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 : 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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