橄榄绿链霉菌Streptomyces olivaceoviridis A1所产木聚糖酶的纯化、酶基因的克隆及表达
摘要
木聚糖酶可将木聚糖降解成低聚糖和木糖。木聚糖酶在饲料、制浆造纸、食品、能源工业中都显示出广阔的应用前景。许多微生物可产生木聚糖酶。我们选择的实验材料是一株橄榄绿链霉菌A1(Streptomyces olivaceoviridis A1)。此菌株能胞外分泌木聚糖酶。从它的培养液中分离纯化出两种木聚糖酶：43kD的XYNA及23kD的XYNB。XYNA的最适温度为60℃；最适pH为5.6；在55℃下以4-O-Me-D-glucurono-D-xylan的K_m值和V_(max)分别是3.32(g/Kg)和15.72(μmol／ml·min)；SDS、EDTA对它有轻微的抑制作用；XYNA无纤维素酶活性；胃蛋白酶、胰蛋白酶处理30分钟，对酶活性无影响。XYNB的最适pH为5.2，最适温度为60℃，在55℃下以4-O-Me-D-glucurono-D-xylan为底物的K_m值和V_(max)分别为22.1(g/Kg)和105.26μmol／ml·min。Mg~(2+)、Cr~(3+)对XYNB有轻微的激活作用。XYNB无纤维素酶活性。胃蛋白酶、胰蛋白酶37℃下处理30分钟，对酶活性无影响。将纯化出的XYNA及XYNB进行N端序列测定。结果XYNAN端十个氨基酸序列与已发表的木聚糖酶FXYN的100％同源，所以直接根据所发表的编码FXYN的基因全序列资料，设计和合成了二段引物，利用PCR技术，克隆出xynA1全序列，测序结果与发表的编码FXYN的基因序列完全一样，再分别克隆出不含信号肽的xynA1-w和含有信号肽的xynA1-s序列，并分别将两段基因克隆进大肠杆菌表达载体pET-22b(+)上，在大肠杆菌BL21(DE3)中都得到特异性表达，并且都能检测到木聚糖酶活性。将xynA1-w序列克隆进酵母表达载体上，在毕赤酵母GS115中得到高效特异性表达。XYNB N端十个氨基酸序列与已发表的最高同源性为90％，根据N端序列测定结果及C端保守序列分别设计了两段引物，经PCR扩增出编码XYNB成熟蛋白的核苷酸序列。将此序列克隆进表达载体pET-22b(+)上，在大肠杆菌BL21(DE3)中也得到特异性表达，并有正常的生物学活性，证明了基因xynB1的生物学功能。将xynB1序列克隆进酵母表达载体pPIC9α上，在毕赤酵母GS115中得到高效特异性表达。对xynA1与xynB1的核苷酸序列进行分析，发现它们之间的同源性仅为40％，并可以断定XYNA属于F／10组木聚糖酶，而XYNB属于G／11组木聚糖酶。
将所测出的基因xynB1的成熟蛋白部分核苷酸序列与GeneBank上的木聚糖酶基因序列进行同源比较，最高同源性为86％，氨基酸序列最高同源性也为86％，并且与之同源性最高的基因所表达的酶酶学性质与XYNB酶学性质有较大差异。综合这些因素，可证明xynB1为新基因。此序列已被EMBL收录，收录号：AJ292317。
Xylanases can hydrolyze xylans into xylooligosaccharides and D-xylose. They have
wide commercial aPplications in industrial processes, such as feed, paPer and pulp,
foodstuff and energy industry. Stroptomyces olivaceovirch Al from soil produce two
extracellular xylanase XYNA of the molecular weigh 43kD and XYNB of the molecular
weight 23kD. Optimal pH Value and temperature of XYNA fOr its activity were 5.6 and 60
OC, respectively. The Km Values and Vm. of XYNA for 4- O - Me - D - gl ucurono -D - xyl an
under 55C were 3.32g/Kg and l5.72pmol/ml. min, respectively. The activity of XYNA
could be decreased slighly by SDS and EDTA. Optimal pH Value and temperatUre of
XYNB for its activity were 5.2 and 60aC, respectively. The Km Values and Vm. of XYNB
for 4- O -Me-D-glucurono-D-xylan under 55C were 22.lg/Kg and l05.26pmoUml' min,
respectively. Mg'+ and Cr3+ can increased the activity of XYNB slighly. Both XYNA and
XYNB didn't display cellulase activity Both XYNA and XYNB were treated by pepsin
or trypsin fOr 30 min under 37C, their activity weren't decreased. The N-terminal amino
acids sequences of XYNB and XYNA were determined by axnino acids sequence analysis.
The result show that the ten N-terminal amino acids sequences of XYNA are same as those
of FXYN published by Atsushi. According to the published data, three primers had been
desighed and synthesized, xyndj-s including siginal sequence and xynA!-w
non-including siginal sequence had been cloned by PCR. According to the determined
N-terminal amino acids sequence of XYNB and the C-terminal conservative amino acids
sequence of G/ll family xylanase. Two primers were desighed and synthesized. XynBj
coding nature protein of XYNB have been cloned by PCR. Then xynd1-s nynA]-w and
xynB1 were all cloned into the plasmid vector pET-22b(+), and were all expressed in E coli.
rynAI-w and xynB1 were all cloned into the plasmid vector pPIC9 Q,and were all
overexpressed in pichia pastoris GS l 1 5.
The nucleic acids sequence and amino acids sequence of xynBi were homologously
analysed with the sequences coding fOr xylanases published in GeneBank. The result
indicated that xynB] was the highest similarity to the gene xyli from sm ptomyces sP.
Strain S38, up to 86%. And the properties of XYLl was different in those of XYNB. So,it
proves that xynBi is a new gene encoding xylanase. The sequence of xynB] has been
accepted by EMBL(Accession number is AJ2923 l7).
将所测出的基因xynB1的成熟蛋白部分核苷酸序列与GeneBank上的木聚糖酶基因序列进行同源比较，最高同源性为86％，氨基酸序列最高同源性也为86％，并且与之同源性最高的基因所表达的酶酶学性质与XYNB酶学性质有较大差异。综合这些因素，可证明xynB1为新基因。此序列已被EMBL收录，收录号：AJ292317。
Xylanases can hydrolyze xylans into xylooligosaccharides and D-xylose. They have
wide commercial aPplications in industrial processes, such as feed, paPer and pulp,
foodstuff and energy industry. Stroptomyces olivaceovirch Al from soil produce two
extracellular xylanase XYNA of the molecular weigh 43kD and XYNB of the molecular
weight 23kD. Optimal pH Value and temperature of XYNA fOr its activity were 5.6 and 60
OC, respectively. The Km Values and Vm. of XYNA for 4- O - Me - D - gl ucurono -D - xyl an
under 55C were 3.32g/Kg and l5.72pmol/ml. min, respectively. The activity of XYNA
could be decreased slighly by SDS and EDTA. Optimal pH Value and temperatUre of
XYNB for its activity were 5.2 and 60aC, respectively. The Km Values and Vm. of XYNB
for 4- O -Me-D-glucurono-D-xylan under 55C were 22.lg/Kg and l05.26pmoUml' min,
respectively. Mg'+ and Cr3+ can increased the activity of XYNB slighly. Both XYNA and
XYNB didn't display cellulase activity Both XYNA and XYNB were treated by pepsin
or trypsin fOr 30 min under 37C, their activity weren't decreased. The N-terminal amino
acids sequences of XYNB and XYNA were determined by axnino acids sequence analysis.
The result show that the ten N-terminal amino acids sequences of XYNA are same as those
of FXYN published by Atsushi. According to the published data, three primers had been
desighed and synthesized, xyndj-s including siginal sequence and xynA!-w
non-including siginal sequence had been cloned by PCR. According to the determined
N-terminal amino acids sequence of XYNB and the C-terminal conservative amino acids
sequence of G/ll family xylanase. Two primers were desighed and synthesized. XynBj
coding nature protein of XYNB have been cloned by PCR. Then xynd1-s nynA]-w and
xynB1 were all cloned into the plasmid vector pET-22b(+), and were all expressed in E coli.
rynAI-w and xynB1 were all cloned into the plasmid vector pPIC9 Q,and were all
overexpressed in pichia pastoris GS l 1 5.
The nucleic acids sequence and amino acids sequence of xynBi were homologously
analysed with the sequences coding fOr xylanases published in GeneBank. The result
indicated that xynB] was the highest similarity to the gene xyli from sm ptomyces sP.
Strain S38, up to 86%. And the properties of XYLl was different in those of XYNB. So,it
proves that xynBi is a new gene encoding xylanase. The sequence of xynB] has been
accepted by EMBL(Accession number is AJ2923 l7).
引文
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