

Asp Gly Thr Asp Gln Trp Ile Gly Arg Arg Asp Thr Leu Phe Ala Thr

145 150 155 160

Leu His Ser Asn Pro Lys Ala Lys Phe Val Thr Arg Val Val Gln Phe

165 170 175

Gly Ser Glu Pro Leu Phe Asp Asp Val Leu Pro His Gln Gln Leu Ala

180 185 190

Glu Gln Val Ile Leu Ala Lys Gln Asn Leu Ser Ser Leu His Ile Asn

195 200 205

Val Thr Val Ser Glu Leu Ala Tyr Gly Tyr Gln Glu Arg Gly Gly Ala

210 215 220

Glu Asp Val Leu Ser Ala Ile Asp Ser Ile Asn Ala His Met Leu Pro

225 230 235 240

Phe Phe Ala Gln Asn Ala Ser Thr Ala Phe Asn Ser Trp Pro Ser Val

245 250 255

Leu Asp Asp Met Asp Trp Phe Val Asn Asn Gly Gln Gly Lys Lys Ile

260 265 270

Tyr Phe Asp Glu Asn Gly Trp Pro Ser Val Thr Ser Pro Ser Val Gln

275 280 285

Asn Asn Ser Ile Tyr Ala Val Ala Asp Val Gln Gln Glu Gln Asp Tyr

290 295 300

Phe Leu Leu Leu Glu Ser Lys Cys Glu Tyr Leu Arg Asp Asn Ala Pro

305 310 315 320

Val Gly Gly Val Gly Trp Phe Ala His Ile Tyr Ser Asp Asn Met Glu

325 330 335

Pro Gly Tyr Gly Ile Tyr Asp Thr Ser Gly Gln Met Lys Phe Pro Phe

340 345 350

Asn Pro Lys Pro His Cys

355

<210> 14

<211> 253

<212> PRT

<213> Sclerotium rolfsii WSH-G01

<400> 14

Met Lys Leu Thr Thr Ser His Val Leu Ala Leu Leu Ala Ala Ala Thr

1 5 10 15

Gly Ala Ser Ala Arg Gln Ile Thr Val Tyr Asn Gly Cys Pro Phe Thr

20 25 30

Ile Trp Pro Ala Met Phe Thr Gly Val Leu Ser Thr Ala Pro Ser Tyr

35 40 45

Thr Thr Gly Trp Glu Ala Ala Ala Tyr Thr His Val Thr Phe Asp Val

50 55 60

Pro Asp Asn Trp Thr Ala Gly Arg Ile Trp Gly Arg Arg Asp Cys Asp

65 70 75 80

Phe Ser Ser Val Gln Gly Pro Thr Ser Cys Leu Asp Gly Gly Cys Asn

85 90 95

Gly Gly Leu Val Cys Asp Leu Ser Thr Gly Thr Gly Val Pro Pro Ala

100 105 110

Thr Leu Ala Glu Phe Thr Leu Ser Thr Ser Ile Asp Asn Tyr Asp Val

115 120 125

Ser Leu Val Asp Gly Tyr Asn Leu Pro Met Arg Ile Asp Asn Thr Asn

130 135 140

Gly Cys Gly Val Ala Ser Cys Pro Val Asp Leu Gly Pro Asp Cys Pro

145 150 155 160

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

165 170 175

Lys Ser Ala Cys Asp Ala Asn Leu Ser Gly Asp Pro Ser Asn Ser Pro

180 185 190

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

195 200 205

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

210 215 220

Ala Tyr Ala Tyr Asp Glu Ser Ser Gly Thr Ala Leu Trp Thr Cys Thr

225 230 235 240

Gly Leu Pro Asn Tyr Thr Val Thr Phe Cys Pro Ala Ser

245 250

<210> 15

<211> 269

<212> PRT

<213> Sclerotium rolfsii WSH-G01

<400> 15

Met Leu Phe Lys Ser Leu Phe Ala Val Val Ser Leu Ser Val Ser Ala

1 5 10 15

Val Ser Ala Gly Lys Arg Gly Leu Ala Trp Pro Trp Tyr Asn Ser Pro

20 25 30

Leu Asp Pro Gly Val Phe Asn Asn Gly Asp Gly Glu Val Val Ala Ile

35 40 45

Tyr Asp Trp Glu Thr Tyr Ala Pro Pro Ser Thr Asn Gly Asn Gly Gly

50 55 60

Leu Gly Phe Ile Gly Thr Gln Arg Cys Met Asp Cys Ser Ser Ser Pro

65 70 75 80

Ile Asp Glu Leu Gln Ser Arg Trp Lys Ala Gln Gly Trp Ala Thr Val

85 90 95

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

100 105 110

Ala Ser Trp Tyr Ile Glu Tyr Ile Asn Pro Leu Asp Ile Lys Lys Ala

115 120 125

Leu Pro Ala Ile Ser Ser Ser Thr Ser Ser Gly Gln Gly Leu Ser Trp

130 135 140

Leu Ala Glu Met Val Ser Ala Cys Ala Gly Ala Cys Tyr Ala Asp Tyr

145 150 155 160

Ile Asn Leu His Trp Tyr Gly Asn Ser Phe Ser Glu Phe Glu Ser Tyr

165 170 175

Val Thr Glu Ala His Asn Gln Phe Pro Gln Tyr Thr Val Val Val Ser

180 185 190

Glu Phe Ala Leu Gln Asn Pro Ser Gly Gly Ala Thr Ala Gln Tyr Asp

195 200 205

Phe Phe Gln Gln Ala Phe Ala Trp Leu Asp Asp Gln Glu Trp Val Thr

210 215 220

Leu Tyr Phe Pro Phe Val Ala Thr Ser Pro Ser Leu Met Gln Ala Asn

225 230 235 240

Asp Ala Ser Ser Tyr Ser Tyr Val Gly Thr Gly Ser Cys Leu Phe Thr

245 250 255

Asn Ala Gly Gln Pro Ser Ser Val Gly Asp Leu Met Tyr

260 265

Claims

1. A sclerotium rolfsii hydrolase has an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the sclerotium rolfsii hydrolase according to claim 1.

3. An expression vector carrying the gene of claim 2.

4. A microbial cell expressing the sclerotium rolfsii hydrolase according to claim 1.

5. A recombinant Pichia pastoris that expresses the sclerotium rolfsase of claim 1.

6. The recombinant Pichia pastoris of claim 5, wherein the Sclerotinia sclerotiorum hydrolase of claim 1 is expressed with Pichia pastoris GS115 as a host and pPIC9K as a vector.

7. Use of a sclerotium rolfsii hydrolase according to claim 1 for the preparation of low molecular weight sclerotium rolfsii.

8. A method for hydrolyzing sclerotium rolfsii gum, characterized in that the sclerotium rolfsii gum hydrolase according to claim 1 is added into a system containing sclerotium rolfsii gum, and the reaction is carried out for at least 2 hours at 28-30 ℃.

9. An enzyme preparation comprising the sclerotium rolfsii hydrolase according to claim 1.

10. Use of the sclerotium rolfsii hydrolase according to claim 1 in the fields of food, biomedicine, petrochemical industry and cosmetics.