CN114807094A - 甲壳素酶SvChiAJ54及其编码基因与应用 - Google Patents
甲壳素酶SvChiAJ54及其编码基因与应用 Download PDFInfo
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- CN114807094A CN114807094A CN202210480284.3A CN202210480284A CN114807094A CN 114807094 A CN114807094 A CN 114807094A CN 202210480284 A CN202210480284 A CN 202210480284A CN 114807094 A CN114807094 A CN 114807094A
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- CN
- China
- Prior art keywords
- chitinase
- svchiaj54
- chitosan
- chitobiose
- chitotriose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明公开了一种甲壳素酶SvChiAJ54,属于功能酶技术领域,其氨基酸序列如SEQ ID NO.1所示,其编码基因的核苷酸序列如SEQ ID NO.2所示。本发明还公开了该甲壳素酶在降解胶质甲壳素或甲壳寡糖中的应用,在制备甲壳二糖和甲壳三糖中的应用。本发明还公开了一种制备甲壳二糖和甲壳三糖的方法。本发明的甲壳素酶SvChiAJ54能完全降解胶质甲壳素、甲壳寡糖,最小的降解单位是甲壳四糖,可将甲壳五糖、甲壳六糖降解为甲壳二糖和甲壳三糖。本发明的甲壳素酶SvChiAJ54降解效率高,适合于工业应用,在酶法制备甲壳二糖和甲壳三糖中具有重要的工业应用价值和经济价值。
Description
技术领域
本发明涉及一种甲壳素酶SvChiAJ54,及其编码基因,以及在降解胶质甲壳素或甲壳寡糖中的应用,在制备甲壳二糖和甲壳三糖中的应用,属于功能酶技术领域。
背景技术
甲壳寡糖是通过β-1,4糖苷键将N-乙酰氨基葡萄糖连接成低聚合度的糖类,它是自然界中含量丰富的天然高分子多糖甲壳素的降解产物,在医药、食品、农业、饲料和化妆品领域具有广泛的前景。甲壳寡糖是低热量的甜味剂,也是肠道有益微生物菌群的增值因子,可以作为食品添加剂。甲壳寡糖能激活植物免疫防御***,同时能作为生长调节剂促进植物生长,可以用于农作物抗病、抗虫等防治病虫害。甲壳寡糖结构中含有较多亲水基团,同时具有抑菌的作用,可以作为化妆品的原料。此外,甲壳二糖除了具有较好的抗菌性,还有螯合的作用;甲壳三糖对人类溶菌酶淀粉样变性具有治疗作用。
甲壳寡糖的常用制备法是化学酸解法,即用浓酸处理断裂甲壳素长链分子间的β-1,4糖苷键,制备出不同分子量的甲壳寡糖,这种制备方法对生产设备的要求高,后期需要大量的水冲洗至中性,会造成水资源的浪费,并且产物分子量不均一,纯化单一产物难度大。酶法制备甲壳寡糖有反应条件温和、反应过程可控性强、反应产物纯度高的优势,能实现在工业生产中甲壳寡糖的绿色制备。目前甲壳素酶的酶解产物主要是甲壳二糖和N-乙酰氨基葡萄糖,缺少能制备其他甲壳寡糖的特异性甲壳素酶。
发明内容
针对上述现有技术,本发明提供了一种新的甲壳素酶——甲壳素酶SvChiAJ54,本发明还提供了其编码基因,以及其在降解胶质甲壳素或甲壳寡糖中的应用,在制备甲壳二糖(GlcNAc)2和和甲壳三糖(GlcNAc)3中的应用。
本发明是通过以下技术方案实现的:
甲壳素酶SvChiAJ54,其氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1:
MRTRLIGLLTAAASALTLSVLALGPTAQADTRAAADDFIVSEGQFESMFPNRNSFYTYSGLTDALSAYPGFTGTGSDTTRKQEAAAFLANVSHETGGLVHIVEQNEANYPHYCDSTQPFGCPAGNDKYYGRGPVQLSWNYNYKAAGDALGIDLLNNPDLVQNDASVAWKTGLWFWNTQSGAGRMTPHDAIVNGAGFGETIRSINGSIECDGGNPGQVQSRVDLYERFTDVLGVEPGDNLSC。
上述甲壳素酶SvChiAJ54的编码基因,其核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.2:
5’-ATGCGTACCCGTCTGATCGGGCTGCTGACCGCGGCCGCGTCCGCGCTGACGCTCTCCGTGCTGGCCCTCGGGCCCACCGCGCAGGCCGATACCCGCGCCGCCGCCGACGACTTCATCGTCAGCGAAGGCCAGTTCGAGTCGATGTTCCCGAACCGGAACTCCTTCTACACCTACTCGGGCCTCACCGACGCGCTCAGCGCCTACCCGGGCTTCACCGGCACCGGCAGCGACACCACACGCAAGCAGGAGGCCGCGGCCTTCCTCGCCAACGTCAGCCACGAGACGGGCGGCCTCGTCCACATCGTCGAGCAGAACGAGGCGAACTACCCGCACTACTGCGACAGCACCCAGCCCTTCGGCTGCCCCGCCGGAAACGACAAGTACTACGGACGCGGGCCGGTCCAGCTCAGCTGGAACTACAACTACAAGGCCGCGGGCGACGCCCTCGGAATCGATCTGCTGAACAATCCCGATCTCGTCCAGAATGACGCCTCGGTCGCCTGGAAAACCGGCCTCTGGTTCTGGAACACCCAGTCCGGAGCGGGCCGTATGACCCCGCACGACGCGATCGTGAACGGTGCCGGATTCGGCGAGACCATTCGCTCCATCAACGGCAGCATCGAATGCGACGGCGGAAATCCCGGCCAGGTCCAGAGCCGTGTCGACCTCTACGAGAGGTTCACCGACGTCCTCGGCGTCGAGCCCGGCGACAACCTCAGCTGCTGA-3’。
所述甲壳素酶SvChiAJ54在降解胶质甲壳素或甲壳寡糖中的应用。所述甲壳寡糖选自甲壳四糖、甲壳五糖、甲壳六糖中的任意一种或两种以上。
所述甲壳素酶SvChiAJ54在制备甲壳二糖和甲壳三糖中的应用。
一种制备甲壳二糖和甲壳三糖的方法:采用上述甲壳素酶SvChiAJ54酶解胶质甲壳素或甲壳寡糖,制备得到甲壳二糖和甲壳三糖;所述甲壳寡糖选自甲壳四糖、甲壳五糖、甲壳六糖中的任意一种或两种以上。
进一步地,所述甲壳素酶SvChiAJ54的酶解条件为:酶解温度25℃~80℃,酶解pH3.0~10.0,酶解时间30分钟~72小时。
更进一步地,酶解温度40℃~50℃,酶解pH 5.0~7.0,酶解时间3~24小时。
更进一步地,酶解温度45℃、70℃、75℃或80℃;酶解pH 6.0、9.0或10.0;酶解时间24小时。
一种酶制剂,其有效成分为甲壳素酶SvChiAJ54。该酶制剂在降解胶质甲壳素或甲壳寡糖中的应用,在制备甲壳二糖和甲壳三糖中的应用。
一种重组表达载体,其携带有上述甲壳素酶SvChiAJ54的编码基因。
一种重组工程菌,其基因组中包含有上述甲壳素酶SvChiAJ54的编码基因,能表达甲壳素酶SvChiAJ54。
所述重组表达载体在制备壳素酶SvChiAJ54中的应用。
所述重组工程菌在制备壳素酶SvChiAJ54中的应用。
本发明的甲壳素酶SvChiAJ54,能完全降解胶质甲壳素、甲壳寡糖,最小的降解单位是甲壳四糖,可将甲壳五糖降解为甲壳二糖和甲壳三糖,可将甲壳六糖降解为甲壳二糖和甲壳三糖。本发明的壳素酶SvChiAJ54,反应温度为25℃~80℃,相对酶活保持在70%以上,反应温度可控范围大;在45℃放置24小时残余酶活在50%以上;在35℃、40℃放置24小时残余酶活在75%以上,温度稳定性良好。在pH 3.0~10.0条件下相对酶活保持在60%以上,适应性广;在pH 3.0~10.0缓冲液中放置72小时后残余酶活仍可达70%以上,pH稳定性良好。在45℃、pH6.0时酶活可达184.31 U/mg,降解效率高,适合于工业应用,在酶法制备甲壳二糖和甲壳三糖中具有重要的工业应用价值和经济价值。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1:SDS-PAGE电泳图,其中,M:Marker;1:20 mM咪唑溶液洗脱的蛋白;2~5:40 mM咪唑溶液洗脱的蛋白;6~9:60 mM咪唑溶液洗脱的目的蛋白。
图2:pH变化对相对酶活的影响示意图。
图3:甲壳素酶SvChiAJ54的pH稳定性示意图。
图4:温度变化对相对酶活的影响示意图。
图5:甲壳素酶SvChiAJ54的温度稳定性示意图。
图6:甲壳素酶SvChiAJ54降解甲壳寡糖(DP 2~6)的产物的高效液相色谱图(甲壳二糖、甲壳三糖、甲壳四糖)。
图7:甲壳素酶SvChiAJ54降解甲壳寡糖(DP 2~6)的产物的高效液相色谱图(甲壳五糖、甲壳六糖)。
图8:甲壳素酶SvChiAJ54降解胶质甲壳素的产物的高效液相色谱图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域技术人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料,可通过正规商业途径获得。下述实施例中所涉及的实验方法、检测方法,若无特别说明,均为现有技术中已有的常规实验方法、检测方法。
本发明所涉及的菌种Streptomyces violascens,来自于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC 4.1923,该菌种是由他人保藏的,本发明是从中国微生物菌种保藏管理委员会普通微生物中心购买得到的。
本发明首次克隆并表达了甲壳素酶SvChiAJ54,并探究其酶学性质和水解产物。本发明在已有的菌种资源中做了全面的筛选,选出具有特色的此酶进行研究。
实施例1 甲壳素酶SvChiAJ54的编码基因的克隆
提取菌种Streptomyces violascens的基因组DNA,以其为模板,进行PCR扩增,得目的基因片段——甲壳素酶SvChiAJ54的编码基因,如SEQ ID NO.2所示,包含726个碱基,其表达的甲壳素酶SvChiAJ54包含241个氨基酸,如SEQ ID NO.1所示。
克隆目的基因时去除自身信号肽,利用载体信号肽。PCR扩增所采用特异性引物如下所示:
上游引物:5’-ctcagctgcaGATACCCGCGCCGCCGCCGACGACTTCATC-3’,如SEQ ID NO.3所示。
下游引物:5’-catcatcagtggtggtggtggtggtgGCAGCTGAGGTTGTCGCCGGG-3’,如SEQID NO.4所示。
PCR扩增的反应体系为:2×PCR Buffer 25 μl,dNTP 10 μl,无菌水10 μl,上下游引物各1.5 μl,模板1 μl,KOD Fx酶1 μl,总体系50 μl。
PCR扩增的反应条件为:94℃预变性5 min,95℃变性20 s,72℃退火30 s,72℃延伸60 s,反应30个循环,72℃后延伸10 min。
实施例2 甲壳素酶SvChiAJ54的表达与纯化
将目的基因片段与pP43NMK载体进行无缝连接,将重组质粒转入枯草芽孢杆菌Bacillus subtilis WB800感受态细胞中,使用含卡那霉素的LB培养基固体平板筛选得到阳性转化子,进行菌落PCR验证后,挑取阳性克隆测序,提取测序正确的重组质粒,命名为pP43NMK- SvChiAJ54。
将含有重组质粒的Bacillus subtilis WB800菌株接种到LB液体培养基中,37℃培养12小时,然后按1%的接种量接入50 mL的LB液体培养基中,30℃、220 r/min摇床培养45小时,得培养液。培养液于4℃、8000 r/min离心15 min,收集上清液,即为粗酶液。
使用Ni-NTA柱亲和层析纯化粗酶液,首先使用平衡缓冲液(10 mM咪唑,500 mMNaCl,50 mM Tris-HCl)平衡柱子,然后依次用咪唑20 mM溶液(20 mM咪唑,500 mM NaCl,50mM Tris-HCl)和咪唑40 mM溶液(40 mM咪唑,500 mM NaCl,50 mM Tris-HCl)洗脱结合力弱的杂蛋白,再用60 mM咪唑溶液(60 mM咪唑,500 mM NaCl,50 mM Tris-HCl)洗脱目的蛋白,得洗脱液,浓缩至蛋白浓度为0.0153 mg/mL(使用Bradford法测定酶液中的蛋白浓度),得纯酶液。对纯酶液进行SDS-PAGE检测,结果如图1所示,所得目的蛋白的分子量约为26.72kDa,与预测相一致,证明该目的蛋白即为SEQ ID NO.1所示的蛋白。同时采用DNS验证所获蛋白是否具有酶活(实施例3)。
实施例3 甲壳素酶SvChiAJ54的酶活测定
甲壳素酶SvChiAJ54粗酶的酶活和纯酶的比酶活测定
取20 μL的粗酶液、纯酶液,分别加入到180 μL的50 mM磷酸盐缓冲液(pH6.0)配制的胶质甲壳素溶液(胶质甲壳素的浓度为0.01 g/ml)中,在45℃下反应10 min,煮沸5 min终止反应,加入300 μL的DNS试剂沸水浴10 min进行显色,冰上冷却5 min,加入1mL去离子水,取200 μL在OD540nm下检测其吸光度值。酶活力定义为在标准条件下每min产生1 μmol还原糖所需要的酶量。经测定,粗酶液的酶活为4700 U/L,纯酶液的酶活为184.31 U/mg。
实施例4 甲壳素酶SvChiAJ54的最适反应pH和pH稳定性
最适pH值测定方法为:将20μL的纯酶液加入到180 μL的不同pH的缓冲液配制的胶质甲壳素溶液(胶质甲壳素的浓度为0.01 g/ml)中,不同pH的缓冲液分别为:柠檬酸缓冲液(pH 3.0~6.0)、磷酸盐缓冲液(pH 6.0~8.0)、Tris-HCl缓冲液(pH 7.0~9.0)和甘氨酸-NaOH缓冲液(pH 9.0~10.0),在45℃下反应30 min。DNS法测定纯酶的活性,结果如图2所示,最适反应pH为pH 6.0(磷酸盐缓冲液),pH 3.0~10.0范围内的相对酶活均在60%以上。
pH稳定性测定方法为:将20μL的纯酶液加入到180 μL的不同pH的缓冲液中,不同pH的缓冲液分别为:柠檬酸缓冲液(pH 3.0~6.0)、磷酸盐缓冲液(pH 6.0~8.0)、Tris-HCl缓冲液(pH 7.0~9.0)和甘氨酸-NaOH缓冲液(pH 9.0~10.0),在25℃下放置 0、12、24、36、48、60、72小时。加入胶质甲壳素(胶质甲壳素的浓度为0.01 g/ml),在45℃下反应30 min,测定残留酶活,结果如图3所示,在pH 3.0~10.0缓冲液中,72小时后残余酶活仍可达70%以上。
实施例5 甲壳素酶SvChiAJ54的最适反应温度和温度稳定性
最适温度测定方法为:将20 μL的纯酶液加入到180 μL的50 mM磷酸盐缓冲液(pH6.0)配制的胶质甲壳素溶液(胶质甲壳素的浓度为0.01 g/ml)中,在不同温度(25、30、35、40、45、50、55、60、65、70、75、80℃)下反应30 min,DNS法测定酶的活性,结果如图4所示,最适反应温度为45℃。
热稳定性测定方法为:将20 μL的纯酶液加入到180 μL的50 mM磷酸盐缓冲液(pH6.0)中,在不同温度(35、40、45、50℃)下放置0、3、6、9、12、15、18、21、24 小时。加入胶质甲壳素(加入后胶质甲壳素的浓度为0.01 g/ml),在45℃下反应30 min,测定残留酶活,结果如图5所示,在45℃放置24小时,残余酶活在50%以上;在35℃、40℃放置24小时,残余酶活在75%以上。
实施例6 甲壳素酶SvChiAJ54降解甲壳寡糖(DP 2~6)
取甲壳寡糖(DP 2~6),加入到50 mM磷酸盐缓冲液(pH6.0)中,配制标准品溶液(标准品溶液中,甲壳二糖、甲壳三糖、甲壳四糖、甲壳五糖、甲壳六糖的浓度均为20 mg/mL);取标准品溶液20 μL,加入180μL的纯酶液,45℃反应24小时,底物甲壳寡糖(DP 2~6)完全反应。取反应液20μL,上样进行高效液相色谱检测,色谱柱为Sugar Pak I(6.5× 300mm),泵的流量0.5 mL/min,50 mg/L的流动相乙二胺四乙酸钙二钠,柱温75℃,检测器为示差检测器。结果如图6、图7所示,由图可见,甲壳素酶SvChiAJ54最小的酶切单位是甲壳四糖,甲壳四糖完全降解产物是甲壳二糖,甲壳五糖和甲壳六糖完全降解产物是甲壳二糖和甲壳三糖。
实施例7 甲壳素酶SvChiAJ54降解胶质甲壳素
将20 μL的纯酶液加入到180 μL的50 mM磷酸盐缓冲液(pH6.0)配制的胶质甲壳素溶液(胶质甲壳素的浓度为0.01 g/ml)中,45℃反应0 min、2 min、15 min、30 min、60 min、24小时,煮沸终止反应。离心取上清,过0.22μm滤膜,上样20μL进行高效液相色谱检测,色谱柱为Sugar Pak I(6.5× 300 mm),泵的流量0.5 mL/min,50 mg/L的流动相乙二胺四乙酸钙二钠,柱温75℃,检测器为示差检测器。结果如图7所示,甲壳素酶SvChiAJ54降解胶质甲壳素的产物为甲壳二糖和甲壳三糖。经进一步高效液相色谱法检测降解24小时的产物中,甲壳二糖占64%,甲壳三糖占36%。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
序列表
<110> 中国海洋大学
<120> 甲壳素酶SvChiAJ54及其编码基因与应用
<141> 2022-05-03
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 241
<212> PRT
<213> Streptomyces violascens
<400> 1
Met Arg Thr Arg Leu Ile Gly Leu Leu Thr Ala Ala Ala Ser Ala Leu
1 5 10 15
Thr Leu Ser Val Leu Ala Leu Gly Pro Thr Ala Gln Ala Asp Thr Arg
20 25 30
Ala Ala Ala Asp Asp Phe Ile Val Ser Glu Gly Gln Phe Glu Ser Met
35 40 45
Phe Pro Asn Arg Asn Ser Phe Tyr Thr Tyr Ser Gly Leu Thr Asp Ala
50 55 60
Leu Ser Ala Tyr Pro Gly Phe Thr Gly Thr Gly Ser Asp Thr Thr Arg
65 70 75 80
Lys Gln Glu Ala Ala Ala Phe Leu Ala Asn Val Ser His Glu Thr Gly
85 90 95
Gly Leu Val His Ile Val Glu Gln Asn Glu Ala Asn Tyr Pro His Tyr
100 105 110
Cys Asp Ser Thr Gln Pro Phe Gly Cys Pro Ala Gly Asn Asp Lys Tyr
115 120 125
Tyr Gly Arg Gly Pro Val Gln Leu Ser Trp Asn Tyr Asn Tyr Lys Ala
130 135 140
Ala Gly Asp Ala Leu Gly Ile Asp Leu Leu Asn Asn Pro Asp Leu Val
145 150 155 160
Gln Asn Asp Ala Ser Val Ala Trp Lys Thr Gly Leu Trp Phe Trp Asn
165 170 175
Thr Gln Ser Gly Ala Gly Arg Met Thr Pro His Asp Ala Ile Val Asn
180 185 190
Gly Ala Gly Phe Gly Glu Thr Ile Arg Ser Ile Asn Gly Ser Ile Glu
195 200 205
Cys Asp Gly Gly Asn Pro Gly Gln Val Gln Ser Arg Val Asp Leu Tyr
210 215 220
Glu Arg Phe Thr Asp Val Leu Gly Val Glu Pro Gly Asp Asn Leu Ser
225 230 235 240
Cys
<210> 3
<211> 726
<212> DNA
<213> Streptomyces violascens
<400> 3
atgcgtaccc gtctgatcgg gctgctgacc gcggccgcgt ccgcgctgac gctctccgtg 60
ctggccctcg ggcccaccgc gcaggccgat acccgcgccg ccgccgacga cttcatcgtc 120
agcgaaggcc agttcgagtc gatgttcccg aaccggaact ccttctacac ctactcgggc 180
ctcaccgacg cgctcagcgc ctacccgggc ttcaccggca ccggcagcga caccacacgc 240
aagcaggagg ccgcggcctt cctcgccaac gtcagccacg agacgggcgg cctcgtccac 300
atcgtcgagc agaacgaggc gaactacccg cactactgcg acagcaccca gcccttcggc 360
tgccccgccg gaaacgacaa gtactacgga cgcgggccgg tccagctcag ctggaactac 420
aactacaagg ccgcgggcga cgccctcgga atcgatctgc tgaacaatcc cgatctcgtc 480
cagaatgacg cctcggtcgc ctggaaaacc ggcctctggt tctggaacac ccagtccgga 540
gcgggccgta tgaccccgca cgacgcgatc gtgaacggtg ccggattcgg cgagaccatt 600
cgctccatca acggcagcat cgaatgcgac ggcggaaatc ccggccaggt ccagagccgt 660
gtcgacctct acgagaggtt caccgacgtc ctcggcgtcg agcccggcga caacctcagc 720
tgctga 726
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 3
ctcagctgca gatacccgcg ccgccgccga cgacttcatc 40
<210> 4
<211> 47
<212> DNA
<213> Artificial Sequence
<400> 4
catcatcagt ggtggtggtg gtggtggcag ctgaggttgt cgccggg 47
Claims (10)
1. 甲壳素酶SvChiAJ54,其氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述的甲壳素酶SvChiAJ54的编码基因,其核苷酸序列如SEQ ID NO.2所示。
3.权利要求1所述的甲壳素酶SvChiAJ54在降解胶质甲壳素或甲壳寡糖中的应用,其特征在于:所述甲壳寡糖选自甲壳四糖、甲壳五糖、甲壳六糖中的任意一种或两种以上。
4.权利要求1所述的甲壳素酶SvChiAJ54在制备甲壳二糖和甲壳三糖中的应用。
5.一种制备甲壳二糖和甲壳三糖的方法,其特征在于:采用权利要求1所述的甲壳素酶SvChiAJ54酶解胶质甲壳素或甲壳寡糖,制备得到甲壳二糖和甲壳三糖;所述甲壳寡糖选自甲壳四糖、甲壳五糖、甲壳六糖中的任意一种或两种以上。
6.根据权利要求5所述的制备甲壳二糖和甲壳三糖的方法,其特征在于:所述甲壳素酶SvChiAJ54的酶解条件为:酶解温度为25℃~80℃,酶解pH为3.0~10.0,酶解时间为30分钟~72小时。
7.根据权利要求6所述的制备甲壳二糖和甲壳三糖的方法,其特征在于:酶解温度为40℃~50℃,酶解pH为5.0~7.0,酶解时间为3~24小时;
或:酶解温度为45℃、70℃、75℃或80℃;酶解pH为6.0、9.0或10.0;酶解时间为24小时。
8.酶制剂在降解胶质甲壳素或甲壳寡糖中的应用,在制备甲壳二糖和甲壳三糖中的应用,其特征在于:所述酶制剂的有效成分为甲壳素酶SvChiAJ54。
9.一种重组表达载体,其特征在于:其携带有权利要求2所述的甲壳素酶SvChiAJ54的编码基因。
10.一种重组工程菌,其特征在于:其基因组中包含有权利要求2所述的甲壳素酶SvChiAJ54的编码基因,能表达权利要求1所述的甲壳素酶SvChiAJ54。
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