酶工程-2微生物发酵产酶

Chapter

2

微生物发酵产酶The

production

of

Enzyme

byFermentation

ofMicroorganismContents

ofchapter

22.3、酶生产过程的动力学2.4、酶的生物

及调节（>、产酶微生物、酶的发酵工艺条件及控制动物、植物生产酶制剂的缺陷：植物由于生长地域、季节、气候等的影响，生产酶制剂的产量、质量都不稳定。动物产生的酶主要从屠宰牲畜的腺体中提取，来源有限。2.1.1

产酶微生物的概述微生物的优势种类繁多生长周期短繁殖快培养相对简单较强的适应性和突变能力产酶微生物来源土壤水体空气环境环境微生物嗜冷菌嗜酸菌嗜盐菌产酶微生物种类细菌bacteria青霉素酰化酶溶菌酶

淀粉酶放线菌actinomycetes蛋白酶

葡萄糖异构酶抗生素酵母菌yeast糖化酶淀粉酶蛋白酶霉菌mold淀粉酶果胶酶水解酶产酶微生物的基本要求不是致病菌，不产生毒素及有害物质；发酵周期短，生产成本低，产酶量高；遗传性稳定，不易变异

，不

噬菌体；最好是产生胞外酶的菌种，利于分离纯化；对和食品用酶，还应考虑安全性；工程菌必须符合安全性要求。2.1.2

常见产酶微生物类别菌名产酶用途细大肠杆菌青霉素酰化酶谷氨酸脱羧酶半乳糖苷酶半

青霉素或头孢霉素调味剂分解乳糖枯草杆菌淀粉酶蛋白酶、啤酒及洗涤剂等生丝脱胶、皮革脱毛、酱油酿造菌乳酸杆菌葡萄糖异构酶由葡萄糖制果糖芽胞杆菌碱性蛋白酶皮革脱毛、洗涤剂霉点青霉葡萄糖氧化酶食品保鲜、去氧黑曲霉酸性蛋白酶啤酒澄清、毛皮软化、菌河内根霉葡萄糖苷酶制葡萄糖里氏木霉纤维素酶单糖、糊精、能源化酶酵母假丝酵母脂肪酶绢丝脱脂、洗涤剂、

、乳品增香放线菌

白色放线菌

蛋白酶皮革脱毛(1)大肠杆菌操作、生长迅大肠杆菌的优势主要因它易于在速，而且营养要求低。应用：大肠杆菌也是最早用作 工程的宿主菌；工业上生产谷氨酸脱羧酶、天冬酰胺酶、青霉素酰化酶、半乳糖苷酶等；DNA聚合酶、连接酶、限制性内切酶等；大多数是胞内酶，需进行细胞破碎。(2)醋酸杆菌（Acetobacter）菌体从椭圆至杆状，单个、成对或成链，革兰氏，运动（周毛）或不运动，不生芽孢。好气。含糖、乙醇和酵母膏的培养基上生长良好。应用：有机酸(食醋等）葡萄糖异构酶(高果糖浆)山梨糖(维C

）。(3)枯草芽孢杆菌（Bacillus

subtilis）直状、近直状的杆菌，周生或侧生鞭毛，革兰氏阳性，无荚膜，芽孢0.5×1.51.8m。枯草芽孢杆菌是工业发酵的重要菌种之一。生产淀粉酶、蛋白酶、某些氨基酸及核苷、碱性磷酸酶。胞外酶啤酒、洗涤剂、生丝脱胶、酱油酿造等(4)链霉菌(Actinomycetes)应用：生产葡萄糖异构酶；青霉素酰化酶、纤维素酶、几丁质酶等。(5)根霉(Rhizopus)分布于土壤、空气中，常见于淀粉食品上，可引起霉腐变质和水果、蔬菜的腐烂。代表种：米根霉（R.oryzae)、黑根霉(R.nigrican)等。应用：产生如淀粉酶、果胶酶、脂肪酶等。在酿酒工业上常用做糖化菌。有些根霉还能产生乳酸延胡索酸等有机酸。(6)曲霉(Aspergillus)分类：多数属于子囊菌亚门，少数属于半知菌亚门。分布：广泛分布于土壤、空气和谷物上，可引起食物、谷物和果蔬的霉腐变质，有的可产生性的黄曲霉毒素。代表种：黑曲霉Asp.Niger、黄曲霉

Asp.flavus应用：是制酱、酿酒、制醋的主要菌种。是生产酶制剂（蛋白酶淀粉酶、果胶酶）的菌种。生产有机酸（如柠檬酸、葡萄糖酸等）。农业上用作生产糖化饲料的菌种。应用：啤酒、酒类生产；生产转化酶、酸脱羧酶、醇脱氢酶等。(7)酿酒酵母(Saccharomyces

cerevisiae)蛋白酶59%糖化酶13%5%葡萄糖异构酶6%淀粉酶果胶酶3%纤维素酶1%脂肪酶3%与分析研究用酶10%不同类型工业酶的比例研究及查阅资料平板分离设计试验方案原种斜面目标菌株初筛复筛

再复筛生产性试验毒性试验

菌种鉴定符合工业生产要求符合安全性要求

菌种保藏单株纯种分离生产2.1.3

产酶微生物的分离与筛选（1）含菌样品获得能降解某物质的产酶菌，可从该物质分布丰富的地方寻找；纤维素酶：腐烂纤维素或堆肥中果胶酶：腐烂的水果、蔬菜胞外酶的稳定性和最适条件与菌的最适生长条件接近。（2）菌的分离纯化平板划线稀释分离（3）产酶性能测定。初筛：选出含有目的酶菌株，平板培养透明圈法；复筛：选出产酶量高、性能更符合要求的菌株22酶的催化作用受到底物浓度、酶浓度、温度、pH值、激活剂浓度、抑制剂浓度等诸多因素的影响。在酶的应用过程中，必须控制好各种环境条件，以充分发挥酶的催化功能。影响酶催化的各种因素23温度最优温度活力％温度/℃酶活性的最佳温度pH以pH值为变量的几种酶的活性函数24pH活力胃蛋白酶蔗糖酶胰岛素251、酶2、酶与酶反应速度；单位1961年国际生物化学与分子生物合会规定：在特定条件下（温度可采用25℃或其它选用的温度，pH等条件均采用最适条件），每1min催化1μmol的底物转化为产物的酶量定义为1个酶单位。这个单位称为国际单位（U）。3、酶

测定方法反应体系及底物选择最适反应条件的确定反应终止及活性检测酶测定26吸光度法辐射线测定发光法测定荧光光度法电位测定法电导测定法旋光测定法固定化酶量热法酶分析方法27酶制剂的质量评估质量指标：活度、纯度、稳定性、配方和包装比活：与其蛋白含量相关的催化活性（U/mg）蛋白质测定污染活性：原料酶中含有的其他酶量性能测试氨基酸分析和蛋白质序列分析稳定性酶制剂的剂型：根据应用采用不同的剂型。2.1.4

产酶微生物优良菌种的选育诱变育种0.2%

LiCl+

UV120S+

2mg/ml

NTG(40min)原生

融合育种工程育种洗牌法（gene

shuffling）定向进化（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

酶的发酵工艺条件与控制发酵生产的优势发酵罐及培养基发酵条件及控制提高产酶的措施发酵方法1、固体培养发酵设备简单、污染少、霉菌2、液体 发酵纯度高、产率高、易控制、机械化高、回收率高3、固定化细胞发酵产酶高、稳定性好、可重复、易纯化4、固定化原生 发酵2.2.1微生物发酵生产法的优点酶的品种齐全酶的产量高生产成本低便于提高酶制品获得率2.2.1保藏细胞细胞活化细胞扩大培养发酵分离纯化酶固定化细胞原生固定化原生培养基预培养无菌空气微生物发酵产酶的工艺流程2.2.1发酵灌的类型：传统的、自吸式、气生式、内外循环等2.2.2：碳源、氮源、无机盐、生长因子、水培养基（medium）是人工配制的，适合微生物生长繁殖或产生代谢产物的营养基质。培养基几乎 对微生物进行研究和利用工作的基础。任何培养基都应该具备微生物生长所需要五大营养要素2.2.2对营养的需求：培养基组成异养微生物自养微生物碳源糖、醇、有机酸等、碳酸盐等氮源蛋白质及其降解物、有机氮化物、无机氮化物、氮无机氮化物、氮生长因子有些需要维生素等生长因子不需要无机元素无机盐无机盐水分水水能源与碳源同氧化无机物或利用日光能1、选择适宜的营养物质2、营养物的浓度及配比合适3、物理、化学条件适宜4、经济节约5、精心设计、试验比较培养不同的微生物必须采用不同的培养条件；培养目的不同，原料的选择和配比不同；不同阶段，培养条件也有所差异。培养基的设计原则2.2.2水水是微生物最基本的组成分（７０％—９０％）；水是微生物体内和体外的溶剂（吸收营养成分和代谢废物）；水是细胞质组分，直接参与各种代谢活动；调节细胞温度和保持环境温度的稳定（比热高，传热快）。碳源营养物质、能量及酶的组成大多数产酶微生物以淀粉或其水解物为碳源；异养微生物：糖类是最好碳源（葡萄糖最为通用）。碳源对酶

的生物调节：阻遏、诱导。氮源构成细胞物质和代谢产物中氮素（不能用作能源）有机氮源

蛋白胨、酵母膏、牛肉膏氮源无机氮源

铵盐、硝酸盐需要注意合适的碳氮比无机盐参与酶的组成、构成酶活性基、激活酶活性维持细胞结构的稳定性调节细胞渗透压控制细胞的氧化还原电位有时可作某些微生物生长的能源物质常用：硫酸盐、磷酸盐、氯化物以及含有钾、钠、钙、镁、铁等元素的化合物。生长因子生长因子是指某些微生物不能用普通的碳源、氮源物质进行

，而必须另外加入少量的生长需求的有机物质。分类：化学结构分成维生素、氨基酸、嘌呤（或嘧啶）及其衍生物和类脂成分等四类功能：以辅酶与辅基的形式参与代谢中的酶促反应中常用酵母膏、蛋白胨、牛肉膏等作为各种生长因子的的需要，麦芽汁、米曲汁等天然培养基中本身含有各种生长因子的常用培养基细

菌:

牛肉膏蛋白胨培养基放线菌：高氏1号

培养基培养；酵母菌：麦芽汁培养基；霉

菌：查氏

培养基.2.2.3发酵条件及控制（1）pH值对产酶的影响与调节控制不同类型微生物的生长繁殖或产生代谢产物要求不同pH的培养基。通常培养条件：细菌与放线菌：pH6.5-8.0

酵母菌和霉菌：pH4.5-6细胞发酵产酶的最适pH值与生长最适pH值往往有所不同。细胞生产某种酶的最适pH值通常接近于该酶催化反应的最适pH值。枯草杆菌碱性磷酸酶：pH9.5最适催化；pH7.4最适生长有些细胞可以同时产生若干种酶，在生产过程中，通过控制培养基的pH值，往往可以改变各种酶之间的产量比例。黑曲霉：pH7.0-淀粉酶；pH5-6-糖化酶维持培养基pH的方法改变培养基组分和比例；

在培养基中加入pH缓冲剂；在进行工业发酵时补加酸、碱；最成功的是补料稳定pH、解除产物的阻遏作用、补充营养物质。通常在生物学范围内每升高10℃，生长速度就加快一倍，所以温度直接影响酶反应，对于微生物来说，温度直接影响其生长和酶。有些细胞发酵产酶的最适温度与细胞生长最适温度有所不同，而且往往低于生长最适温度。这是由于在较低的温度条件下，可以提高酶所对应的mRNA的稳定性，增加酶生物的延续时间，从而提高酶的产量。（2）温度的影响与调控枯草杆菌的最适生长温度为34～37℃黑曲霉的最适生长温度为28～32

℃调节温度的方法热水升温冷水降温热交换装置：排管、蛇管、喷淋管等。随时准备冷、热水。（3）溶解氧的控制在酶的发酵生产过程中，处于不同生长阶段的细胞，其细胞浓度和细胞呼吸强度各不相同，致使耗氧速率有很大的差别。因此必须根据耗氧量的不同，不断供给适量的溶解氧。培养液中溶解氧的量，决定于在一定条件下氧气的溶解速度。溶氧速率与通气量、氧气分压、气液接触时间、气液接触面积以及培养液的性质等有密切关系。调节通气量调节氧的分压调节气液接触时间调节气液接触面积改变培养液的性质控制溶解氧方法临界氧浓度（4）的影响排除阻碍影响氧溶解发酵液外溢限制装料量选不易产菌种、调节培养基成分、机械消泡或化学消泡2.2.4

提高酶产量的措施（1）添加诱导物对于诱导酶的发酵生产，在发酵过程中某个适宜的

时机，添加适宜的诱导物，可以显著提高酶的产量。乳糖诱导β-半乳糖苷酶纤维二糖诱导纤维素酶诱导物一般可以分为3类酶的作用底物——纤维二糖酶的催化反应产物——半乳糖醛酸作用底物的类似物——IPTG（2）控制阻遏物的浓度为了减少或者解除分解代谢物阻遏作用，应当控制培养基中葡萄糖等容易利用的碳源的浓度。采用其他较难利用的碳源，如淀粉等采用补料、分次流加碳源添加一定量的环腺苷酸（cAMP）对于受代谢途径末端产物阻遏的酶，可以通过控制末端产物的浓度的方法使阻遏解除。（3）添加表面活性剂表面活性剂可以与细胞膜相互作用，增加细胞的透过性，有利于胞外酶的

，从而提高酶的产量。将适量的非离子型表面活性剂，如吐温（Tween）、特里顿(Triton)等添加到培养基中，可以加速胞外酶的，而使酶的产量增加。由于离子型表面活性剂对细胞

害作用，对细胞的毒性较大，不能在酶的发酵生产中添加到培养基中。（4）添加产酶促进剂产酶促进剂是指可以促进产酶、但是作用机理未阐明清楚的物质。植酸钙镁，可使霉菌蛋白酶或者桔青霉磷酸二酯酶的产量提高1～20倍；聚乙烯醇可以提高糖化酶的产量。产酶促进剂对不同细胞、不同酶的作用效果各不相同，现在还没有规律可循，要通过试验确定所添加的产酶促进剂的种类和浓度。微生物发酵生产法中尚待解决的问题消除毒性优良产酶菌种的筛选、培育回本节2.3酶生产过程的动力学酶生物

的模式酶生产过程中细胞生长