酶工程-2微生物發(fā)酵產(chǎn)酶

Chapter

2

微生物發(fā)酵產(chǎn)酶The

production

of

Enzyme

byFermentation

ofMicroorganismContents

ofchapter

22.3、酶生產(chǎn)過程的動力學2.4、酶的生物

及調(diào)節(jié)（>、產(chǎn)酶微生物、酶的發(fā)酵工藝條件及控制動物、植物生產(chǎn)酶制劑的缺陷：植物由于生長地域、季節(jié)、氣候等的影響，生產(chǎn)酶制劑的產(chǎn)量、質(zhì)量都不穩(wěn)定。動物產(chǎn)生的酶主要從屠宰牲畜的腺體中提取，來源有限。2.1.1

產(chǎn)酶微生物的概述微生物的優(yōu)勢種類繁多生長周期短繁殖快培養(yǎng)相對簡單較強的適應(yīng)性和突變能力產(chǎn)酶微生物來源土壤水體空氣環(huán)境環(huán)境微生物嗜冷菌嗜酸菌嗜鹽菌產(chǎn)酶微生物種類細菌bacteria青霉素酰化酶溶菌酶

淀粉酶放線菌actinomycetes蛋白酶

葡萄糖異構(gòu)酶抗生素酵母菌yeast糖化酶淀粉酶蛋白酶霉菌mold淀粉酶果膠酶水解酶產(chǎn)酶微生物的基本要求不是致病菌，不產(chǎn)生毒素及有害物質(zhì)；發(fā)酵周期短，生產(chǎn)成本低，產(chǎn)酶量高；遺傳性穩(wěn)定，不易變異

，不

噬菌體；最好是產(chǎn)生胞外酶的菌種，利于分離純化；對和食品用酶，還應(yīng)考慮安全性；工程菌必須符合安全性要求。2.1.2

常見產(chǎn)酶微生物類別菌名產(chǎn)酶用途細大腸桿菌青霉素?；腹劝彼崦擊让赴肴樘擒彰赴?/p>

青霉素或頭孢霉素調(diào)味劑分解乳糖枯草桿菌淀粉酶蛋白酶、啤酒及洗滌劑等生絲脫膠、皮革脫毛、醬油釀造菌乳酸桿菌葡萄糖異構(gòu)酶由葡萄糖制果糖芽胞桿菌堿性蛋白酶皮革脫毛、洗滌劑霉點青霉葡萄糖氧化酶食品保鮮、去氧黑曲霉酸性蛋白酶啤酒澄清、毛皮軟化、菌河內(nèi)根霉葡萄糖苷酶制葡萄糖里氏木霉纖維素酶單糖、糊精、能源化酶酵母假絲酵母脂肪酶絹絲脫脂、洗滌劑、

、乳品增香放線菌

白色放線菌

蛋白酶皮革脫毛(1)大腸桿菌操作、生長迅大腸桿菌的優(yōu)勢主要因它易于在速，而且營養(yǎng)要求低。應(yīng)用：大腸桿菌也是最早用作 工程的宿主菌；工業(yè)上生產(chǎn)谷氨酸脫羧酶、天冬酰胺酶、青霉素?；浮肴樘擒彰傅?；DNA聚合酶、連接酶、限制性內(nèi)切酶等；大多數(shù)是胞內(nèi)酶，需進行細胞破碎。(2)醋酸桿菌（Acetobacter）菌體從橢圓至桿狀，單個、成對或成鏈，革蘭氏，運動（周毛）或不運動，不生芽孢。好氣。含糖、乙醇和酵母膏的培養(yǎng)基上生長良好。應(yīng)用：有機酸(食醋等）葡萄糖異構(gòu)酶(高果糖漿)山梨糖(維C

）。(3)枯草芽孢桿菌（Bacillus

subtilis）直狀、近直狀的桿菌，周生或側(cè)生鞭毛，革蘭氏陽性，無莢膜，芽孢0.5×1.51.8m?？莶菅挎邨U菌是工業(yè)發(fā)酵的重要菌種之一。生產(chǎn)淀粉酶、蛋白酶、某些氨基酸及核苷、堿性磷酸酶。胞外酶啤酒、洗滌劑、生絲脫膠、醬油釀造等(4)鏈霉菌(Actinomycetes)應(yīng)用：生產(chǎn)葡萄糖異構(gòu)酶；青霉素?；浮⒗w維素酶、幾丁質(zhì)酶等。(5)根霉(Rhizopus)分布于土壤、空氣中，常見于淀粉食品上，可引起霉腐變質(zhì)和水果、蔬菜的腐爛。代表種：米根霉（R.oryzae)、黑根霉(R.nigrican)等。應(yīng)用：產(chǎn)生如淀粉酶、果膠酶、脂肪酶等。在釀酒工業(yè)上常用做糖化菌。有些根霉還能產(chǎn)生乳酸延胡索酸等有機酸。(6)曲霉(Aspergillus)分類：多數(shù)屬于子囊菌亞門，少數(shù)屬于半知菌亞門。分布：廣泛分布于土壤、空氣和谷物上，可引起食物、谷物和果蔬的霉腐變質(zhì)，有的可產(chǎn)生性的黃曲霉毒素。代表種：黑曲霉Asp.Niger、黃曲霉

Asp.flavus應(yīng)用：是制醬、釀酒、制醋的主要菌種。是生產(chǎn)酶制劑（蛋白酶淀粉酶、果膠酶）的菌種。生產(chǎn)有機酸（如檸檬酸、葡萄糖酸等）。農(nóng)業(yè)上用作生產(chǎn)糖化飼料的菌種。應(yīng)用：啤酒、酒類生產(chǎn)；生產(chǎn)轉(zhuǎn)化酶、酸脫羧酶、醇脫氫酶等。(7)釀酒酵母(Saccharomyces

cerevisiae)蛋白酶59%糖化酶13%5%葡萄糖異構(gòu)酶6%淀粉酶果膠酶3%纖維素酶1%脂肪酶3%與分析研究用酶10%不同類型工業(yè)酶的比例研究及查閱資料平板分離設(shè)計試驗方案原種斜面目標菌株初篩復篩

再復篩生產(chǎn)性試驗毒性試驗

菌種鑒定符合工業(yè)生產(chǎn)要求符合安全性要求

菌種保藏單株純種分離生產(chǎn)2.1.3

產(chǎn)酶微生物的分離與篩選（1）含菌樣品獲得能降解某物質(zhì)的產(chǎn)酶菌，可從該物質(zhì)分布豐富的地方尋找；纖維素酶：腐爛纖維素或堆肥中果膠酶：腐爛的水果、蔬菜胞外酶的穩(wěn)定性和最適條件與菌的最適生長條件接近。（2）菌的分離純化平板劃線稀釋分離（3）產(chǎn)酶性能測定。初篩：選出含有目的酶菌株，平板培養(yǎng)透明圈法；復篩：選出產(chǎn)酶量高、性能更符合要求的菌株22酶的催化作用受到底物濃度、酶濃度、溫度、pH值、激活劑濃度、抑制劑濃度等諸多因素的影響。在酶的應(yīng)用過程中，必須控制好各種環(huán)境條件，以充分發(fā)揮酶的催化功能。影響酶催化的各種因素23溫度最優(yōu)溫度活力％溫度/℃酶活性的最佳溫度pH以pH值為變量的幾種酶的活性函數(shù)24pH活力胃蛋白酶蔗糖酶胰島素251、酶2、酶與酶反應(yīng)速度；單位1961年國際生物化學與分子生物合會規(guī)定：在特定條件下（溫度可采用25℃或其它選用的溫度，pH等條件均采用最適條件），每1min催化1μmol的底物轉(zhuǎn)化為產(chǎn)物的酶量定義為1個酶單位。這個單位稱為國際單位（U）。3、酶

測定方法反應(yīng)體系及底物選擇最適反應(yīng)條件的確定反應(yīng)終止及活性檢測酶測定26吸光度法輻射線測定發(fā)光法測定熒光光度法電位測定法電導測定法旋光測定法固定化酶量熱法酶分析方法27酶制劑的質(zhì)量評估質(zhì)量指標：活度、純度、穩(wěn)定性、配方和包裝比活：與其蛋白含量相關(guān)的催化活性（U/mg）蛋白質(zhì)測定污染活性：原料酶中含有的其他酶量性能測試氨基酸分析和蛋白質(zhì)序列分析穩(wěn)定性酶制劑的劑型：根據(jù)應(yīng)用采用不同的劑型。2.1.4

產(chǎn)酶微生物優(yōu)良菌種的選育誘變育種0.2%

LiCl+

UV120S+

2mg/ml

NTG(40min)原生

融合育種工程育種洗牌法（gene

shuffling）定向進化（directed

evolution）Sugar

production

of

mutantstrains406080120140020406080100BC8C4YEN100Time,

hSpecific

capability

of

sugar

production,

mg

mg-1020406080100??gDNASequenceRNA-seq

ProfilesFunctional

GenesElucidation

ofthepossiblelignocellulosicdegradation

mechanismwith

thegDNAandRNA-sequencing

from

Penicillium

expansiumPART

IGenome

Sequencing

andAnnotationKaryotypegDNA

ExtractionM:

lamda

DNA

;1

,

2:

crude

gDNA

samples;

3,

4:

gDNA

samples

after

gel

purificationFigure

1

The

result

of

electrophoresisOU-FGS-1OU-FGS-2260/2801.941.92260/2301.981.92ng/μl198.3089138.0788Volume(μl)100100Total(μg)19.813.8Sequencing

SummaryPlatformRead

LengthReads

NumberTotal

Bases454329bp

average1,438,311473,315,737IlluminaGAIIx150bp

single

end19,447,8482,917,177,200Illumina

Hiseq100bp

paired

end99,252,6549,925,265,400TotalN/A120,138,813~13.3GbGenomeSequencing

(>400

coverage)35Overview

of

Genome

CharacteristicsNucleargenomeP.expensiumP.chrysogenumGeneral

informationSize

(Mb)32.2732.2G+C

content

(mole%)50.6348.9Coding

(%)51.5856.6Number

ofsupercontigs5949Supercontig

length106>100kb14Largest

supercontig9697kb6,367kbNumber

ofgenes8,98912,943Mean

gene

length

(bp)15841515Genes

with

intron

(%)7,447(82.8)10,812(83.5)ExonsMean

number

per

gene3.33Mean

length

(bp)477434IntronsMean

number

per

gene2.32.2Mean

length

(bp)408.887.436structure

and

biogenesis3%RNA

processing

andTranslation,

ribosomal

modification3%Transcription3%Replication,bination

andrepair2%Chromatin

structure

anddynamics1%Cell

cycle

control,

cell

division,chromosome

partitioning2%Nuclearstructure0%Cell

wall/membranbiogenesis1%Signal

transduction4%Cytoskeleton1%Intracellulartrafficking,secretion,3%Energyproduction3%Carbohydratemetabolism3%Nucleotide

transportand

metabolism1%Coenzyme

transportand

metabolism1%Lipid

transport

andmetabolism3%Inorganic

ion

transport

andmetabolism2%Secondary

metabolitesbiosynthesis,

transport

andcatabolism3%General

functionpredictiononly10%Functionunknown3%No

Metabolic

Hits3%No

HitsFound34%Functional

Classification(KOG)glucanase16%Cellulase2%glucosidase10%glycosyl

hydrolase10%glycosyltransferase12%xylanase9%mannosidase20%galactosidase10%chitinase11%laccase0%The

functional

genes

associatedwith

cell-walldegradationTotal 217

genesGenome

Structureysis

inComparing

with

Trichoderma

reesei39Penicilium

spp.

YT02Trichoderma

reeseiGenome

size32.26M(Estimated

77.7%)33.9M(99%)Genenumber8,9899,129%

coding51.5840.40%

GC50.6352.0Carbohydrate

transportand

metabolism292346Cellulolytic

enzymes3410Hemicellulose

degradingenzymes8816(no

feruloyl

esterase)Part

IIRNA-seq

for

Different

Expressing

ProfilesConstructionEnzymes

Activity

with

Different

Substrates0

0G

A

X

S

S'The

result

of

enzyme

activity

and

saccharification

ratio

with

different

substrates5101520253035FPaseXylanaseCMCaseSaccharificationratioSaccharification

ratio,

%Enzyme

activities,

IU

mg-15101520253035Glucose(G)Xylan(X)Avicel(A)Switchgrass(S1)Switchgrass(S2)Substrates2%2%2%2%2%Time(h)6060606080FlowchartRNA

ExtractmRNAIsolationOligo(dT)

cDNA

synthesiscDNAfragmentationAdd adapters

and

amplificationSize

selection(100-300bp)Illumina

sequencingRandom

hexamer

primedcDNA

synthesisty

of

the

dscDNAS

lThety

of

dscDNA

samples

by

picogreenAGXS1S2Volume(μl)505010010050Concentration(ng/μl)186.49172.5451.2880.80102.42Total

cDNA

(μg)9.3258.6255.1288.0805.120General

Characteristics

of

Datasets

from

FiveDifferentConditionsSampleReads

NumberTotal

BasesTotal

ContigsLargest

ContigADD31,602,6201,137,694,32025,8215,712GDB45,610,6281,641,982,60823,2476,725SDD49,910,8121,796,789,23222,5906,818SDF46,153,5641,661,528,30425,4099,405XDA73,523,2962,646,838,65626,5516,971TOTAL246,800,9208,884,833,12098,20912,150Full

Transcriptome

ProfilesPCAysisCorrelation

of

Five

ConditionsGAS1S2XG-0.88780.79760.80410.8489A0.3137-0.88250.87250.9084S10.24700.9305-0.94350.9276S20.41800.87370.9337-0.9558X0.47630.80090.90230.9413-y

=0.9708x+0.2815R2

=0.9558012345601234567SDFXDAy

=0.8555x+0.4178R2

=0.79760123456012456GDB3SDDSDF/XDA

SDD/GDB7

7Construction

of

different

expressing

profiles……….Uniquegenes188/A173/G106/S2140/S153/X8616403020100xylanasemannanasearabinaserhamnosidasegalactosidaseferuloylesteraseglucanaseglucosidasecellobioseglycoside

hydrolaseglycosyl

transferasechitinaseacetylglucosaminidaselaccasemonooxygenaseHemicellulose

degradationgenes(111)cellolosedegradationgenes(119)chitindegradationgenes(24)lignindegradationgenes(20)The

functional

genes

associated

with

lignocellulosedegradation

fromRNA-seq

datasetsHighest

redundant

genes

(Glucose

1)Gene

IDFunctional

DescriptionReadsLength(bp)Trans.Noorf_28861438_28862285_FGlutathione

S-transferase1093748474714641orf_17498192_17498692_FMolecular

chaperone574286450111462orf_3736573_3737355_RNo

Hits

Found55201326548440orf_28870698_28871560_FPutative

transcriptional

regulator

DJ-6200orf_9040901_9043160_FPredicted

transporter766317616534635orf_28271460_28272867_FGAPDH466171211134188orf_30067947_30069076_RUbiquitin

and

ubiquitin-like

proteins36645129183991orf_13275344_13277812_RPhosphoenolpyruvate

carboxykinase695361618023858orf_1248397_1249824_FAspartyl

protease416768411943490orf_27495603_27497324_FAlcohol

dehydrogenase,

class

V484822814463352orf_29915011_29916732_FAlcohol

dehydrogenase,

class

V482954414463339orf_28880634_28882247_RPredicted

transporter514832416143189orf_19768542_19770105_REnolase358347613172720orf_2326463_2328432_Facyl-CoA

dehydrogenase393948017642233orf_12556572_12558305_Fcell

wall

glucanase

(Scw11)344102417341984orf_8024672_8026816_FThiamine

pyrophosphate

enzyme344005218361873orf_13090822_13092729_RMolecularchaperonesHSP70/HSC70343648819081801orf_17149181_17152281_FPlasma

membrane

ATPase454032029791524orf_13288691_13294018_RUnnamed

protein637459246621367orf_2041227_2044073_FPeroxisomal

beta-oxidatase351266427001300Highest

redundant

genes

(Avicel

13)Gene

IDFunctional

DescriptionReadsLength(bp)Trans.Noorf_3983888_3985736_Rcellobiohydrolase30763188137422389orf_3822669_3824306_Fexo-cellobiohydrolase36101556163822040orf_3779111_3780538_Fendoglucanase

I14625324142810241orf_9040901_9043160_FMFS

lactose

permease,

putative1645743616539956orf_14906718_14908209_Rendo-1,4-beta-D-glucanase1202940012639524orf_3776299_3778439_Rendoglucanase

GH4567940649697011orf_27809763_27811540_Fbeta-glucosidase664326014524575orf_11505519_11507382_Rendo-1,4-beta-xylanase

precursor610869614584189orf_12261978_12262633_FGPI

anchored

serine-rich

protein24058445914070orf_17480803_17482708_Rswollenin472618815003150orf_17485802_17486874_Fendo-1,4-beta-xylanase28168569333019orf_28271460_28272867_FGAPDH307411211132762orf_1248397_1249824_Fasparticendopeptidase

Pep2296773211942485orf_21457706_21459650_Fhexose

transporter

protein336013216502036orf_18068062_18070612_Fextracellularendoglucanase357440419321850orf_22655921_22657540_Rmannanase241642813561782orf_30741653_30743241_Ftranslationelongation

factor

eEF-1242636413831754orf_8054963_8056551_Rtranslationelongationfactor241207213831744orf_13275344_13277812_Rphosphoenolpyruvate

carboxykinase255200418031415orf_9143836_9149018_Fxylulose-5-phosphate

phosphoketase30764164269720Highestredundant

genes(Switchgrass60h-13)Gene

IDFunctional

DescriptionReadsLength(bp)Trans.Noorf_3983888_3985736_Rcellobiohydrolase51397992137437407orf_3822669_3824306_Fexo-cellobiohydrolase44363484163827083orf_3779111_3780538_Fendoglucanase

I23303700142816319orf_3776299_3778439_Rendoglucanase

GH451221314496912603orf_11505519_11507382_Rendo-1,4-beta-xylanase

precursor18254556145812520orf_14906718_14908209_Rendo-1,4-beta-D-glucanase13034124126310319orf_9040901_9043160_FPredicted

transporter1631257216539868orf_2684073_2684942_FAcetylxylan

esterase66129127059380orf_17485802_17486874_Fendo-1,4-beta-xylanase65097729336977orf_17480803_17482708_RA

Expansin905965215006039orf_2154456_2155288_Fendoglucanase,

putative44351287505913orf_24780385_24781661_Farabinofuranosidase

precursor649627211885468orf_9562997_9564789_Rendo-1,4-beta-xylanase

A529336810714942orf_1248397_1249824_FAspartyl

protease469202411943929orf_21457706_21459650_FPredicted

transporter633150016503837orf_14757638_14758510_Racetylglucosaminyltransferase27338047713545orf_30067947_30069076_RUbiquitin

and

ubiquitin-like

proteins26996409182940orf_13275344_13277812_RPhosphoenolpyruvate

carboxykinase455767218022529orf_18068062_18070612_Fextracellularendoglucanase383994019321987orf_11159299_11160969_FSubtilisin-related

protease276602415121829Highestredundant

genes(Switchgrass80h-10)Gene

IDFunctional

DescriptionReadsLength(bp)Trans.Noorf_3983888_3985736_Rcellobiohydrolase26038548137418950orf_3822669_3824306_Fexo-cellobiohydrolase28153620163817187orf_3779111_3780538_Fendoglucanase

I14381532142810071orf_11505519_11507382_Rendo-1,4-beta-xylanase

precursor1206932414588278orf_3776299_3778439_Rendoglucanase

GH4574527929697691orf_9040901_9043160_FPredicted

transporter1091584816536603orf_14757638_14758510_RN-acetylglucosaminyltransferase48866407716338orf_14906718_14908209_Rendo-1,4-beta-D-glucanase798098412636319orf_1513792_1515133_FBranched

chain

aminotransferase583142410115767orf_17480803_17482708_RA

Expansin41895369694323orf_4534221_4535610_F4-hydroxydioxygenase494859612064103orf_13275344_13277812_RPhosphoenol

carboxykinase712177218023952orf_1248397_1249824_FAspartyl

protease464713211943892orf_17485802_17486874_Fendo-1,4-beta-xylanase30301929333247orf_21457706_21459650_FPredicted

transporter502372816503044orf_27809763_27811540_FBeta-glucosidase,

la

e433188014522983orf_1516192_1518079_Rcytochrome

P450,

putative420595215662685orf_2326463_2328432_Facyl-CoA

dehydrogenase430808417642442orf_18486616_18488944_RH+/oligopeptide

symporter440823618572373orf_1518774_1523957_FFatty

acid

synthase44691124908910Highest

redundant

genes

(Xylan

10)Gene

IDFunctional

DescriptionReadsLength(bp)Trans.Noorf_3983888_3985736_Rcellobiohydrolase31373280137422833orf_3822669_3824306_Fexo-cellobiohydrolase33463764163820429orf_2684073_2684942_FAcetylxylan

esterase1021219270514485orf_11505519_11507382_Rendo-1,4-beta-xylanase

precursor18190872145812476orf_3779111_3780538_Fendoglucanase

I17301888142812116orf_17498192_17498692_FMolecular

chaperone567378050111324orf_21457706_21459650_FPredicted

transporter1512104416509164orf_9040901_9043160_FPredicted

transporter1417525216538575orf_14906718_14908209_Rendo-1,4-beta-D-glucanase936684012637416orf_3776299_3778439_Rendoglucanase

GH4563047529696506orf_14757638_14758510_Rbeta-1,6-N-acetylglucosaminyltransferase44887687715822orf_17485802_17486874_Fendo-1,4-beta-xylanase53613369335746orf_1248397_1249824_FAspartyl

protease678664811945683orf_17480803_17482708_RA

Expansin54568089695631orf_13275344_13277812_RPhosphoenolpyruvate

carboxykinase

(ATP)893016018024955orf_28271460_28272867_FGlyceraldehyde

3-phosphate

dehydrogenase473356811134252orf_24780385_24781661_Falpha-L-arabinofuranosidase

precursor435657611883667orf_27495603_27497324_FAlcohol

dehydrogenase,

class

V514630814463558orf_29915011_29916732_FAlcohol

dehydrogenase,

class

V513831614463553orf_2326463_2328432_FVery-long-chainacyl-CoAdehydrogenase515354417642921Full

map

of

lignocellulose

degradation

genesCellulose

degradation

genesXylan

degradationgenesChitin

and

lignin

degradationgenesXylandegradation

genesCellulose

degradationgenesComparison

of

Two

Timepoints

with

Switchgrass

Substratey

=

0.9509x

+0.1103R2

=0.94350123456701234567SDFSDDThe

comparing

result

of

SDD/SDF024681012orf_3503601_3504248_R

No

Hits

Foundorf_25524577_25525863_F

UDP-glucose4-epimeraseorf_22655921_22657540_R

mannanaseorf_3691285_3692106_F

SAM-dependent

methyltransferasesorf_28601867_28603591_F

No

Hits

Foundorf_5745718_5747713_RCystathionine

beta-lyasesorf_29232884_29234354_F

Copper

transporterorf_3776299_3778439_R

endoglucanase

GH45orf_26249386_26251934_F

No

Hits

Foundorf_11505519_11507382_R

endo-1,4-beta-xylanase

precursororf_28672194_28674108_F

Beta-glucocerebrosidaseorf_23810372_23811330_F

No

Hits

Foundorf_26888257_26889804_R

SPAC2E1P3.05corf_27809763_27811540_F

Beta-glucosidase,

la e

phlorizinhyorf_18068062_18070612_F

extracellular

endoglucanaseorf_17480803_17482708_R

AExpansinorf_23808488_23809499_R

Unnamedproteinorf_14906718_14908209_R

endo-1,4-beta-D-glucanaseorf_2154456_2155288_F

endoglucanase,

putativeorf_3983888_3985736_R

cellobiohydrolaseorf_21301994_21303242_R

endoglucanase/cellulase,

putativeorf_3222755_3224637_R

galactan

1,3-beta-galactosidaseTop

20

up-regulated

gene

profile/Avicel024681012orf_2482178_2483467_R

glucan

endo-1,3-beta-glucosidaseorf_4534221_4535610_F

4-hydroxyphenylpyruvate

dioxygenaseorf_4846315_4848420_R

Predicted

transporteorf_3691285_3692106_F

SAM-dependent

methyltransferasesorf_232163_233206_R

xylosidaseorf_5059812_5061456_F

Chitinaseorf_1613471_1614506_R

Dehydrogenasesorf_21457706_21459650_F

Predicted

transporterorf_18068062_18070612_F

extracellular

endoglucanaseorf_3482108_3482931_R

Manganese

superoxidedismutaseorf_26888257_26889804_R

SPAC2E1P3.05corf_3854576_3856302_F

Kynurenine3-monooxygenaseorf_17480803_17482708_R

AExpansinorf_29232884_29234354_F

Copper

transporterorf_5745718_5747713_R

Cystathionine

beta-lyasesorf_14094792_14095541_F

NoHitsFoundorf_3983888_3985736_R

cellobiohydrolaseorf_3287909_3289519_R

endo-1,6-beta-D-glucanase

BGN16orf_2684073_2684942_F

Acetylxylanesteraseorf_3222755_3224637_R

galactan1,3-beta-galactosidase.orf_2154456_2155288_F

endoglucanase,

putativeTop

20

up-regulated

gene

profile/xylanXylan

metabolism6.05.04.03.02.01.00.0-1.0-2.0-3.0Xylosidase

expressing

profilesADD/GSDD/GSDF/GXDA/Gcell

wall

glucanase,

putativeendoglucanase/cellulaseendoglucanase

IV

precursorADD/GSDD/GSDF/GXDA/G6.05.04.03.02.01.00.0ADD/GSDD/GSDF/GXDA/GComparasion6.04.02.00.0-2.04.0-6.0-8.0Glucosidase

Expressing

ProfilesADD/GSDD/GSDF/G

XDA/Gexo-cellobiohydrolaseexo-cellobiohydrolasecellobiohydrolasePartIIIGene

Cloning

and

ExpressingTheCandidate

Genes

forCloning

andExpressingGeneIDFunction

AnnotationAA

numbersFGENESH:

34879exon

(s)endo-1,4-beta-xylanase

A329aaFGENESH:

37142exon

(s)endo-1,4-beta-xylanase360aaFGENESH:

68502exon

(s)xylanase

I335aaFGENESH:

85581

exon

(s)xylosidase631aaFGENESH:

26133exon

(s)endoglucanase

I746aaFGENESH:

41104exon

(s)endoglucanase

II813aaFGENESH:

2453exon

(s)exo-cellobiohydrolase453aaFGENESH:

61274exon

(s)glucan

1,4-alpha-glucosidase974aaPrimer

DesignsGeneIDFunctionAnnotationPrimers

withoutSignal

PeptidesFGENESH:

3714 2

exon

(s)endo-1,4-beta-xylanaseEBXYL-P:CGCGAATCCCACAATGTGG

ACCTCAATAAGFGENESH:

6850 2

exon

(s)xylanase

IXy

l-P: GAC

GAATTCGCCCCCACTC

CCGAGCTGGCTFGENESH:

8558 1

exon

(s)xylosidaseXYO

L-P:

CGT

GAATTC

ATGGATCCCA

AACCCCTCGT

CAFGENESH:

2613 3

exon

(s)endoglucanase

IEGI

L-P:

CGC

GAATTC

CAGCAGCCGG

CGGTTGCGAACFGENESH:

2453

exon

(s)exo-cellobiohydrolaseCBHI

L-P:

CGC

GAATTC

CAGCAGGTTG

GAACTCAGAAGFGENESH:

6127 4

exon

(s)glucan

1,4-alpha-glucosidaseEG

IIL-P:

CTC

GAATTC

GCTCCTCAGC

TGTCTCCTCGTCloning

andExpression

ofFunctionalGenesAssociated

with

Lignocellulose

DegradationCulture

with

various

substratescDNA

synthesiscDNA

cloning

andysisConstruction

of

expression

vectorsTransformation

to

P.

pastorisConfirmation

ofgenesExpression

iden.

ofgenesSDS-PAGEPurification

ofsecreted

proteinEnzyme

activityassayRealtime

PCRXylosidase(8558)

Genes

Cloning

andysisXylanase

I(6850)

GenesCloningandysis>contig03310

length=12240Candidate

functionalgenesxylosidase

gene(XYO1,

XYO2)

with

length

of1005bp;endoglucanase

gene(EGI1

and

EGI2)

with

length

of1431bpxylanase

(EB1,

EB2)

1080bp;xylanase

gene(XY1

andXY2)

657bpexo-cellobiohydrolase

CBH1,

CBH2)

1359bp;beta-glucosidase

(EG1

and

EG2)

2483bp;glucan

1,4-alpha-glucosidase

(BG1

and

BG2

)

1893bp;Vector

MapVector

Construction

andIdentificationM:

Ikb

ladder;

1,

6,

9

and

12:

control

of

emptyvector;binant

vector

from

8558;2

and

3:

double

enzyme

digestof4

and

5:

double

enzyme

digestof7

and8:

double

enzyme

digestof10

and

11:

double

enzyme

digest

ofbinant

vector

from

6850;binant

vector

from

2613;binant

vector

from

6127;Identification

of

YeastbinantsM:

Ikb

ladder;

Candidate

yeast

positive binants

of

8558,

6850,3714,2613,

and

6127

respectively

by

genome

PCREnzyme

Assay

and

SDS-PAGEResultThe

result

of

SDS-PAGE

ofXYO8558200180160140120100806040200

00

1

2

3

4

5

6

7

8

9Time,

dXylosidase,

IU

mg-120018016014012010080604020Optimal

conditions20

30

40

50

60

70

80100908070605040302010Xylosidase,

IU

mg-1Temperature,

oCThe

enzyme

activity

of

8558

at

differenttempretures10090807060504030201002

41501351201059075604530156

8

10pHXylosidase,

IU

mg-1150135120105907560453015The

enzyme

activity

of

8558

at

different

pHsSummary8,989

geneswerepredicated

from

gDNA

sequence

dataset

and

217

geneswereassociated

with

lignocellulose

degradation;Genes

of

hemicelluloseand

cellulosedegradation

werehighly

overexpressedfrom

four

treatment

conditions

by

the

RNA-seq

elucidation;Comparing

to

the

Trichoderma

reesei

genome

dataset,

P.expansium

YT02

havemore

completeand

complex

lignicellulose

degradation

enzymesystem,especially

hemicellulose

degradation

genes;Different

expressingprofilesindicated

P.expansiumpreferentially

utilizedcellulose

as

substrates

when

cultured

with

switchgrass;Comparingto

other

fungi,

P.

expansium

onlyhave

one

wayto

degrade

thexylan

by

1,4-endoxylanase

metabolism

pathway;Seven

functionalgeneswere

clonedand

expressedin

Pich

toris

forenzymes

products2.2

酶的發(fā)酵工藝條件與控制發(fā)酵生產(chǎn)的優(yōu)勢發(fā)酵罐及培養(yǎng)基發(fā)酵條件及控制提高產(chǎn)酶的措施發(fā)酵方法1、固體培養(yǎng)發(fā)酵設(shè)備簡單、污染少、霉菌2、液體 發(fā)酵純度高、產(chǎn)率高、易控制、機械化高、回收率高3、固定化細胞發(fā)酵產(chǎn)酶高、穩(wěn)定性好、可重復、易純化4、固定化原生 發(fā)酵2.2.1微生物發(fā)酵生產(chǎn)法的優(yōu)點酶的品種齊全酶的產(chǎn)量高生產(chǎn)成本低便于提高酶制品獲得率2.2.1保藏細胞細胞活化細胞擴大培養(yǎng)發(fā)酵分離純化酶固定化細胞原生固定化原生培養(yǎng)基預(yù)培養(yǎng)無菌空氣微生物發(fā)酵產(chǎn)酶的工藝流程2.2.1發(fā)酵灌的類型：傳統(tǒng)的、自吸式、氣生式、內(nèi)外循環(huán)等2.2.2：碳源、氮源、無機鹽、生長因子、水培養(yǎng)基（medium）是人工配制的，適合微生物生長繁殖或產(chǎn)生代謝產(chǎn)物的營養(yǎng)基質(zhì)。培養(yǎng)基幾乎 對微生物進行研究和利用工作的基礎(chǔ)。任何培養(yǎng)基都應(yīng)該具備微生物生長所需要五大營養(yǎng)要素2.2.2對營養(yǎng)的需求：培養(yǎng)基組成異養(yǎng)微生物自養(yǎng)微生物碳源糖、醇、有機酸等、碳酸鹽等氮源蛋白質(zhì)及其降解物、有機氮化物、無機氮化物、氮無機氮化物、氮生長因子有些需要維生素等生長因子不需要無機元素無機鹽無機鹽水分水水能源與碳源同氧化無機物或利用日光能1、選擇適宜的營養(yǎng)物質(zhì)2、營養(yǎng)物的濃度及配比合適3、物理、化學條件適宜4、經(jīng)濟節(jié)約5、精心設(shè)計、試驗比較培養(yǎng)不同的微生物必須采用不同的培養(yǎng)條件；培養(yǎng)目的不同，原料的選擇和配比不同；不同階段，培養(yǎng)條件也有所差異。培養(yǎng)基的設(shè)計原則2.2.2水水是微生物最基本的組成分（７０％—９０％）；水是微生物體內(nèi)和體外的溶劑（吸收營養(yǎng)成分和代謝廢物）；水是細胞質(zhì)組分，直接參與各種代謝活動；調(diào)節(jié)細胞溫度和保持環(huán)境溫度的穩(wěn)定（比熱高，傳熱快）。碳源營養(yǎng)物質(zhì)、能量及酶的組成大多數(shù)產(chǎn)酶微生物以淀粉或其水解物為碳源；異養(yǎng)微生物：糖類是最好碳源（葡萄糖最為通用）。碳源對酶

的生物調(diào)節(jié)：阻遏、誘導。氮源構(gòu)成細胞物質(zhì)和代謝產(chǎn)物中氮素（不能用作能源）有機氮源

蛋白胨、酵母膏、牛肉膏氮源無機氮源

銨鹽、硝酸鹽需要注意合適的碳氮比無機鹽參與酶的組成、構(gòu)成酶活性基、激活酶活性維持細胞結(jié)構(gòu)的穩(wěn)定性調(diào)節(jié)細胞滲透壓控制細胞的氧化還原電位有時可作某些微生物生長的能源物質(zhì)常用：硫酸鹽、磷酸鹽、氯化物以及含有鉀、鈉、鈣、鎂、鐵等元素的化合物。生長因子生長因子是指某些微生物不能用普通的碳源、氮源物質(zhì)進行

，而必須另外加入少量的生長需求的有機物質(zhì)。分類：化學結(jié)構(gòu)分成維生素、氨基酸、嘌呤（或嘧啶）及其衍生物和類脂成分等四類功能：以輔酶與輔基的形式參與代謝中的酶促反應(yīng)中常用酵母膏、蛋白胨、牛肉膏等作為各種生長因子的的需要，麥芽汁、米曲汁等天然培養(yǎng)基中本身含有各種生長因子的常用培養(yǎng)基細

菌:

牛肉膏蛋白胨培養(yǎng)基放線菌：高氏1號

培養(yǎng)基培養(yǎng)；酵母菌：麥芽汁培養(yǎng)基；霉

菌：查氏

培養(yǎng)基.2.2.3發(fā)酵條件及控制（1）pH值對產(chǎn)酶的影響與調(diào)節(jié)控制不同類型微生物的生長繁殖或產(chǎn)生代謝產(chǎn)物要求不同pH的培養(yǎng)基。通常培養(yǎng)條件：細菌與放線菌：pH6.5-8.0

酵母菌和霉菌：pH4.5-6細胞發(fā)酵產(chǎn)酶的最適pH值與生長最適pH值往往有所不同。細胞生產(chǎn)某種酶的最適pH值通常接近于該酶催化反應(yīng)的最適pH值?？莶輻U菌堿性磷酸酶：pH9.5最適催化；pH7.4最適生長有些細胞可以同時產(chǎn)生若干種酶，在生產(chǎn)過程中，通過控制培養(yǎng)基的pH值，往往可以改變各種酶之間的產(chǎn)量比例。黑曲霉：pH7.0-淀粉酶；pH5-6-糖化酶維持培養(yǎng)基pH的方法改變培養(yǎng)基組分和比例；

在培養(yǎng)基中加入pH緩沖劑；在進行工業(yè)發(fā)酵時補加酸、堿；最成功的是補料穩(wěn)定pH、解除產(chǎn)物的阻遏作用、補充營養(yǎng)物質(zhì)。通常在生物學范圍內(nèi)每升高10℃，生長速度就加快一倍，所以溫度直接影響酶反應(yīng)，對于微生物來說，溫度直接影響其生長和酶。有些細胞發(fā)酵產(chǎn)酶的最適溫度與細胞生長最適溫度有所不同，而且往往低于生長最適溫度。這是由于在較低的溫度條件下，可以提高酶所對應(yīng)的mRNA的穩(wěn)定性，增加酶生物的延續(xù)時間，從而提高酶的產(chǎn)量。（2）溫度的影響與調(diào)控枯草桿菌的最適生長溫度為34～37℃黑曲霉的最適生長溫度為28～32

℃調(diào)節(jié)溫度的方法熱水升溫冷水降溫熱交換裝置：排管、蛇管、噴淋管等。隨時準備冷、熱水。（3）溶解氧的控制在酶的發(fā)酵生產(chǎn)過程中，處于不同生長階段的細胞，其細胞濃度和細胞呼吸強度各不相同，致使耗氧速率有很大的差別。因此必須根據(jù)耗氧量的不同，不斷供給適量的溶解氧。培養(yǎng)液中溶解氧的量，決定于在一定條件下氧氣的溶解速度。溶氧速率與通氣量、氧氣分壓、氣液接觸時間、氣液接觸面積以及培養(yǎng)液的性質(zhì)等有密切關(guān)系。調(diào)節(jié)通氣量調(diào)節(jié)氧的分壓調(diào)節(jié)氣液接觸時間調(diào)節(jié)氣液接觸面積改變培養(yǎng)液的性質(zhì)控制溶解氧方法臨界氧濃度（4）的影響排除阻礙影響氧溶解發(fā)酵液外溢限制裝料量選不易產(chǎn)菌種、調(diào)節(jié)培養(yǎng)基成分、機械消泡或化學消泡2.2.4

提高酶產(chǎn)量的措施（1）添加誘導物對于誘導酶的發(fā)酵生產(chǎn)，在發(fā)酵過程中某個適宜的

時機，添加適宜的誘導物，可以顯著提高酶的產(chǎn)量。乳糖誘導β-半乳糖苷酶纖維二糖誘導纖維素酶誘導物一般可以分為3類酶的作用底物——纖維二糖酶的催化反應(yīng)產(chǎn)物——半乳糖醛酸作用底物的類似物——IPTG（2）控制阻遏物的濃度為了減少或者解除分解代謝物阻遏作用，應(yīng)當控制培養(yǎng)基中葡萄糖等容易利用的碳源的濃度。采用其他較難利用的碳源，如淀粉等采用補料、分次流加碳源添加一定量的環(huán)腺苷酸（cAMP）對于受代謝途徑末端產(chǎn)物阻遏的酶，可以通過控制末端產(chǎn)物的濃度的方法使阻遏解除。（3）添加表面活性劑表面活性劑可以與細胞膜相互作用，增加細胞的透過性，有利于胞外酶的

，從而提高酶的產(chǎn)量。將適量的非離子型表面活性劑，如吐溫（Tween）、特里頓(Triton)等添加到培養(yǎng)基中，可以加速胞外酶的，而使酶的產(chǎn)量增加。由于離子型表面活性劑對細胞

害作用，對細胞的毒性較大，不能在酶的發(fā)酵生產(chǎn)中添加到培養(yǎng)基中。（4）添加產(chǎn)酶促進劑產(chǎn)酶促進劑是指可以促進產(chǎn)酶、但是作用機理未闡明清楚的物質(zhì)。植酸鈣鎂，可使霉菌蛋白酶或者桔青霉磷酸二酯酶的產(chǎn)量提高1～20倍；聚乙烯醇可以提高糖化酶的產(chǎn)量。產(chǎn)酶促進劑對不同細胞、不同酶的作用效果各不相同，現(xiàn)在還沒有規(guī)律可循，要通過試驗確定所添加的產(chǎn)酶促進劑的種類和濃度。微生物發(fā)酵生產(chǎn)法中尚待解決的問題消除毒性優(yōu)良產(chǎn)酶菌種的篩選、培育回本節(jié)2.3酶生產(chǎn)過程的動力學酶生物

的模式酶生產(chǎn)過程中細胞生長