Patent Application: US-201313849465-A

Abstract:
the present invention relates to protein engineering , and concerns especially family g / 11 xylanases , and genes encoding said enzymes . in specific , the invention concerns trichoderma reesei xynii gene , which codes for endo - 1 , 4 - β - xylanase . the invention describes how site - directed mutagenesis can be used to improve the properties of an enzyme to match the industrial conditions where it is used . protein engineering can be used to improve thermoactivity and thermostability of xylanases , as well as to broaden their ph range .

Description:
the family g / 11 xylanases originating from bacteria , yeast and fungi have common molecular structure . examples of such xylanases are : aspergillus niger xyna aspergillus kawachii xync aspergillus tubigensis xyna bacillus circulans xyna bacillus pumilus xyna bacillus subtilis xyna neocallimastix patriciarum xyna streptomyces lividans xynb streptomyces lividans xync streptomyces thermoviolaceus xynii thermomonospora fusca xyna trichoderma harzianum xyn trichoderma reesei xyni , trichoderma reesei xynii trichoderma viride xyn the invention deals with xylanases of the family g / 11 with the following common features : enzymes in which the n - terminal sequence is a part of the double - layered β - sheet ( in the family 11 xylanases the a - and the b - sheet , ( gruber , et al ., 1998 )) and in which the first β - strand ( in t . reesei xynii the amino acids 5 - 10 ) or the n - terminal end can be bound by disulfide bridges either to the adjacent β - strands ( in t . reesei xynii the amino acids 13 - 19 ) or to other neighbouring regions . ( ii ). enzymes in which the c - terminal peptide chain forms a β - strand ( in t . reesei xynii amino acids 183 - 190 ), which is a part of a larger β - sheet and in which the c - terminal region can be bound by disulfide bridges to the adjacent β - strands or by salt bridges to the body of the enzyme . ( iii ). enzymes which have an α - helix on the other side of the enzyme structure with regard to the catalytic canyon , and wherein said α - helix or the neighbouring regions can be bound more tightly by a disulfide bridge to the body of the protein . the t . reesei xylanase ii has the above mentioned properties and in said enzyme thermostability , ph - stability and thermoactivity can be modified based on these properties . the following changes have been made to the xylanase gene ( xynii ) of t . reesei : by site - directed mutagenesis disulfide bridges are formed in the n - terminal region : threonines 2 and 28 are changed to cysteines resulting in a disulfide bridge being formed between them ( t2c and t28c ). proline 5 and asparagine 19 are changed to cysteines resulting in a disulfide bridge being formed between them ( p5c and n19c ). threonine 7 and serine 16 are changed to cysteines resulting in a disulfide bridge being formed between them ( t7c and s16c ). asparagine 10 and asparagine 29 are changed to cysteines resulting in a disulfide bridge being formed between them ( n10c and n29c ). by site - directed mutagenesis , the c - terminus is bound more tightly to the body of the enzyme by adding as a recombinant change one amino acid ( e . g . aspartic acid or glutamic acid ) to the c - terminus of the xylanase , which then forms a salt bridge from the c - terminus to the body of the enzyme . if appropriate , a suitable amino acid replacement can be made in the body of the protein , so as to enable the formation of a salt bridge . an aspartic acid (+ 191d ) is added to the c - terminal serine ( s 190 ). this results in a salt bridge with arginine at position 58 , where wild - type lysine has been replaced by arginine ( k58r ). by site - directed mutagenesis at least one disulfide bridge is formed to stabilise the enzyme in the c - terminal part via the α - helix or a region near the α - helix . leucine 105 and glutamine 162 are changed to cysteines resulting in disulfide bridge between them ( l105c and q162c ). by site - directed mutagenesis point mutations are made to increase the stability of t . reesei xylanase ii : n11d , t26r , g30h , n67r , n97r , a132r , n157r , a160r , t165n , m169h , s186r . production of mutated and recombinant xynii genes were carried out by the following general procedures : t . reesei xylanase ii was produced in e . coli strains xl1 - blue or rv308 using the vector pkktac ( vtt , espoo , finland ) or the vector palk143 ( roal , rajamäki , finland ). t . reesei xynii gene was directly cloned by pcr from the cdna of t . reesei to the vector pkktac ( induction of expression by iptg ). alternatively , the plasmid palk143 was used which contains t . reesei xynii gene . both of the vectors secrete the xylanase into the medium ; the vector pkktac by pectate lyase ( pelb ) signal sequence and the vector palk143 by amylase signal sequence . the production of mutated t . reesei xynii gene used in the examples of this application , was effected as follows : mutations were produced by polymerase chain reaction ( pcr ) using oligonucleotide primers which contained the sequences for the changed codons . examples of the used oligonucleotides are given in fig1 , as well as in the appended sequence listing as sequences 1 to 12 . pcr using the primers ( containing the desired mutation ) was carried out by quick change method ( stratagene , westburg , leusden , the netherlands ) and by generally known methods . pfuturbo was used as dna polymerase ( stratagene , la jolla , calif ., usa ). the cloned e . coli strains were cultivated on plates containing xylan ( birchwood xylan : sigma , steinheim , germany ) coupled to rhemazol brilliant blue . the xylanase activity could be seen as halos around the colonies ( biely et al ., 1985 ). the xylanase activity of enzyme samples was determined by measuring the amount of reducing sugars released from the hydrolysed xylan fibre . the reducing sugars were measured by dns - method in 50 mm citrate - phosphate buffer ( bailey et al ., 1992 ). standard activity determination was carried out at ph 5 and 50 ° c . the stability of the xylanases was tested by measuring the half - life of the modified enzymes at different temperatures . the enzyme was incubated for varying times at 55 or 65 ° c . and the residual activity was measured as described above . the stability at high temperatures was also measured by incubating the enzymes for 10 mm at varying temperatures and subsequently measuring the residual activity by dns - method . the ph - dependent xylanase activity was measured by determining the enzyme activity in varying ph - values . the temperature optimum of the enzyme was determined by measuring the activity at varying temperatures ( 10 mm , ph 5 ). the properties of the mutated enzymes were compared to the wild - type t . reesei xynii enzyme . the three - fold mutations ( t2c , t28c and k58r ) and the recombinant change (+ 191d ) were made in t . reesei xynii by using the methods described above . the mutant enzyme was designated as y5 . said mutant enzyme was expressed in e . coli , which was cultivated at + 37 ° c . in shake flasks using luria broth as growth medium . after cultivation the cells were removed by centrifugation and the xylanase secreted into the medium was characterized in varying conditions , as described above . fig2 shows the effect of the temperature to the enzyme activity when the mutant y5 ( t2c , t28c , k58r , + 191d ) and the wild - type ( t . reesei xynii ) enzyme were incubated for 10 min with birchwood xylan in varying temperatures , and the relative amount of the reducing sugars as released were measured with dns - method . said mutations improved the temperature optimum of xylanase by about 15 ° c . the three - fold mutant xylanase ( t2c , t28c , k58r , + 191d ) described in example 1 was incubated for 10 mm in 1 % birchwood xylan at 50 ° c . in citrate - phosphate buffer in varying ph - values . fig3 shows the relative amount of reducing sugars as released for the mutant and the wild - type xylanases . the mutations broadened slightly the ph - dependent activity of the enzyme to alkaline direction . the mutant enzyme was more active than the wild - type enzyme at ph 7 - 8 ; the activity of mutant enzyme was about 20 % higher at ph 8 ( 50 ° c .). the above - mentioned three - fold mutant ( t2c , t28c , k58r , + 191d ) and the wild - type enzyme were incubated for 10 min at varying temperatures . after the incubation the samples were cooled and the residual activity was determined in standard conditions . the wild - type enzyme was completely inactivated already at 55 - 60 ° c . the mutant enzyme retained about 50 % of its activity even at 65 ° c . ( fig4 ). table 1 below shows the half - lives ( t½ ) of the mutant ( y5 ) and the wild - type xylanase at 55 ° c . and 65 ° c . with the above - mentioned methods a disulfide bridge was made ( l105c and q162c ) to bind the c - terminus of the α - helix to the neighbouring β - strand . the enzyme was produced in e . coli and its properties were determined . fig5 shows the inactivation of the mutant enzyme at different temperatures compared to the wild - type enzyme . at 55 ° c . the stability of the mutated enzyme increased about 20 - fold , with regard to the wild - type enzyme , whereby the half - life increased from 5 min ( the wild - type enzyme ) up to about 1 . 5 hours ( the mutated enzyme ). arase , a ., yomo , t ., urabe , i ., hata , y ., katsube , y . & amp ; okada , h . ( 1993 ). stabilization of xylanase by random mutagenesis . febs letters 316 , 123 - 7 . bailey , j . m ., biely , p . & amp ; poutanen , k . 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